Publications by year
In Press
Lin R, Clowsley AH, Lutz T, Baddeley D, Soeller C (In Press). 3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.
Abstract:
3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples
AbstractAssessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time-and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.
Abstract.
Hartwich TMP, Hin Chung KK, Schroeder L, Bewersdorf J, Soeller C, Baddeley D (In Press). A stable, high refractive index, switching buffer for super-resolution imaging.
Abstract:
A stable, high refractive index, switching buffer for super-resolution imaging
AbstractdSTORM super-resolution imaging relies on switching buffers to enable dye molecules to enter and exit a metastable dark state. Current buffers have a very limited shelf life of approximately 1 day and poorly match sample refractive index, impacting negatively on measurement reproducibility and image fidelity. We present a buffer based on chemical, rather than enzymatic, oxygen scavenging which exhibits dramatically improved stability, switching speed, contrast, and index matching.
Abstract.
Hunt H, Tilunaite A, Bass G, Soeller C, Roderick HL, Rajagopal V, Crampin EJ (In Press). Ca2+ release via IP3 receptors shapes the cytosolic Ca2+ transient for. hypertrophic signalling in ventricular cardiomyocytes.
Abstract:
Ca2+ release via IP3 receptors shapes the cytosolic Ca2+ transient for. hypertrophic signalling in ventricular cardiomyocytes
Calcium (Ca2+) plays a central role in mediating both contractile function
and hypertrophic signalling in ventricular cardiomyocytes. L-type Ca2+ channels
trigger release of Ca2+ from ryanodine receptors (RyRs) for cellular
contraction, while signalling downstream of Gq coupled receptors stimulates
Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP3Rs), engaging
hypertrophic signalling pathways. Modulation of the amplitude, duration, and
duty cycle of the cytosolic Ca2+ contraction signal, and spatial localisation,
have all been proposed to encode this hypertrophic signal. Given current
knowledge of IP3Rs, we develop a model describing the effect of functional
interaction (cross-talk) between RyR and IP3R channels on the Ca2+ transient,
and examine the sensitivity of the Ca2+ transient shape to properties of IP3R
activation. A key result of our study is that IP3R activation increases Ca2+
transient duration for a broad range of IP3R properties, but the effect of IP3R
activation on Ca2+ transient amplitude is dependent on IP3 concentration.
Furthermore we demonstrate that IP3-mediated Ca2+ release in the cytosol
increases the duty cycle of the Ca2+ transient, the fraction of the cycle for
which [Ca2+] is elevated, across a broad range of parameter values and IP3
concentrations. When coupled to a model of downstream transcription factor
(NFAT) activation, we demonstrate that there is a high correspondence between
the Ca transient duty cycle and the proportion of activated NFAT in the
nucleus. These findings suggest increased cytosolic Ca2+ duty cycle as a
plausible mechanism for IP3-dependent hypertrophic signalling via
Ca2+-sensitive transcription factors such as NFAT in ventricular
cardiomyocytes.
Abstract.
Author URL.
Tilūnaitė A, Ladd D, Hunt H, Soeller C, Roderick HL, Crampin EJ, Rajagopal V (In Press). Ca2+ release via IP3Rs increases RyR mediated Ca2+ spark frequency in ventricular cardiomyocytes without altering spark amplitude and duration.
Abstract:
Ca2+ release via IP3Rs increases RyR mediated Ca2+ spark frequency in ventricular cardiomyocytes without altering spark amplitude and duration
AbstractCalcium plays critical roles in cardiac cells, coupling electrical excitation to mechanical contraction with each heartbeat, while simultaneously mediating biochemical signals that regulate cell growth. While ryanodine receptors (RyRs) are fundamental to generation of elementary calcium release events (sparks) and global calcium elevations that underlie excitation-contraction coupling (ECC), calcium release via inositol 1,4,5-trisphosphate receptors (IP3Rs) is also reported in cardiomyocytes. IP3R calcium release modifies ECC as well as contributing to downstream regulation of hypertrophic gene expression. Recent studies suggest that proximal localisation of IP3Rs with RyRs contributes to their ability to modify Ca2+ handling during ECC. Here we aim to determine the mechanism by which IP3Rs modify Ca2+ handling in cardiomyocytes. We develop a mathematical model incorporating the stochastic behaviour of receptor opening that allows for the parametric tuning of the system to reveal the impact of IP3Rs on spark activation. By testing multiple spark initiation mechanisms, we find that Ca2+ release via IP3Rs result in increased propensity for spark initiation within the cardiac dyad. Our simulations suggest that opening of IP3Rs elevates Ca2+ within the dyad, which increase the probability of spark initiation. Finally, we find that while increasing the number of IP3Rs increases the probability of spark formation, it has little effect on spark amplitude, duration, or overall shape. Our study therefore suggests that IP3R play a critical role in modulating Ca2+ signaling for excitation contraction couplingAuthor summaryWhile Ca2+ release through ryanodine receptors (RyRs) initiates contraction in cardiomyocytes, the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) in cardiomyocytes is less clear with Ca2+ release through these channels being invoked in regulating ECC and hypertrophic signalling. RyRs generate cytosolic Ca2+ signals through elemental Ca2+ release events called sparks. The mechanisms by which IP3Rs influence cytosolic Ca2+ are not well understood. We created a 1D model of calcium spark formation in a cardiomyocyte dyad—the primary site of elemental RyR-based calcium release. We investigated possible behaviours of IP3Rs and their interaction with RyRs in generating Ca2+ sparks. We show that for high IP3 concentration, a large number of IP3Rs and high IP3R affinity are required to noticeably affect spark shape. At lower IP3 concentration IP3Rs can increase Ca2+ spark activity, but do not significantly alter the spark shape. Finally our simulations suggest that spark frequency can be reliably increased when IP3Rs activity is such that a small continuous Ca2+ flux is introduced to the dyad to elevate Ca2+, and not via brief but high Ca2+ release from these receptors.
Abstract.
Ladd D, Tilunaite A, Soeller C, Roderick HL, Crampin E, Rajagopal V (In Press). Detecting RyR clusters with CaCLEAN: influence of spatial distribution and structural heterogeneity.
Abstract:
Detecting RyR clusters with CaCLEAN: influence of spatial distribution and structural heterogeneity
AbstractIn cardiomyocytes, the coordinated release of calcium ions from intracellular stores through ryanodine receptor (RyR) clusters is key to the generation of a calcium transient and induction of contraction. Recently, a deconvolution algorithm from radio astronomy was adapted and applied to analysis of calcium fluorescence imaging of cardiomyocytes. The algorithm, CaCLEAN, showed potential in revealing both the spatial locations of RyR clusters and their functional response in living cells. However, whether the RyR clusters identified by CaCLEAN analysis of the imaging data were true or false positives remained unvalidated in the absence of ground truth values. In this work, a structurally realistic finite element model was developed to simulate reaction-diffusion of calcium emanating from RyR clusters during the rising phase (first 30 ms) of the calcium transient. The effect of two sets of factors were examined with the model: (1) the number and spacing of simulated RyR clusters and (2) the effect of mitochondria acting as barriers to diffusion. Confocal fluorescence microscopy images were simulated from the model results and analysed using CaCLEAN. The performance of CaCLEAN was found to be sensitive to cluster spacing and distance from the imaging plane. In a case with sparsely-packed clusters, detection recall and precision were 0.82 for clusters up to 610 nm from the imaging plane; in a densely-packed cluster case, recall and precision were 0.69 for clusters up to 280 nm from the imaging plane. Users interested in applying CaCLEAN to their data should therefore consider the likely density of cluster distributions and the trade-off between precision and recall when determining the maximum relevant depth of RyR clusters in their application.
Abstract.
Clowsley AH, Kaufhold WT, Lutz T, Meletiou A, Michele LD, Soeller C (In Press). Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy.
Abstract:
Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy
ABSTRACTInteractions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in-situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present Proximity-Dependent PAINT (PD-PAINT), a method for sub-diffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA labels anchored to the target species. Labeled protein pairs are then imaged with high contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution and the ability to image quantitatively.
Abstract.
Wakefield J, Soeller C, Jeynes J, Tariq A (In Press). In vitro reconstitution of branching microtubule nucleation.
eLife,
8 Full text.
Marin Z, Graff M, Barentine AES, Soeller C, Chung KKH, Fuentes LA, Baddeley D (In Press). PYMEVisualize: an open-source tool for exploring 3D super-resolution data.
Abstract:
PYMEVisualize: an open-source tool for exploring 3D super-resolution data
ABSTRACTLocalization-based super-resolution microscopy techniques such as PALM, STORM, and PAINT are increasingly critical tools for biological discovery. These methods generate lists of single fluorophore positions that capture nanoscale structural details of subcellular organisation, but to develop biological insight, we must post-process and visualize this data in a meaningful way. A large number of algorithms have been developed for localization post-processing, transforming point data into representations which approximate traditional microscopy images, and performing specific quantitative analysis directly on points. Implementations of these algorithms typically stand in isolation, necessitating complex workflows involving multiple different software packages. Here we present PYMEVisualize, an open-source tool for the interactive exploration and analysis of 3D, multicolor, single-molecule localization data. PYMEVisualize brings together a broad range of the most commonly used post-processing, density mapping, and direct quantification tools in an easy-to-use and extensible package. This software is one component of the PYthon Microscopy Environment (python-microscopy.org), an integrated application suite for light microscopy acquisition, data storage, visualization, and analysis built on top of the scientific Python environment.
Abstract.
Clowsley AH, Kaufhold WT, Lutz T, Meletiou A, Di Michele L, Soeller C (In Press). Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy.
Abstract:
Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy
AbstractDNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA ‘imagers’ to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.
Abstract.
Hofer M, Soeller C, Brasselet S, Bertolotti J (In Press). Wide field fluorescence epi-microscopy behind a scattering medium. enabled by speckle correlations.
Opt. Express,
26, 9866-9881.
Abstract:
Wide field fluorescence epi-microscopy behind a scattering medium. enabled by speckle correlations
Fluorescence microscopy is widely used in biological imaging, however
scattering from tissues strongly limits its applicability to a shallow depth.
In this work we adapt a methodology inspired from stellar speckle
interferometry, and exploit the optical memory effect to enable fluorescence
microscopy through a turbid layer. We demonstrate efficient reconstruction of
micrometer-size fluorescent objects behind a scattering medium in
epi-microscopy, and study the specificities of this imaging modality
(magnification, field of view, resolution) as compared to traditional
microscopy. Using a modified phase retrieval algorithm to reconstruct
fluorescent objects from speckle images, we demonstrate robust reconstructions
even in relatively low signal to noise conditions. This modality is
particularly appropriate for imaging in biological media, which are known to
exhibit relatively large optical memory ranges compatible with tens of
micrometers size field of views, and large spectral bandwidths compatible with
emission fluorescence spectra of tens of nanometers widths.
Abstract.
Author URL.
Full text.
2021
Clowsley AH, Kaufhold WT, Lutz T, Meletiou A, Di Michele L, Soeller C (2021). Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy.
Nature Communications,
12(1).
Abstract:
Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy
AbstractDNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA ‘imagers’ to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.
Abstract.
2020
Lin R, Clowsley AH, Lutz T, Baddeley D, Soeller C (2020). 3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.
Methods,
174, 56-71.
Abstract:
3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.
Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time- and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.
Abstract.
Author URL.
Clowsley AH, Kaufhold WT, Lutz T, Meletiou A, Di Michele L, Soeller C (2020). Detecting Nanoscale Distribution of Protein Pairs by Proximity-Dependent Super-resolution Microscopy.
J Am Chem Soc,
142(28), 12069-12078.
Abstract:
Detecting Nanoscale Distribution of Protein Pairs by Proximity-Dependent Super-resolution Microscopy.
Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively.
Abstract.
Author URL.
Dawes ML, Soeller C, Scholpp S (2020). Studying molecular interactions in the intact organism: fluorescence correlation spectroscopy in the living zebrafish embryo.
HISTOCHEMISTRY AND CELL BIOLOGY,
154(5), 507-519.
Author URL.
Soeller C (2020). Super-resolution microscopy illuminates cardiac structure, function and pathology at the nanoscale. Microscopy and Microanalysis
Novillo CS, Oliveras A, Comes N, Soeller C, Felipe A (2020). The Cardiac K(v)7.1-KCNE1 Channel Assembles at ER-PM Junctions before Translocated to the Plasma Membrane.
Author URL.
Oliveras A, Serrano-Novillo C, Moreno C, de la Cruz A, Valenzuela C, Soeller C, Comes N, Felipe A (2020). The unconventional biogenesis of Kv7.1-KCNE1 complexes.
SCIENCE ADVANCES,
6(14).
Author URL.
2019
Shen X, van den Brink J, Hou Y, Colli D, Le C, Kolstad TR, MacQuaide N, Carlson CR, Kekenes-Huskey PM, Edwards AG, et al (2019). 3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes.
J Physiol,
597(2), 399-418.
Abstract:
3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes.
KEY POINTS: Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non-dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z-axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. ABSTRACT: Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z-axis and obscured detection by 2D techniques. Non-overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single 'rogue' RyR. Internal CRUs were more tightly packed along z-lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t-tubules, which enabled quantification of dyadic and non-dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.
Abstract.
Author URL.
Ladd D, Tilūnaitė A, Roderick HL, Soeller C, Crampin EJ, Rajagopal V (2019). Assessing Cardiomyocyte Excitation-Contraction Coupling Site Detection from Live Cell Imaging Using a Structurally-Realistic Computational Model of Calcium Release.
Frontiers in Physiology,
10Abstract:
Assessing Cardiomyocyte Excitation-Contraction Coupling Site Detection from Live Cell Imaging Using a Structurally-Realistic Computational Model of Calcium Release
© Copyright © 2019 Ladd, Tilūnaitė, Roderick, Soeller, Crampin and Rajagopal. Calcium signaling plays a pivotal role in cardiomyocytes, coupling electrical excitation to mechanical contraction of the heart. Determining locations of active calcium release sites, and how their recruitment changes in response to stimuli and in disease states is therefore of central interest in cardiac physiology. Current algorithms for detecting release sites from live cell imaging data are however not easily validated against a known “ground truth,” which makes interpretation of the output of such algorithms, in particular the degree of confidence in site detection, a challenging task. Computational models are capable of integrating findings from multiple sources into a consistent, predictive framework. In cellular physiology, such models have the potential to reveal structure and function beyond the temporal and spatial resolution limitations of individual experimental measurements. Here, we create a spatially detailed computational model of calcium release in an eight sarcomere section of a ventricular cardiomyocyte, using electron tomography reconstruction of cardiac ultrastructure and confocal imaging of protein localization. This provides a high-resolution model of calcium diffusion from intracellular stores, which can be used as a platform to simulate confocal fluorescence imaging in the context of known ground truth structures from the higher resolution model. We use this capability to evaluate the performance of a recently proposed method for detecting the functional response of calcium release sites in live cells. Model permutations reveal how calcium release site density and mitochondria acting as diffusion barriers impact the detection performance of the algorithm. We demonstrate that site density has the greatest impact on detection precision and recall, in particular affecting the effective detectable depth of sites in confocal data. Our findings provide guidance on how such detection algorithms may best be applied to experimental data and give insights into limitations when using two-dimensional microscopy images to analyse three-dimensional cellular structures.
Abstract.
Ladd D, Tilunaite A, Roderick HL, Soeller C, Crampin E, Rajagopal V (2019). Detecting RyR Clusters with CaCLEAN: Validation and Influence of Spatial Heterogeneity.
Author URL.
Gulácsy CE, Meade R, Catici DAM, Soeller C, Pantos GD, Jones DD, Alibhai D, Jepson M, Valev VK, Mason JM, et al (2019). Excitation-Energy-Dependent Molecular Beacon Detects Early Stage Neurotoxic Aβ Aggregates in the Presence of Cortical Neurons.
ACS Chem Neurosci,
10(3), 1240-1250.
Abstract:
Excitation-Energy-Dependent Molecular Beacon Detects Early Stage Neurotoxic Aβ Aggregates in the Presence of Cortical Neurons.
There is now crucial medical importance placed on understanding the role of early stage, subvisible protein aggregation, particularly in neurodegenerative disease. While there are strategies for detecting such aggregates in vitro, there is no approach at present that can detect these toxic species associated with cells and specific subcellular compartments. We have exploited excitation-energy-dependent fluorescence edge-shift of recombinant protein labeled with a molecular beacon, to provide a sensitive read out for the presence of subvisible protein aggregates. To demonstrate the potential utility of the approach, we examine the major peptide associated with the initiation of Alzheimer's disease, amyloid β-protein (Aβ) at a patho-physiologically relevant concentration in mouse cortical neurons. Using our approach, we find preliminary evidence that subvisible Aβ aggregates are detected at specific subcellular regions and that neurons drive the formation of specific Aβ aggregate conformations. These findings therefore demonstrate the potential of a novel fluorescence-based approach for detecting and imaging protein aggregates in a cellular context, which can be used to sensitively probe the association of early stage toxic protein aggregates within subcellular compartments.
Abstract.
Author URL.
Full text.
Lutz T (2019). New optical super-resolution imaging approaches involving DNA nanotechnology.
Abstract:
New optical super-resolution imaging approaches involving DNA nanotechnology
With recent advances in optical super-resolution microscopy, biological structures can be imaged with single-nanometre resolution using visible light. One implementation thereof, DNA-PAINT (Point Accumulation for Imaging in Nano-scale Topography), is based on the highly specific and transient binding of fluorescently labelled oligonucleotides, the "imager strands", to complementary strands with which the targets are labelled, the "docking strands". The imager-docking binding events are detected as fluorescence blinking and can be localised with single-nanometre precision. From the set of localised events a super-resolution image can be assembled. DNA-PAINT has multiple advantages over other imaging methods, e.g. high photon yields resulting in high resolution, a free choice of fluorophores while being effectively free from photobleaching, straightforward implementation on a conventional fluorescence microscope and the possibility of temporally multiplexed and quantitative imaging.
In this thesis, a test sample based on functionalised microspheres is developed, which allows for optimisation of various DNA-PAINT imaging parameters and for the characterisation and testing of new variations and modifications of DNA-PAINT. One such method which was developed for this thesis, Quencher-Exchange-PAINT, facilitates temporally multiplexed imaging, which is based on the sequential exchange of imager strands targeting different docking strands. The exchange step is replaced by addition of competitive quencher-strands, allowing for rapid, low-crosstalk imager exchange even in biological samples with limited diffusion. Additionally, Proximity-Dependent PAINT is introduced, which enables the imaging of the nanoscale distribution of protein pairs by interaction of two proximity probes which activates DNA-PAINT type binding. The technique is demonstrated both on the microsphere assay as well as in biological samples. Finally, approaches for enhancing the signal-to-noise ratio are explored.
Abstract.
Full text.
Cannell MB, Soeller C, Baddeley D (2019). Practical Aspects of Localization Microscopy. In (Ed) Fundamentals of Fluorescence Imaging, Jenny Stanford Publishing, 347-382.
Harrison C (2019). Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and. DNA PAINT single molecule localisation microscopy.
Abstract:
Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and. DNA PAINT single molecule localisation microscopy
Aberrant Ryanodine receptor behaviour is highly implicated in cardiovascular disease. Post-translational modifications are used widely in the body to control the dynamics of proteins to respond to acute and chronic demands. Phosphorylation is a key, highly tuneable modification used by cells by the reversible enzymatic addition of a phosphate group to single amino acids within protein structures. Common cardiac diseases such as arrhythmia, hypertension, and heart failure have been linked to excessive ryanodine receptor phosphorylation and transgenic constitutive phosphorylation has shown disease aetiology in disease models.
Phosphorylation is typically measured en masse by use of Western blots, or more recently phosphoproteomics, however the spatial distribution has remained a mystery. Ryanodine receptors are found in clusters and their influence tightly controlled spatially. As phosphorylation increases their range of influence, it is of great interest to observe the pattern of phosphorylation within and between ryanodine receptor clusters.
Ryanodine receptor clusters have been well characterised in electron microscopy and the fluorescence based super resolution microscopy, achieving single receptor resolution. This thesis details the validation pipeline for translation phosphorylation-state specific antibodies from Western blot through to super resolution microscopy. The phosphorylation distribution was compared between isolated ventricular cardiomyocytes and ventricular tissue sections for Ser 2808 and Ser 2814 phosphorylation. Strong reductions in basal phosphorylation caused by the isolation procedure were observed for Ser 2808 but not Ser 2814. These differences in Ser¬ 2808 phosphorylation were then investigated in dual channel STORM super resolution microscopy, highlighting stark contracts in colocalisation between the confocal and STORM techniques.
This population and sub population experiment was then translated into the new DNA PAINT technology and a direct comparison of performance between STORM and DNA PAINT was discussed. The data described in this thesis shows a methodological approach to enabling other biophysicists to perform quantitative super resolution microscopy to determine the extent and spatial distribution pattern of phosphorylation of a protein of interest at the nanoscale. Important differences were observed in the phosphorylation state due to cardiomyocyte isolation procedures that are of interest to a wide audience of cardiovascular researchers.
DNA-PAINT is emerging as the progression of SMLM from STORM microscopy due to the greater control of imaging parameters it affords. Parallel experiments of Ryanodine receptor Ser-2808 phosphorylation were performed in tissue sections. Comparisons between dual channel STORM and DNA-PAINT were evaluated. Open questions about DNA PAINT are also highlighted and discussed.
Abstract.
Harrison C (2019). Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and. DNA-PAINT single molecule localisation microscopy.
Abstract:
Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and. DNA-PAINT single molecule localisation microscopy
Aberrant Ryanodine receptor behaviour is highly implicated in cardiovascular disease. Post-translational modifications are used widely in the body to control the dynamics of proteins to respond to acute and chronic demands. Phosphorylation is a key, highly tuneable modification used by cells by the reversible enzymatic addition of a phosphate group to single amino acids within protein structures. Common cardiac diseases such as arrhythmia, hypertension, and heart failure have been linked to excessive ryanodine receptor phosphorylation and transgenic constitutive phosphorylation has shown disease aetiology in disease models.
Phosphorylation is typically measured en masse by use of Western blots, or more recently phosphoproteomics, however the spatial distribution has
remained a mystery. Ryanodine receptors are found in clusters and their
influence tightly controlled spatially. As phosphorylation increases their range of influence, it is of great interest to observe the pattern of phosphorylation within and between ryanodine receptor clusters.
Ryanodine receptor clusters have been well characterised in electron
microscopy and the fluorescence based super-resolution microscopy,
achieving single receptor resolution. This thesis details the validation pipeline for translation phosphorylation-state specific antibodies from Western blot through to super-resolution microscopy. The phosphorylation distribution was compared between isolated ventricular cardiomyocytes and ventricular tissue sections for Ser-2808 and Ser-2814 phosphorylation. Strong reductions in basal phosphorylation caused by the isolation procedure were observed for Ser-2808 but not Ser-2814. These differences in Ser-2808 phosphorylation were then investigated in dual channel STORM super-resolution microscopy, highlighting stark contracts in colocalisation between the confocal and STORM techniques.
This population and sub-population experiment was then translated into the new DNA-PAINT technology and a direct comparison of performance between STORM and DNA-PAINT was discussed. The data described in this thesis shows a methodological approach to enabling other biophysicists to perform quantitative super-resolution microscopy to determine the extent and spatial distribution pattern of phosphorylation of a protein of interest at the nanoscale. Important differences were observed in the phosphorylation state due to cardiomyocyte isolation procedures that are of interest to a wide audience of cardiovascular researchers.
DNA-PAINT is emerging as the progression of SMLM from STORM
microscopy due to the greater control of imaging parameters it affords.
Parallel experiments of Ryanodine receptor Ser-2808 phosphorylation were performed in tissue sections. Comparisons between dual channel STORM and DNA-PAINT were evaluated. Open questions about DNA-PAINT are also highlighted and discussed.
Abstract.
Harrison C (2019). Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and DNA PAINT single molecule localisation microscopy.
Abstract:
Super-resolution imaging of cardiac immuno-markers: Defining quality criteria for use in dual colour STORM and DNA PAINT single molecule localisation microscopy
Aberrant Ryanodine receptor behaviour is highly implicated in cardiovascular disease. Post-translational modifications are used widely in the body to control the dynamics of proteins to respond to acute and chronic demands. Phosphorylation is a key, highly tuneable modification used by cells by the reversible enzymatic addition of a phosphate group to single amino acids within protein structures. Common cardiac diseases such as arrhythmia, hypertension, and heart failure have been linked to excessive ryanodine receptor phosphorylation and transgenic constitutive phosphorylation has shown disease aetiology in disease models.
Phosphorylation is typically measured en masse by use of Western blots, or more recently phosphoproteomics, however the spatial distribution has remained a mystery. Ryanodine receptors are found in clusters and their influence tightly controlled spatially. As phosphorylation increases their range of influence, it is of great interest to observe the pattern of phosphorylation within and between ryanodine receptor clusters.
Ryanodine receptor clusters have been well characterised in electron microscopy and the fluorescence based super resolution microscopy, achieving single receptor resolution. This thesis details the validation pipeline for translation phosphorylation-state specific antibodies from Western blot through to super resolution microscopy. The phosphorylation distribution was compared between isolated ventricular cardiomyocytes and ventricular tissue sections for Ser 2808 and Ser 2814 phosphorylation. Strong reductions in basal phosphorylation caused by the isolation procedure were observed for Ser 2808 but not Ser 2814. These differences in Ser¬ 2808 phosphorylation were then investigated in dual channel STORM super resolution microscopy, highlighting stark contracts in colocalisation between the confocal and STORM techniques.
This population and sub population experiment was then translated into the new DNA PAINT technology and a direct comparison of performance between STORM and DNA PAINT was discussed. The data described in this thesis shows a methodological approach to enabling other biophysicists to perform quantitative super resolution microscopy to determine the extent and spatial distribution pattern of phosphorylation of a protein of interest at the nanoscale. Important differences were observed in the phosphorylation state due to cardiomyocyte isolation procedures that are of interest to a wide audience of cardiovascular researchers.
DNA-PAINT is emerging as the progression of SMLM from STORM microscopy due to the greater control of imaging parameters it affords. Parallel experiments of Ryanodine receptor Ser-2808 phosphorylation were performed in tissue sections. Comparisons between dual channel STORM and DNA-PAINT were evaluated. Open questions about DNA PAINT are also highlighted and discussed.
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Sheard TMD, Hurley ME, Colyer J, White E, Norman R, Pervolaraki E, Narayanasamy KK, Hou Y, Kirton HM, Yang Z, et al (2019). Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy.
ACS Nano,
13(2), 2143-2157.
Abstract:
Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy.
Nanodomains are intracellular foci which transduce signals between major cellular compartments. One of the most ubiquitous signal transducers, the ryanodine receptor (RyR) calcium channel, is tightly clustered within these nanodomains. Super-resolution microscopy has previously been used to visualize RyR clusters near the cell surface. A majority of nanodomains located deeper within cells have remained unresolved due to limited imaging depths and axial resolution of these modalities. A series of enhancements made to expansion microscopy allowed individual RyRs to be resolved within planar nanodomains at the cell periphery and the curved nanodomains located deeper within the interiors of cardiomyocytes. With a resolution of ∼ 15 nm, we localized both the position of RyRs and their individual phosphorylation for the residue Ser2808. With a three-dimensional imaging protocol, we observed disturbances to the RyR arrays in the nanometer scale which accompanied right-heart failure caused by pulmonary hypertension. The disease coincided with a distinct gradient of RyR hyperphosphorylation from the edge of the nanodomain toward the center, not seen in healthy cells. This spatial profile appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a β-adrenergic agonist. Simulations of RyR arrays based on the experimentally determined channel positions and phosphorylation signatures showed how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in nanodomain excitability which may arise from such RyR reorganization.
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2018
Shen X, van den Brink J, Kolstad TR, Norden E, Edwards AG, Frisk M, Sjaastad I, Soeller C, Louch WE (2018). 3D dSTORM Imaging Reveals Disassembly of Ryanodine Receptor Clusters in Failing Cardiomyocytes.
Author URL.
Jayasinghe I, Clowsley AH, Soeller C (2018). Advances in the Visualization of Molecular Assemblies Within Cellular Signaling Nanodomains: Insights from a Decade of Mapping of Ryanodine Receptor Clusters.
Advances in Biomembranes and Lipid Self-Assembly,
28, 167-197.
Abstract:
Advances in the Visualization of Molecular Assemblies Within Cellular Signaling Nanodomains: Insights from a Decade of Mapping of Ryanodine Receptor Clusters
© 2018 Elsevier Inc. Nanodomains are naturally assembled signaling stations, which facilitate fast and highly regulated signaling within and between cells. Calcium (Ca2+) nanodomains known as junctional membrane complexes (JMCs) transduce fast and highly synchronized intracellular signals, which are required by a variety of cell types. Common to most such nanodomains are clustered assemblies of the principal intracellular Ca2+ release channels, ryanodine receptors (RyRs). JMCs found in cardiac muscle cells have been studied extensively as self-assembled clusters of RyR. While known to form crystalline arrays in vitro, the organization of RyRs in situ within the JMCs has been less clear. The development of single-molecule localization microscopy (SMLM or super-resolution) optical methods has transformed our ability to visualize and accurately quantify the spatial geometries and sizes of RyR clusters. The recent application of the novel DNA-PAINT super-resolution technology has exploited an unprecedented optical resolution of 10–15 nm to visualize the natural arrays of RyRs within JMCs. In this chapter, we review the key insights into the in situ RyR assembly within cardiac nanodomains that have been gained over the last decade with the utility of super-resolution microscopy and the major considerations in interpreting and validating such image data.
Abstract.
Sassmann S, Rodrigues C, Milne SW, Nenninger A, Allwood E, Littlejohn GR, Talbot NJ, Soeller C, Davies B, Hussey PJ, et al (2018). An Immune-Responsive Cytoskeletal-Plasma Membrane Feedback Loop in Plants.
Current Biology,
28(13), 2136-2144.e7.
Abstract:
An Immune-Responsive Cytoskeletal-Plasma Membrane Feedback Loop in Plants
© 2018 the Authors Cell wall appositions (CWAs) are produced reactively by the plant immune system to arrest microbial invasion through the local inversion of plant cell growth. This process requires the controlled invagination of the plasma membrane (PM) in coordination with the export of barrier material to the volume between the plant PM and cell wall. Plant actin dynamics are essential to this response, but it remains unclear how exocytosis and the cytoskeleton are linked in space and time to form functional CWAs. Here, we show that actin-dependent trafficking to immune response sites of Arabidopsis thaliana delivers membrane-integrated FORMIN4, which in turn contributes to local cytoskeletal dynamics. Total internal reflection fluorescence (TIRF) microscopy combined with controlled induction of FORMIN4-GFP expression reveals a dynamic population of vesicular bodies that accumulate to form clusters at the PM through an actin-dependent process. Deactivation of FORMIN4 and its close homologs partially compromises subsequent defense and alters filamentous actin (F-actin) distribution at mature CWAs. The localization of FORMIN4 is stable and segregated from the dynamic traffic of the endosomal network. Moreover, the tessellation of FORMIN4 at the PM with meso-domains of PEN3 reveals a fine spatial segregation of destinations for actin-dependent immunity cargo. Together, our data suggest a model where FORMIN4 is a spatial feedback element in a multi-layered, temporally defined sequence of cytoskeletal response. This positional feedback makes a significant contribution to the distribution of actin filaments at the dynamic CWA boundary and to the outcomes of pre-invasion defense.
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Curl CL, Danes VR, Bell JR, Raaijmakers AJA, Ip WTK, Chandramouli C, Harding TW, Porrello ER, Erickson JR, Charchar FJ, et al (2018). Cardiomyocyte functional etiology in heart failure with preserved ejection fraction is distinctive-a new preclinical model.
Journal of the American Heart Association,
7(11).
Abstract:
Cardiomyocyte functional etiology in heart failure with preserved ejection fraction is distinctive-a new preclinical model
© 2018 the Authors. Background--Among the growing numbers of patients with heart failure, up to one half have heart failure with preserved ejection fraction (HFpEF). The lack of effective treatments for HFpEF is a substantial and escalating unmet clinical need-and the lack of HFpEF-specific animal models represents a major preclinical barrier in advancing understanding of HFpEF. As established treatments for heart failure with reduced ejection fraction (HFrEF) have proven ineffective for HFpEF, the contention that the intrinsic cardiomyocyte phenotype is distinct in these 2 conditions requires consideration. Our goal was to validate and characterize a new rodent model of HFpEF, undertaking longitudinal investigations to delineate the associated cardiac and cardiomyocyte pathophysiology. Methods and Results--The selectively inbred Hypertrophic Heart Rat (HHR) strain exhibits adult cardiac enlargement (without hypertension) and premature death (40% mortality at 50 weeks) compared to its control strain, the normal heart rat. Hypertrophy was characterized in vivo by maintained systolic parameters (ejection fraction at 85%-90% control) with marked diastolic dysfunction (increased E/E'). Surprisingly, HHR cardiomyocytes were hypercontractile, exhibiting high Ca2+ operational levels and markedly increased L-type Ca2+ channel current. In HHR, prominent regions of reparative fibrosis in the left ventricle free wall adjacent to the interventricular septum were observed. Conclusions--Thus, the cardiomyocyte remodeling process in the etiology of this HFpEF model contrasts dramatically with the suppressed Ca2+ cycling state that typifies heart failure with reduced ejection fraction. These findings may explain clinical observations, that treatments considered appropriate for heart failure with reduced ejection fraction are of little benefit for HFpEF -and suggest a basis for new therapeutic strategies.
Abstract.
Rajagopal V, Bass G, Ghosh S, Hunt H, Walker C, Hanssen E, Crampin E, Soeller C (2018). Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology.
J Vis Exp(134).
Abstract:
Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology.
With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.
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Munro ML, Shen X, Ward M, Ruygrok PN, Crossman DJ, Soeller C (2018). Highly variable contractile performance correlates with myocyte content in trabeculae from failing human hearts.
Sci Rep,
8(1).
Abstract:
Highly variable contractile performance correlates with myocyte content in trabeculae from failing human hearts.
Heart failure (HF) is defined by compromised contractile function and is associated with changes in excitation-contraction (EC) coupling and cardiomyocyte organisation. Tissue level changes often include fibrosis, while changes within cardiomyocytes often affect structures critical to EC coupling, including the ryanodine receptor (RyR), the associated protein junctophilin-2 (JPH2) and the transverse tubular system architecture. Using a novel approach, we aimed to directly correlate the influence of structural alterations with force development in ventricular trabeculae from failing human hearts. Trabeculae were excised from explanted human hearts in end-stage failure and immediately subjected to force measurements. Following functional experiments, each trabecula was fixed, sectioned and immuno-stained for structural investigations. Peak stress was highly variable between trabeculae from both within and between failing hearts and was strongly correlated with the cross-sectional area occupied by myocytes (MCSA), rather than total trabecula cross-sectional area. At the cellular level, myocytes exhibited extensive microtubule densification which was linked via JPH2 to time-to-peak stress. Trabeculae fractional MCSA variability was much higher than that in adjacent free wall samples. Together, these findings identify several structural parameters implicated in functional impairment in human HF and highlight the structural variability of ventricular trabeculae which should be considered when interpreting functional data.
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Hunt H, Bass G, Roderick L, Soeller C, Rajagopal V, Crampin E (2018). Mixed Signals: Interaction between RyR and IP3R Mediated Calcium Release Shapes the Calcium Transient for Hypertrophic Signalling in Cardiomyocytes.
Author URL.
Jayasinghe ID, Clowsley AH, Lin R, Lutz T, Harrison C, Green EM, Baddeley D, Di Michele L, Soeller C (2018). Molecular Scale Visualisation of Variable Clustering Properties of the Cardiac Ryanodine Receptor.
Author URL.
Hou Y, Le C, Soeller C, Louch W (2018). Protocol for the Isolation and Super-resolution dSTORM. Imaging of RyR2 in Cardiac Myocytes. BIO-PROTOCOL, 8(15).
Soeller C, Jayasinghe ID (2018). Quantitative Super-Resolution Microscopy of Cardiomyocytes. In (Ed) Microscopy of the Heart, 37-73.
Wang W, Landstrom AP, Wang Q, Munro ML, Beavers D, Ackerman MJ, Soeller C, Wehrens XH (2018). Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice (vol 307, pg H1317, 2014).
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY,
314(5), H1115-H1115.
Author URL.
Soeller C (2018). Ryanodine receptor cluster size sets the tone in cerebral smooth muscle.
Proc Natl Acad Sci U S A,
115(41), 10195-10197.
Author URL.
Jayasinghe I, Clowsley AH, de langen O, Sali SS, Crossman DJ, Soeller C (2018). Shining New Light on the Structural Determinants of Cardiac Couplon Function: Insights from Ten Years of Nanoscale Microscopy.
FRONTIERS IN PHYSIOLOGY,
9 Author URL.
Jayasinghe I, Clowsley AH, Lin R, Lutz T, Harrison C, Green E, Baddeley D, Di Michele L, Soeller C (2018). True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors.
Cell Rep,
22(2), 557-567.
Abstract:
True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors.
Signaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites of cardiomyocytes where ryanodine receptors (RyRs) are clustered with their molecular partners. Localization microscopy has been crucial to visualizing these nanodomains but has been limited by brightness of markers, restricting the resolution and quantification of individual proteins clustered within. Harnessing the remarkable localization precision of DNA-PAINT (
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Lutz T, Clowsley AH, Lin R, Pagliara S, Di Michele L, Soeller C (2018). Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT.
Nano Research,
11(12), 6141-6154.
Abstract:
Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT
© 2018, the author(s). The optical super-resolution technique DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) provides a flexible way to achieve imaging of nanoscale structures at ∼10-nanometer resolution. In DNA-PAINT, fluorescently labeled DNA “imager” strands bind transiently and with high specificity to complementary target “docking” strands anchored to the structure of interest. The localization of single binding events enables the assembly of a super-resolution image, and this approach effectively circumvents photobleaching. The solution exchange of imager strands is the basis of Exchange-PAINT, which enables multiplexed imaging that avoids chromatic aberrations. Fluid exchange during imaging typically requires specialized chambers or washes, which can disturb the sample. Additionally, diffusional washout of imager strands is slow in thick samples such as biological tissue slices. Here, we introduce Quencher-Exchange-PAINT—a new approach to Exchange-PAINT in regular open-top imaging chambers—which overcomes the comparatively slow imager strand switching via diffusional imager washout. Quencher-Exchange-PAINT uses “quencher” strands, i.e. oligonucleotides that prevent the imager from binding to the targets, to rapidly reduce unwanted single-stranded imager concentrations to negligible levels, decoupled from the absolute imager concentration. The quencher strands contain an effective dye quencher that reduces the fluorescence of quenched imager strands to negligible levels. We characterized Quencher-Exchange-PAINT when applied to synthetic, cellular, and thick tissue samples. Quencher-Exchange-PAINT opens the way for efficient multiplexed imaging of complex nanostructures, e.g. in thick tissues, without the need for washing steps. [Figure not available: see fulltext.].
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2017
Lin R, Clowsley AH, Jayasinghe ID, Baddeley D, Soeller C (2017). Algorithmic corrections for localization microscopy with sCMOS cameras - Characterisation of a computationally efficient localization approach.
Optics Express,
25(10), 11701-11716.
Abstract:
Algorithmic corrections for localization microscopy with sCMOS cameras - Characterisation of a computationally efficient localization approach
© 2017, OSA - the Optical Society. All rights reserved. Modern sCMOS cameras are attractive for single molecule localization microscopy (SMLM) due to their high speed but suffer from pixel non-uniformities that can affect localization precision and accuracy. We present a simplified sCMOS non-uniform noise model that incorporates pixel specific read-noise, offset and sensitivity variation. Using this model we develop a new weighted least squared (WLS) fitting method designed to remove the effect of sCMOS pixel non-uniformities. Simulations with the sCMOS noise model, performed to test under which conditions sCMOS specific localization corrections are required, suggested that pixel specific offsets should always be removed. In many applications with thick biological samples photon fluxes are sufficiently high that corrections of read-noise and sensitivity correction may be neglected. When correction is required, e.g. during fast imaging in thin samples, our WLS fit procedure recovered the performance of an equivalent sensor with uniform pixel properties and the fit estimates also attained the Cramer-Rao lower bound. Experiments with sub-resolution beads and a DNA origami test sample confirmed the results of the simulations. The WLS localization procedure is fast to converge, compatible with 2D, 3D and multi-emitter localization and thus provides a computationally efficient sCMOS localization approach compatible with most SMLM modalities.
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Jones RA, Harrison C, Eaton SL, Hurtado ML, Graham LC, Alkhammash L, Oladiran OA, Gale A, Lamont DJ, Simpson H, et al (2017). Cellular and Molecular Anatomy of the Human Neuromuscular Junction.
CELL REPORTS,
21(9), 2348-2356.
Author URL.
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Soeller C, Hou Y, Jayasinghe ID, Baddeley D, Crossman D (2017). Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.
Methods Mol Biol,
1663, 205-217.
Abstract:
Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.
Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.
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Crossman DJ, Shen X, Jüllig M, Munro M, Hou Y, Middleditch M, Shrestha D, Li A, Lal S, Dos Remedios CG, et al (2017). Increased collagen within the transverse tubules in human heart failure.
Cardiovasc Res,
113(8), 879-891.
Abstract:
Increased collagen within the transverse tubules in human heart failure.
Aims: in heart failure transverse-tubule (t-tubule) remodelling disrupts calcium release, and contraction. T-tubules in human failing hearts exhibit increased labelling by wheat germ agglutinin (WGA), a lectin that binds to the dystrophin-associated glycoprotein complex. We hypothesized changes in this complex may explain the increased WGA labelling and contribute to t-tubule remodelling in the failing human heart. In this study we sought to identify the molecules responsible for this increased WGA labelling. Methods and results: Confocal and super-resolution fluorescence microscopy and proteomic analyses were used to quantify left ventricle samples from healthy donors and patients with idiopathic dilated cardiomyopathy (IDCM). Confocal microscopy demonstrated both WGA and dystrophin were located at t-tubules. Super-resolution microscopy revealed that WGA labelling of t-tubules is largely located within the lumen while dystrophin was restricted to near the sarcolemma. Western blots probed with WGA reveal a 5.7-fold increase in a 140 kDa band in IDCM. Mass spectrometry identified this band as type VI collagen (Col-VI) comprised of α1(VI), α2(VI), and α3(VI) chains. Pertinently, mutations in Col-VI cause muscular dystrophy. Western blotting identified a 2.4-fold increased expression and 3.2-fold increased WGA binding of Col-VI in IDCM. Confocal images showed that Col-VI is located in the t-tubules and that their diameter increased in the IDCM samples. Super-resolution imaging revealed Col-VI was restricted to the t-tubule lumen where increases were associated with displacement in the sarcolemma as identified from dystrophin labelling. Samples were also labelled for type I, III, and IV collagen. Both confocal and super-resolution imaging identified that these collagens were also present within t-tubule lumen. Conclusion: Increased expression and labelling of collagen in IDCM samples indicates fibrosis may contribute to t-tubule remodelling in human heart failure.
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Goodman L, Baddeley D, Ambroziak W, Waites CL, Garner CC, Soeller C, Montgomery JM (2017). N-terminal SAP97 isoforms differentially regulate synaptic structure and postsynaptic surface pools of AMPA receptors.
Hippocampus,
27(6), 668-682.
Abstract:
N-terminal SAP97 isoforms differentially regulate synaptic structure and postsynaptic surface pools of AMPA receptors.
The location and density of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is controlled by scaffolding proteins within the postsynaptic density (PSD). SAP97 is a PSD protein with two N-terminal isoforms, α and β, that have opposing effects on synaptic strength thought to result from differential targeting of AMPA receptors into distinct synaptic versus extrasynaptic locations, respectively. In this study, we have applied dSTORM super resolution imaging in order to localize the synaptic and extrasynaptic pools of AMPA receptors in neurons expressing α or βSAP97. Unexpectedly, we observed that both α and βSAP97 enhanced the localization of AMPA receptors at synapses. However, this occurred via different mechanisms: αSAP97 increased PSD size and consequently the number of receptor binding sites, whilst βSAP97 increased synaptic receptor cluster size and surface AMPA receptor density at the PSD edge and surrounding perisynaptic sites without changing PSD size. αSAP97 also strongly enlarged presynaptic active zone protein clusters, consistent with both presynaptic and postsynaptic enhancement underlying the previously observed αSAP97-induced increase in AMPA receptor-mediated currents. In contrast, βSAP97-expressing neurons increased the proportion of immature filopodia that express higher levels of AMPA receptors, decreased the number of functional presynaptic terminals, and also reduced the size of the dendritic tree and delayed the maturation of mushroom spines. Our data reveal that SAP97 isoforms can specifically regulate surface AMPA receptor nanodomain clusters, with βSAP97 increasing extrasynaptic receptor domains at peri-synaptic and filopodial sites. Moreover, βSAP97 negatively regulates synaptic maturation both structurally and functionally. These data support diverging presynaptic and postsynaptic roles of SAP97 N-terminal isoforms in synapse maturation and plasticity. As numerous splice isoforms exist in other major PSD proteins (e.g. Shank, PSD95, and SAP102), this alternative splicing may result in individual PSD proteins having divergent functional and structural roles in both physiological and pathophysiological synaptic states.
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Author URL.
Jeynes JCG, Geraki K, Jeynes C, Zhaohong M, Bettiol AA, Latorre E, Harries LW, Soeller C (2017). Nanoscale Properties of Human Telomeres Measured with a Dual Purpose X-ray Fluorescence and Super Resolution Microscopy Gold Nanoparticle Probe.
ACS Nano,
11(12), 12632-12640.
Abstract:
Nanoscale Properties of Human Telomeres Measured with a Dual Purpose X-ray Fluorescence and Super Resolution Microscopy Gold Nanoparticle Probe.
Techniques to analyze human telomeres are imperative in studying the molecular mechanism of aging and related diseases. Two important aspects of telomeres are their length in DNA base pairs (bps) and their biophysical nanometer dimensions. However, there are currently no techniques that can simultaneously measure these quantities in individual cell nuclei. Here, we develop and evaluate a telomere "dual" gold nanoparticle-fluorescent probe simultaneously compatible with both X-ray fluorescence (XRF) and super resolution microscopy. We used silver enhancement to independently visualize the spatial locations of gold nanoparticles inside the nuclei, comparing to a standard QFISH (quantitative fluorescence in situ hybridization) probe, and showed good specificity at ∼90%. For sensitivity, we calculated telomere length based on a DNA/gold binding ratio using XRF and compared to quantitative polymerase chain reaction (qPCR) measurements. The sensitivity was low (∼10%), probably because of steric interference prohibiting the relatively large 10 nm gold nanoparticles access to DNA space. We then measured the biophysical characteristics of individual telomeres using super resolution microscopy. Telomeres that have an average length of ∼10 kbps, have diameters ranging between ∼60-300 nm. Further, we treated cells with a telomere-shortening drug and showed there was a small but significant difference in telomere diameter in drug-treated vs control cells. We discuss our results in relation to the current debate surrounding telomere compaction.
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Crossman DJ, Jayasinghe ID, Soeller C (2017). Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?.
Biophys Rev,
9(6), 919-929.
Abstract:
Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?
Transverse (t)-tubules are invaginations of the plasma membrane that form a complex network of ducts, 200-400 nm in diameter depending on the animal species, that penetrates deep within the cardiac myocyte, where they facilitate a fast and synchronous contraction across the entire cell volume. There is now a large body of evidence in animal models and humans demonstrating that pathological distortion of the t-tubule structure has a causative role in the loss of myocyte contractility that underpins many forms of heart failure. Investigations into the molecular mechanisms of pathological t-tubule remodelling to date have focused on proteins residing in the intracellular aspect of t-tubule membrane that form linkages between the membrane and myocyte cytoskeleton. In this review, we shed light on the mechanisms of t-tubule remodelling which are not limited to the intracellular side. Our recent data have demonstrated that collagen is an integral part of the t-tubule network and that it increases within the tubules in heart failure, suggesting that a fibrotic mechanism could drive cardiac junctional remodelling. We examine the evidence that the linkages between the extracellular matrix, t-tubule membrane and cellular cytoskeleton should be considered as a whole when investigating the mechanisms of t-tubule pathology in the failing heart.
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2016
Munro ML, Soeller C (2016). Early transverse tubule development begins in utero in the sheep heart.
Journal of Muscle Research and Cell Motility,
37(6), 195-202.
Abstract:
Early transverse tubule development begins in utero in the sheep heart
© 2017, Springer International Publishing Switzerland. The ventricular cardiomyocytes of adult mammals contain invaginations of the plasma membrane known as transverse (t)-tubules. These regular structures are essential for the synchronisation of excitation–contraction (EC) coupling throughout the cell, which is a vital process for cardiac function. T-tubules form a close association with the sarcoplasmic reticulum (SR) to form junctions, where several key proteins involved in EC coupling are localised, including the SR calcium release channels—the ryanodine receptors (RyR). The lipophilic SR protein junctophilin-2 (JPH2) has been implicated in the development of both the junctions and t-tubules. Several studies have identified that t-tubules develop only postnatally in rodents, while historical electron microscopy data indicate that this is not the case in larger mammals, including humans. We have performed, to our knowledge, the first fluorescent, target-specific study to characterise t-tubule development in the large mammalian fetal heart, focussing on the sheep. T-tubules were present in fetal sheep hearts from 114 days gestation (with term being 145 days), with occurrence progressively increasing with gestational age, and further maturation after birth. This was accompanied by an increasing intracellular localisation of JPH2, which progressively increased its association with RyR within the cardiomyocytes as they undergo hypertrophy. These findings indicate that large mammalian hearts exhibit a significantly different temporal pattern of development compared to that of the rodent. Our findings have potential implications for human cardiac development, including the future investigation of congenital heart disease.
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Munro ML, Jayasinghe ID, Wang Q, Quick A, Wang W, Baddeley D, Wehrens XHT, Soeller C (2016). Junctophilin-2 in the nanoscale organisation and functional signalling of ryanodine receptor clusters in cardiomyocytes.
Journal of Cell Science,
129(23), 4388-4398.
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Munro ML, Shen X, Ward M, Crossman DJ, Soeller C (2016). Relationship between Multi-Scale Cardiomyocyte Organization and Function in Trabeculae of the Failing Human Heart.
Author URL.
Lin R, Clowsley A, Baddeley D, Jayasinghe I, Soeller C (2016). Single Molecule Localisation Microscopy with sCMOS Cameras.
Author URL.
Jayasinghe I, Clowsley A, Lutz T, Green E, Lin R, di Michele L, Soeller C (2016). TRUE MOLECULAR SCALE ANALYSIS OF THE CALCIUM RELEASE MACHINERY OF THE HEART WITH ENHANCED SUPER-RESOLUTION IMAGING.
Author URL.
2015
Crossman DJ, Hou Y, Jayasinghe I, Baddeley D, Soeller C (2015). Combining confocal and single molecule localisation microscopy: a correlative approach to multi-scale tissue imaging.
Methods,
88, 98-108.
Abstract:
Combining confocal and single molecule localisation microscopy: a correlative approach to multi-scale tissue imaging.
Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described.
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Author URL.
Rajagopal V, Bass G, Walker CG, Crossman DJ, Petzer A, Hickey A, Siekmann I, Hoshijima M, Ellisman MH, Crampin EJ, et al (2015). Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes.
PLoS Comput Biol,
11(9).
Abstract:
Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes.
Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils. We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i ≈1 μM; F/F0≈5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structure-induced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes.
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Author URL.
Full text.
Hou Y, Jayasinghe I, Crossman DJ, Baddeley D, Soeller C (2015). Nanoscale analysis of ryanodine receptor clusters in dyadic couplings of rat cardiac myocytes.
J Mol Cell Cardiol,
80, 45-55.
Abstract:
Nanoscale analysis of ryanodine receptor clusters in dyadic couplings of rat cardiac myocytes.
The contractile properties of cardiac myocytes depend on the calcium (Ca(2+)) released by clusters of ryanodine receptors (RyRs) throughout the myoplasm. Accurate quantification of the spatial distribution of RyRs has previously been challenging due to the comparatively low resolution in optical microscopy. We have combined single-molecule localisation microscopy (SMLM) in a super-resolution modality known as dSTORM with immunofluorescence staining of tissue sections of rat ventricles to resolve a wide, near-exponential size distribution of RyR clusters that lined on average ~57% of the perimeter of each myofibril. The average size of internal couplons is ~63 RyRs (nearly 4 times larger than that of peripheral couplons) and the largest clusters contain many hundreds of RyRs. Similar to previous observations in peripheral couplons, we observe many clusters with one or few receptors; however ≥80% of the total RyRs were detected in clusters containing ≥100 receptors. ~56% of all clusters were within an edge-to-edge distance sufficiently close to co-activate via Ca(2+)-induced Ca(2+) release (100nm) and were grouped into 'superclusters'. The co-location of superclusters with the same or adjacent t-tubular connections in dual-colour super-resolution images suggested that member sub-clusters may be exposed to similar local luminal Ca(2+) levels. Dual-colour dSTORM revealed high co-localisation between the cardiac junctional protein junctophilin-2 (JPH2) and RyR clusters that confirmed that the majority of the RyR clusters observed are dyadic. The increased sensitivity of super-resolution images revealed approximately twice as many RyR clusters (2.2clusters/μm(3)) compared to previous confocal measurements. We show that, in general, the differences of previous confocal estimates are largely attributable to the limited spatial resolution of diffraction-limited imaging. The new data can be used to inform the construction of detailed mechanistic models of cardiac Ca(2+) signalling.
Abstract.
Author URL.
Crossman DJ, Ruygrok PN, Hou YF, Soeller C (2015). Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.
Heart Failure Reviews,
20(2), 203-214.
Abstract:
Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy
© 2014, Springer Science+Business Media New York. Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.
Abstract.
Crossman DJ, Ruygrok PN, Hou YF, Soeller C (2015). Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.
Heart Fail Rev,
20(2), 203-214.
Abstract:
Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.
Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.
Abstract.
Author URL.
Jayasinghe ID, Clowsley AH, Munro M, Hou Y, Crossman DJ, Soeller C (2015). Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess.
European journal of translational myology,
25(1).
Abstract:
Revealing T-Tubules in Striated Muscle with New Optical Super-Resolution Microscopy Techniquess.
The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.
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Sauer M, Soeller C, Hanrahan O (2015). Single-molecule localization microscopy comes of age: from concepts to biological impact. Science, 350(6261).
Crossman DJ, Young AA, Ruygrok PN, Nason GP, Baddelely D, Soeller C, Cannell MB (2015). T-tubule disease: Relationship between t-tubule organization and regional contractile performance in human dilated cardiomyopathy.
J Mol Cell Cardiol,
84, 170-178.
Abstract:
T-tubule disease: Relationship between t-tubule organization and regional contractile performance in human dilated cardiomyopathy.
Evidence from animal models suggest that t-tubule changes may play an important role in the contractile deficit associated with heart failure. However samples are usually taken at random with no regard as to regional variability present in failing hearts which leads to uncertainty in the relationship between contractile performance and possible t-tubule derangement. Regional contraction in human hearts was measured by tagged cine MRI and model fitting. At transplant, failing hearts were biopsy sampled in identified regions and immunocytochemistry was used to label t-tubules and sarcomeric z-lines. Computer image analysis was used to assess 5 different unbiased measures of t-tubule structure/organization. In regions of failing hearts that showed good contractile performance, t-tubule organization was similar to that seen in normal hearts, with worsening structure correlating with the loss of regional contractile performance. Statistical analysis showed that t-tubule direction was most highly correlated with local contractile performance, followed by the amplitude of the sarcomeric peak in the Fourier transform of the t-tubule image. Other area based measures were less well correlated. We conclude that regional contractile performance in failing human hearts is strongly correlated with the local t-tubule organization. Cluster tree analysis with a functional definition of failing contraction strength allowed a pathological definition of 't-tubule disease'. The regional variability in contractile performance and cellular structure is a confounding issue for analysis of samples taken from failing human hearts, although this may be overcome with regional analysis by using tagged cMRI and biopsy mapping.
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Author URL.
2014
Hartwich TMP, Soeller C, Baddeley D (2014). A Simple Chemical Oxygen Scavenging System for Improved dSTORM Tissue Imaging.
Author URL.
Munro M, Wang W, Baddeley D, Wehrens X, Soeller C (2014). Nanoscale Changes in the Organisation of Junctional Proteins in JPH2 Transgenic Mice.
Author URL.
Jayasinghe ID, Munro M, Baddeley D, Launikonis BS, Soeller C (2014). Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging.
J R Soc Interface,
11(99).
Abstract:
Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging.
Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle.
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Author URL.
Wang W, Landstrom AP, Wang Q, Munro ML, Beavers D, Ackerman MJ, Soeller C, Wehrens XHT (2014). Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice.
Am J Physiol Heart Circ Physiol,
307(9), H1317-H1326.
Abstract:
Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice.
Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca(2+) leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca(2+) leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca(2+) release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca(2+) imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca(2+) leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na(+)/Ca(2+)-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd(2+) administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca(2+) leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca(2+). This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca(2+) leak and heart failure development.
Abstract.
Author URL.
Jayasinghe ID, Munro M, Baddeley D, Launikonis BS, Soeller C (2014). Resolving the Calcium Release Machinery of Mammalian Fast- and Slow-Twitch Skeletal Muscle.
Author URL.
Hou Y, Crossman DJ, Rajagopal V, Baddeley D, Jayasinghe I, Soeller C (2014). Super-resolution fluorescence imaging to study cardiac biophysics: α-actinin distribution and Z-disk topologies in optically thick cardiac tissue slices.
Progress in Biophysics and Molecular Biology,
115(2-3), 328-339.
Abstract:
Super-resolution fluorescence imaging to study cardiac biophysics: α-actinin distribution and Z-disk topologies in optically thick cardiac tissue slices
© 2014 Elsevier Ltd. A major motivation for the use of super-resolution imaging methods in the investigation of cardiac biophysics has been the insight from biophysical considerations and detailed mathematical modeling that the spatial structure and protein organisation at the scale of nanometres can have enormous implications for calcium signalling in cardiac muscle. We illustrate the use of dSTORM based super-resolution in optically thick (~10μm) tissue slices of rat ventricular tissue to visualize proteins at the cardiac Z-disk and compare those images with confocal (diffraction-limited) as well as electron microscopy (EM) data which still provides a benchmark in terms of resolution. α-actinin is an abundant protein target that effectively defines the Z-disk in striated muscle and provides a reference structure for other proteins at the Z-line and the transverse tubules. Using super-resolution imaging α-actinin labelling provides very detailed outlines of the contractile machinery which we have used to study the properties of Z-disks and the distribution of α-actinin itself. We determined the local diameters of the myo-fibrillar and non-myofibrillar space using α-actinin labelling. Comparison between confocal and super-resolution based myofibrillar masks suggested that super-resolution data was able to segment myofibrils accurately while confocal approaches were not always able to distinguish neighbouring myofibrillar bundles which resulted in overestimated diameters. The increased resolution of super-resolution methods provides qualitatively new information to improve our understanding of cardiac biophysics. Nevertheless, conventional diffraction-limited imaging still has an important role to play which we illustrate with correlative confocal and super-resolution data.
Abstract.
Hou Y, Crossman DJ, Rajagopal V, Baddeley D, Jayasinghe I, Soeller C (2014). Super-resolution fluorescence imaging to study cardiac biophysics: α-actinin distribution and Z-disk topologies in optically thick cardiac tissue slices.
Prog Biophys Mol Biol,
115(2-3), 328-339.
Abstract:
Super-resolution fluorescence imaging to study cardiac biophysics: α-actinin distribution and Z-disk topologies in optically thick cardiac tissue slices.
A major motivation for the use of super-resolution imaging methods in the investigation of cardiac biophysics has been the insight from biophysical considerations and detailed mathematical modeling that the spatial structure and protein organisation at the scale of nanometres can have enormous implications for calcium signalling in cardiac muscle. We illustrate the use of dSTORM based super-resolution in optically thick (∼10 μm) tissue slices of rat ventricular tissue to visualize proteins at the cardiac Z-disk and compare those images with confocal (diffraction-limited) as well as electron microscopy (EM) data which still provides a benchmark in terms of resolution. α-actinin is an abundant protein target that effectively defines the Z-disk in striated muscle and provides a reference structure for other proteins at the Z-line and the transverse tubules. Using super-resolution imaging α-actinin labelling provides very detailed outlines of the contractile machinery which we have used to study the properties of Z-disks and the distribution of α-actinin itself. We determined the local diameters of the myo-fibrillar and non-myofibrillar space using α-actinin labelling. Comparison between confocal and super-resolution based myofibrillar masks suggested that super-resolution data was able to segment myofibrils accurately while confocal approaches were not always able to distinguish neighbouring myofibrillar bundles which resulted in overestimated diameters. The increased resolution of super-resolution methods provides qualitatively new information to improve our understanding of cardiac biophysics. Nevertheless, conventional diffraction-limited imaging still has an important role to play which we illustrate with correlative confocal and super-resolution data.
Abstract.
Author URL.
2013
Jayasinghe ID, Lo HP, Morgan GP, Baddeley D, Parton RG, Soeller C, Launikonis BS (2013). Examination of the subsarcolemmal tubular system of mammalian skeletal muscle fibers.
Biophys J,
104(11), L19-L21.
Abstract:
Examination of the subsarcolemmal tubular system of mammalian skeletal muscle fibers.
A subsarcolemmal tubular system network (SSTN) has been detected in skeletal muscle fibers by confocal imaging after the removal of the sarcolemma. Here we confirm the existence and resolve the fine architecture and the localization of the SSTN at an unprecedented level of detail by examining extracellularly applied tubular system markers in skeletal muscle fiber preparations with a combination of three imaging modalities: confocal fluorescence microscopy, direct stochastic optical reconstruction microscopy, and tomographic electron microscopy. Three-dimensional reconstructions showed that the SSTN was a dense two-dimensional network within the subsarcolemmal space around the fiber, running ~500-600 nm underneath and parallel to the sarcolemma. The SSTN is composed of tubules ~95 nm in width with ~60% of the tubules directed transversely and >30% directed longitudinally. The deeper regular transverse tubules located at each A-I boundary of the sarcomeres branched from the SSTN, indicating individual transverse tubules that form triads are continuous with, but do not directly contact the sarcolemma. This suggests that the SSTN plays an important role in affecting the exchange of deeper tubule lumina with the extracellular space.
Abstract.
Author URL.
Wong J, Baddeley D, Bushong EA, Yu Z, Ellisman MH, Hoshijima M, Soeller C (2013). Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions.
Biophys J,
104(11), L22-L24.
Abstract:
Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions.
We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²⁺ release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.
Abstract.
Author URL.
Soeller C, Baddeley D (2013). Super-resolution imaging of EC coupling protein distribution in the heart.
Journal of Molecular and Cellular Cardiology,
58(1), 32-40.
Abstract:
Super-resolution imaging of EC coupling protein distribution in the heart
The cardiac ryanodine receptor (RyR) plays a central role in the control of contractile function of the heart. In cardiac ventricular myocytes RyRs and associated Ca2+ handling proteins, including membrane Ca2+ channels, Ca2+ pumps and other sarcolemmal and sarcoplasmic reticulum proteins interact to set the time course and amplitude of the electrically triggered cytosolic Ca2+ transient. It has become increasingly clear that protein distribution and clustering on the nanometer scale is critical in determining the interaction of these proteins and the resulting properties of cardiac Ca2+ handling. Such intricate near-molecular scale detail cannot be visualized with conventional fluorescence microscopy techniques (e.g. confocal microscopy) but it has recently become accessible with optical super-resolution techniques. These techniques retain the advantages of fluorescent marker technology, i.e. high specificity and excellent contrast, but have a spatial resolution approaching 10nm, i.e. objects not much further apart than 10nm can be distinguished, previously only attainable with electron microscopy. We review the use of these novel imaging techniques for the study of protein distribution in cardiac ventricular myocytes and discuss technical considerations as well as recent findings using super-resolution imaging. An emphasis is on single molecule localization based super-resolution approaches and their use to reveal the complexity of RyR cluster morphology, placement and relationship to other excitation-contraction coupling proteins. Super-resolution imaging approaches have already demonstrated their utility for the study of cardiac structure-function relationships and we anticipate that their use will rapidly increase and help improve our understanding of cardiac Ca2+ regulation. This article is part of a Special Issue entitled "Calcium Signaling in Heart". © 2012 Elsevier Ltd.
Abstract.
Soeller C, Baddeley D (2013). Super-resolution imaging of EC coupling protein distribution in the heart.
J Mol Cell Cardiol,
58, 32-40.
Abstract:
Super-resolution imaging of EC coupling protein distribution in the heart.
The cardiac ryanodine receptor (RyR) plays a central role in the control of contractile function of the heart. In cardiac ventricular myocytes RyRs and associated Ca(2+) handling proteins, including membrane Ca(2+) channels, Ca(2+) pumps and other sarcolemmal and sarcoplasmic reticulum proteins interact to set the time course and amplitude of the electrically triggered cytosolic Ca(2+) transient. It has become increasingly clear that protein distribution and clustering on the nanometer scale is critical in determining the interaction of these proteins and the resulting properties of cardiac Ca(2+) handling. Such intricate near-molecular scale detail cannot be visualized with conventional fluorescence microscopy techniques (e.g. confocal microscopy) but it has recently become accessible with optical super-resolution techniques. These techniques retain the advantages of fluorescent marker technology, i.e. high specificity and excellent contrast, but have a spatial resolution approaching 10nm, i.e. objects not much further apart than 10nm can be distinguished, previously only attainable with electron microscopy. We review the use of these novel imaging techniques for the study of protein distribution in cardiac ventricular myocytes and discuss technical considerations as well as recent findings using super-resolution imaging. An emphasis is on single molecule localization based super-resolution approaches and their use to reveal the complexity of RyR cluster morphology, placement and relationship to other excitation-contraction coupling proteins. Super-resolution imaging approaches have already demonstrated their utility for the study of cardiac structure-function relationships and we anticipate that their use will rapidly increase and help improve our understanding of cardiac Ca(2+) regulation.
Abstract.
Author URL.
Baddeley D, Soeller C (2013). Understanding Switching Variability in dSTORM Type Microscopy.
Author URL.
2012
Wong JIS, Baddeley D, Bushong EA, Ellisman M, Hoshijima M, Soeller C (2012). 3D Imaging of Nanoscopic Membrane Systems Regulating Cardiac Excitation-Contraction Coupling: Multi-Color Optical Super-Resolution and Serial Block-Face Scanning Electron Microscopy.
Author URL.
Jayasinghe I, Crossman D, Soeller C, Cannell M (2012). Comparison of the organization of T-tubules, sarcoplasmic reticulum and ryanodine receptors in rat and human ventricular myocardium.
Clin Exp Pharmacol Physiol,
39(5), 469-476.
Abstract:
Comparison of the organization of T-tubules, sarcoplasmic reticulum and ryanodine receptors in rat and human ventricular myocardium.
1. It is apparent from the literature that there are significant differences in excitation-contraction coupling between species, particularly in the density of calcium transporting proteins in the t-system and sarcoplasmic reticulum (SR) Ca(2+). release channels. Unfortunately, there is a lack of information as to how the principal structures that link electrical excitation to the activation of calcium-induced calcium release (CICR) are different between human and animal models (particularly rat). 2. Comparison of wheat germ agglutinin and caveolin-3 labelling revealed a non-uniform distribution of surface membrane glycosylation in the rat, rabbit and human, and that the rat t-system appeared more complex in geometry than the latter species. Analysis of the t-system skeleton showed that the t-system was highly branched in the rat compared with that of the human (0.8 ± 0.08 and 0.2 ± 0.07 branch points per μm(2) , respectively; P
Abstract.
Author URL.
Jayasinghe ID, Baddeley D, Kong CHT, Wehrens XHT, Cannell MB, Soeller C (2012). Nanoscale organization of junctophilin-2 and ryanodine receptors within peripheral couplings of rat ventricular cardiomyocytes.
Biophys J,
102(5), L19-L21.
Abstract:
Nanoscale organization of junctophilin-2 and ryanodine receptors within peripheral couplings of rat ventricular cardiomyocytes.
The peripheral distributions of the cardiac ryanodine receptor (RyR) and a junctional protein, junctophilin-2 (JPH2), were examined using single fluorophore localization-based super-resolution microscopy in rat ventricular myocytes. JPH2 was strongly associated with RyR clusters. Estimates of the colocalizing fraction of JPH labeling with RyR was ~90% within 30 nm of RyR clusters. This is comparable to fractions estimated from confocal data (~87%). Similarly, most RyRs were associated with JPH2 labeling in super-resolution images (~81% within 30 nm of JPH2 clusters). The shape of associated RyR clusters and JPH2 clusters were very similar, but not identical, suggesting that JPH2 is dispersed throughout RyR clusters and that the packing of JPH2 into junctions and the assembly of RyR clusters are tightly linked.
Abstract.
Author URL.
Rajagopal V, Khwaounjoo P, Walker CG, O'Sullivan M, Soeller C (2012). Subcellular Structural Changes in Diabetic Cardiomyopathy and its Impact on Cardiac Cell Calcium Dynamics.
Author URL.
Crossman D, Ruygrok P, Young A, Soeller C, Cannell M (2012). Transverse Tubule Structure is Related to Contractile Function in Human Heart Failure.
Author URL.
Baddeley D, Jayasinghe ID, Kong CHT, Wehrens XHT, Cannell MB, Soeller C (2012). Using Multi-Color Super-Resolution Microscopy to Probe the Organization of Dyadic Proteins within Rat Cardiac Myocytes.
Author URL.
2011
Baddeley D, Jayasinghe I, Kong C, Crossman D, Cheyne J, Montgomery J, Cannell M, Soeller C (2011). 4D spatial-spectral super-resolution imaging of nanoscopic membrane signalling domains.
2011 Int. Quantum Electron. Conf. IQEC 2011 and Conf. Lasers and Electro-Optics, CLEO Pacific Rim 2011 Incorporating the Australasian Conf. Optics, Lasers and Spectroscopy and the Australian Conf., 77-78.
Abstract:
4D spatial-spectral super-resolution imaging of nanoscopic membrane signalling domains
We present a practical implementation of single-molecule based localisation microscopy by combining single laser line excitation and use of conventional dyes. We investigate the organization of membrane proteins into clusters in cardiac and neuronal cells. © 2011 IEEE.
Abstract.
Baddeley D, Jayasinghe I, Kong C, Crossman D, Cheyne J, Montgomery J, Cannell M, Soeller C (2011). 4D spatial-spectral super-resolution imaging of nanoscopic membrane signalling domains.
Optics InfoBase Conference Papers, 77-78.
Abstract:
4D spatial-spectral super-resolution imaging of nanoscopic membrane signalling domains
We present a practical implementation of single-molecule based localisation microscopy by combining single laser line excitation and use of conventional dyes. We investigate the organization of membrane proteins into clusters in cardiac and neuronal cells. © 2011 AOS.
Abstract.
Baddeley D, Crossman D, Rossberger S, Cheyne JE, Montgomery JM, Jayasinghe ID, Cremer C, Cannell MB, Soeller C (2011). 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.
PLoS One,
6(5).
Abstract:
4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.
BACKGROUND: Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. METHODOLOGY/PRINCIPAL FINDINGS: We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to
Abstract.
Author URL.
Full text.
Blumgart EI, Jayasinghe ID, O'Sullivan MJ, Soeller C, Walker CG, Rajagopal V (2011). Cardiac Excitation-Contraction Coupling Proteins: a 3D Spatial Analysis.
Author URL.
Carlson S, Trial J, Soeller C, Entman ML (2011). Cardiac mesenchymal stem cells contribute to scar formation after myocardial infarction.
Cardiovasc Res,
91(1), 99-107.
Abstract:
Cardiac mesenchymal stem cells contribute to scar formation after myocardial infarction.
AIMS: Therapeutic advances in prevention and treatment of myocardial infarction (MI) have decreased patient mortality and increased concern about efficient repair and scar formation, processes that are necessary to attenuate complications such as adverse remodelling and heart failure. Since the rapid accumulation and activity of cardiac fibroblasts is critical for proper scar formation, we hypothesized that infarct fibroblasts are generated by a cardiac-resident progenitor cell population. METHODS AND RESULTS: We found that infarct fibroblasts in C57BL/6 mice are generated by a mesenchymal stem cell (MSC) population that responds robustly to injury by proliferating and accumulating in the infarct. We report that stem cell-derived fibroblasts contribute to the formation of a scar after an infarction by differentiating into matrix-producing fibroblasts closely associated with fibrillar collagen in the infarct. Further characterization of these cells revealed a heterogenous population with expression of both stem cell and canonical cardiac fibroblast markers, suggesting that some have a commitment to the fibroblast phenotype. Our in vitro study of these cells shows that they have extended self-renewal capability and express the primitive marker Nanog. In keeping with these observations, we also report that these cells are multipotent and differentiate readily into fibroblasts as well as other mesenchymal lineages. CONCLUSION: Cells with the properties of MSCs participate in wound healing after MI in the adult heart.
Abstract.
Author URL.
Crossman DJ, Ruygrok PN, Soeller C, Cannell MB (2011). Changes in the organization of excitation-contraction coupling structures in failing human heart.
PLoS One,
6(3).
Abstract:
Changes in the organization of excitation-contraction coupling structures in failing human heart.
BACKGROUND: the cardiac myocyte t-tubular system ensures rapid, uniform cell activation and several experimental lines of evidence suggest changes in the t-tubular system and associated excitation-contraction coupling proteins may occur in heart failure. METHODS AND RESULTS: the organization of t-tubules, L-type calcium channels (DHPRs), ryanodine receptors (RyRs) and contractile machinery were examined in fixed ventricular tissue samples from both normal and failing hearts (idiopathic (non-ischemic) dilated cardiomyopathy) using high resolution fluorescent imaging. Wheat germ agglutinin (WGA), Na-Ca exchanger, DHPR and caveolin-3 labels revealed a shift from a predominantly transverse orientation to oblique and axial directions in failing myocytes. In failure, dilation of peripheral t-tubules occurred and a change in the extent of protein glycosylation was evident. There was no change in the fractional area occupied by myofilaments (labeled with phalloidin) but there was a small reduction in the number of RyR clusters per unit area. The general relationship between DHPRs and RyR was not changed and RyR labeling overlapped with 51±3% of DHPR labeling in normal hearts. In longitudinal (but not transverse) sections there was an ∼30% reduction in the degree of colocalization between DHPRs and RyRs as measured by Pearson's correlation coefficient in failing hearts. CONCLUSIONS: the results show that extensive remodelling of the t-tubular network and associated excitation-contraction coupling proteins occurs in failing human heart. These changes may contribute to abnormal calcium handling in heart failure. The general organization of the t-system and changes observed in failure samples have subtle differences to some animal models although the general direction of changes are generally similar.
Abstract.
Author URL.
Full text.
Crossman DJ, Ruygrok PR, Soeller C, Cannell MB (2011). Correction: Changes in the Organization of Excitation-Contraction Coupling Structures in Failing Human Heart. PLoS ONE, 6(4).
Rajagopal V, Jayasinghe ID, Soeller C (2011). Modelling the Structure and Function of Cardiac Cell Transverse-Axial-Tubules.
Author URL.
Baddeley D, Cannell MB, Soeller C (2011). Three-dimensional sub-100 nm super-resolution imaging of biological samples using a phase ramp in the objective pupil.
Nano Research,
4(6), 589-598.
Abstract:
Three-dimensional sub-100 nm super-resolution imaging of biological samples using a phase ramp in the objective pupil
Localisation microscopy overcomes the diffraction limit by measuring the position of individual molecules to obtain optical images with a lateral resolution better than 30 nm. Single molecule localisation microscopy was originally demonstrated only in two dimensions but has recently been extended to three dimensions. Here we develop a new approach to three-dimensional (3D) localisation microscopy by engineering of the point-spread function (PSF) of a fluorescence microscope. By introducing a linear phase gradient between the two halves of the objective pupil plane the PSF is split into two lateral lobes whose relative position depends on defocus. Calculations suggested that the phase gradient resulting from the very small tolerances in parallelism of conventional slides made from float glass would be sufficient to generate a two-lobed PSF. We demonstrate that insertion of a suitably chosen microscope slide that occupies half the objective aperture combined with a novel fast fitting algorithm for 3D localisation estimation allows nanoscopic imaging with detail resolution well below 100 nm in all three dimensions (standard deviations of 20, 16, and 42 nm in x, y, and z directions, respectively). The utility of the approach is shown by imaging the complex 3D distribution of microtubules in cardiac muscle cells that were stained with conventional near infrared fluorochromes. The straightforward optical setup, minimal hardware requirements and large axial localisation range make this approach suitable for many nanoscopic imaging applications. © 2011 Tsinghua University Press and Springer-Verlag Berlin Heidelberg.
Abstract.
2010
Kjällman THM, Peng H, Soeller C, Travas-Sejdic J (2010). A CdTe nanoparticle-modified hairpin probe for direct and sensitive electrochemical detection of DNA.
Analyst,
135(3), 488-494.
Abstract:
A CdTe nanoparticle-modified hairpin probe for direct and sensitive electrochemical detection of DNA.
Detection of specific sequences of target DNA is of high importance in many fields, especially in medicinal diagnostics. DNA sensors should exhibit fast response to minute concentrations of the target sequence and have the ability to distinguish single-base mismatches from fully complementary target. This study focuses on the response of an electrochemical, CdTe nanoparticle-modified hairpin DNA sensor. The stem-loop structured probes and the blocking poly(ethylene glycol) (PEG) molecules were self-assembled on the gold electrode through S-Au bonding, to form a mixed monolayer employed as the sensing platform. Water-soluble CdTe nanoparticles were covalently attached to the hairpin probes (HPPs) and impedance spectroscopy was used for investigation of the electron transfer processes at a modified gold electrode before and after hybridization with the target DNA. The sensor showed reliable and sensitive detection of 4.7 fM of target. Although the selectivity of the sensor towards one-base mismatch targets needs to be improved, discrimination of non-complementary targets was achieved.
Abstract.
Author URL.
Jayasinghe ID, Crossman DJ, Soeller C, Cannell MB (2010). A new twist in cardiac muscle: dislocated and helicoid arrangements of myofibrillar z-disks in mammalian ventricular myocytes.
J Mol Cell Cardiol,
48(5), 964-971.
Abstract:
A new twist in cardiac muscle: dislocated and helicoid arrangements of myofibrillar z-disks in mammalian ventricular myocytes.
Using deconvolved confocal microscopy of fluorescently labeled markers for z-disks, t-tubules and ryanodine receptors, we have examined sarcomere organization in cardiac myocytes from rat, rabbit and human. We show that sarcomeres exhibit dislocations in registration and occasionally more complex helicoidal topology. This organization was present at both slack ( approximately 1.8 microm) and long sarcomere lengths ( approximately 2.2 microm). Misregistrations in z-disks persisted over 15-20 sarcomere lengths and appeared to arise primarily from variations in fiber direction; particularly as myofibrils pass around nuclei. In addition, myofibrils twist along the cell length. T-tubules generally follow the sarcomere z-disks although additional elements bridging adjacent myofibrils and along the length of the myofibril are present to varying degrees in all cells. Ryanodine receptors (the sarcoplasmic reticulum Ca(2+) release channel) are generally located within 250 nm of the local plane containing t-tubules and z-disks, but a small fraction ( approximately 2%) is found on longitudinal elements of the t-system between z-disks. The results are discussed with respect to the possible role(s) of such complex z-disk organization and z-disk dislocations in the maintenance of cell structure and sarcomere assembly. In addition, the non-planar organization of z-disks may be important in the propagation of local Ca(2+) waves which may have a useful role in helping maintain the uniformity of sarcomere activation in the presence of t-tubule remodeling.
Abstract.
Author URL.
Rossberger S, Baddeley D, Cannell MB, Cremer C, Soeller C (2010). Characterization of the Use of Far-Red Dyes for Localization Microscopy of Biological Samples.
Author URL.
Li P, Wei W, Cai X, Soeller C, Cannell MB, Holden AV (2010). Computational modelling of the initiation and development of spontaneous intracellular Ca2+ waves in ventricular myocytes.
Philos Trans a Math Phys Eng Sci,
368(1925), 3953-3965.
Abstract:
Computational modelling of the initiation and development of spontaneous intracellular Ca2+ waves in ventricular myocytes.
Intracellular Ca(2+) dynamics provides excitation-contraction coupling in cardiac myocytes. Under pathological conditions, spontaneous Ca(2+) release events can lead to intracellular Ca(2+) travelling waves, which can break, giving transitory or persistent intracellular re-entrant Ca(2+) scroll waves. Intracellular Ca(2+) waves can trigger cellular delayed after-depolarizations of membrane potential, which if they occur in a cluster of a few hundred neighbouring myocytes may lead to cardiac arrhythmia. Quantitative prediction of the initiation and propagation of intracellular Ca(2+) waves requires the dynamics of Ca(2+)-induced Ca(2+) release, and the intracellular spatial distribution of Ca(2+) release units (CRUs). The spatial distribution of ryanodine receptor clusters within a few sarcomeres was reconstructed directly from confocal imaging measurements. It was then embedded into a three-dimensional ventricular cell model, with a resting membrane potential and simple stochastic Ca(2+)-induced Ca(2+) release dynamics. Isotropic global Ca(2+) wave propagation can be produced within the anisotropic intracellular architecture, by isotropic local Ca(2+) diffusion, and the branching Z-disc structure providing inter Z-disc pathways for Ca(2+) propagation. The branching Z-disc provides a broader spatial distribution of ryanodine receptor clusters across Z-discs, which reduces the likelihood of wave initiation by spontaneous Ca(2+) releases. Intracellular Ca(2+) dynamics during catecholaminergic polymorphic ventricular tachycardia (CPVT) was simulated phenomenologically by increasing the Ca(2+) sensitivity factor of the CRU, which results in an increased rate of Ca(2+) release events. Flecainide has been shown to prevent arrhythmias in a murine model of CPVT and in patients. The modelled actions of flecainide on the time course of Ca(2+) release events prevented the initiation of Ca(2+) waves.
Abstract.
Author URL.
Jayasinghe ID, Li P, Holden AV, Crossman DJ, Cannell MB, Soeller C (2010). Dislocations and Helicoids in Myofibrillar Z-Disks of Mammalian Ventricular Myocytes and Implications for Calcium Handling.
Author URL.
Cooper PJ, Soeller C, Cannell MB (2010). Excitation-contraction coupling in human heart failure examined by action potential clamp in rat cardiac myocytes.
J Mol Cell Cardiol,
49(6), 911-917.
Abstract:
Excitation-contraction coupling in human heart failure examined by action potential clamp in rat cardiac myocytes.
The effect of the loss of the notch in the human action potential (AP) during heart failure was examined by voltage clamping rat ventricular myocytes with human APs and recording intracellular Ca(2+) with fluorescent dyes. Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca(2+) transient due to reduced ability of the L-type Ca(2+) channel to trigger release. The failing human AP increased non-uniformity of cytosolic Ca(2+), with some cellular regions failing to elicit Ca(2+)-induced Ca(2+) release from the sarcoplasmic reticulum. In addition, there was an increase in the occurrence of late Ca(2+) sparks. Monte-Carlo simulations of spark activation by L-type Ca(2+) current supported the idea that the decreased synchrony of Ca(2+) spark production associated with the loss of the notch could be explained by reduced Ca(2+) influx from open Ca(2+) channels. We conclude that the notch of the AP is critical for efficient and synchronous EC coupling and that the loss of the notch will reduce the SR Ca(2+) release in heart failure, without changes in (for example) SR Ca(2+)-ATPase uptake.
Abstract.
Author URL.
Baddeley D, Jayasinghe ID, Lam L, Rossberger S, Cannell MB, Soeller C (2010). Imaging of the Ryanodine Receptor Distribution in Rat Cardiac Myocytes with Optical Single Channel Resolution.
Author URL.
Baddeley D, Jayasinghe ID, Rossberger S, Cannell MB, Soeller C (2010). Novel Visualisation Techniques for Localisation Microscopy.
Author URL.
Torres NS, Savio-Galimberti E, Goldhaber JI, Soeller C, Bridge JHB, Sachse FB (2010). Ryanodine Receptors Outside of Couplons are Involved in Excitation-Contraction Coupling in Rabbit Ventricular Myocytes.
Author URL.
Spires JB, Peng H, Williams DE, Soeller C, Travas-Sejdic J (2010). Solvent-induced microstructure changes and consequences for electrochemical activity of redox-active conducting polymers.
Electrochimica Acta,
55(9), 3061-3067.
Abstract:
Solvent-induced microstructure changes and consequences for electrochemical activity of redox-active conducting polymers
Electrochemical impedance spectroscopy (EIS) was used to demonstrate solvent-induced collapse of the microstructure of redox-active conducting polymers; the collapse was dependent on the nature of the polymer and had a significant effect on the redox activity of the materials. Conducting polymer films of terthiophene, poly(3-((2′:2″,5″:2‴-terthiophene)-3′′-yl) acrylic acid) (PTAA) and poly(3,4-ethylenedioxythiophene) (PEDOT) were produced in an organic solvent (DCM, dichloromethane) and subsequently immersed in other organic solvents (DCM, acetonitrile) or in an aqueous solution (Tris buffer). Impedance diagrams over time implied shrinkage of the polymer upon solvent change, particularly in Tris buffer, that was more pronounced and more rapid for PTAA than for PEDOT. These changes were correlated with a loss of reversibility of electrochemical cycling and an increase of potential required for ion insertion into the polymer and could simply be understood in terms of the change in solvation of the polymer. © 2010 Elsevier Ltd. All rights reserved.
Abstract.
Baddeley D, Cannell MB, Soeller C (2010). Visualization of localization microscopy data.
Microsc Microanal,
16(1), 64-72.
Abstract:
Visualization of localization microscopy data.
Localization microscopy techniques based on localizing single fluorophore molecules now routinely achieve accuracies better than 30 nm. Unlike conventional optical microscopies, localization microscopy experiments do not generate an image but a list of discrete coordinates of estimated fluorophore positions. Data display and analysis therefore generally require visualization methods that translate the position data into conventional images. Here we investigate the properties of several widely used visualization techniques and show that a commonly used algorithm based on rendering Gaussians may lead to a 1.44-fold loss of resolution. Existing methods typically do not explicitly take sampling considerations into account and thus may produce spurious structures. We present two additional visualization algorithms, an adaptive histogram method based on quad-trees and a Delaunay triangulation based visualization of point data that address some of these deficiencies. The new visualization methods are designed to suppress erroneous detail in poorly sampled image areas but avoid loss of resolution in well-sampled regions. A number of criteria for scoring visualization methods are developed as a guide for choosing among visualization methods and are used to qualitatively compare various algorithms.
Abstract.
Author URL.
2009
Reuter H, Louch WE, Brette F, Sham JSK, Sun H, Yang X-R, Soeller C, Lakatta EG, Balijepalli RC (2009). Commentaries on viewpoint: the cardiac contraction cycle: is Ca2+ going local? Counterpoint.
J Appl Physiol (1985),
107(6), 1985-1987.
Author URL.
Peng H, Zhang L, Soeller C, Travas-Sejdic J (2009). Conducting polymers for electrochemical DNA sensing.
Biomaterials,
30(11), 2132-2148.
Abstract:
Conducting polymers for electrochemical DNA sensing.
Conducting polymers (CPs) are a class of polymeric materials that have attracted considerable interest because of their unique electronic, chemical and biochemical properties, making them suitable for numerous applications such as energy storage, memory devices, chemical sensors, and in electrocatalysis. Conducting polymer-based electrochemical DNA sensors have shown applicability in a number of areas related to human health such as diagnosis of infectious diseases, genetic mutations, drug discovery, forensics and food technology due to their simplicity and high sensitivity. This review paper summarizes the advances in electrochemical DNA sensing based on conducting polymers as active substrates. The various conducting polymers used for DNA detection, along with different DNA immobilization and detection methodologies are presented. Current trends in this field and newly developed applications due to advances in nanotechnology are also discussed.
Abstract.
Author URL.
Rajagopal V, Soeller C, Crampin EJ, Hunter PJ (2009). DEVELOPING a REALISTIC 3D MODEL OF THE VENTRICULAR CARDIOMYOCYTE.
Author URL.
Li P, Wei W, Cai X, Soeller C, Cannell MB, Holden AV (2009). Evolution of intracellular ca<sup>2∈+∈</sup> waves from about 10,000 ryr clusters: Towards solving a computationally daunting task.
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics),
5528, 11-20.
Abstract:
Evolution of intracellular ca2∈+∈ waves from about 10,000 ryr clusters: Towards solving a computationally daunting task
Detailed knowledge of spatial-temporal behaviours of intracellular calcium dynamics is very important in understanding excitation-contraction coupling of cardiac myocytes under both normal and pathological conditions, such as initiation and propagation of spontaneous calcium waves. A full cell simulation integrating about 10,000 Ca2∈+∈ release units (CRUs) in a typical cardiac myocyte is considered a multi-scale, computationally demanding problem. In this paper, we imported an experimentally obtained spatial distribution of Ryanodine Receptor (RyR) clusters into a spatially extended, stochastic model of intracellular Ca2∈+∈ dynamics, to investigate the role of the structural bifurcation of Z-disks on the initiation and propagation of intracellular Ca2∈+∈ waves from spatially symmetric Ca2∈+∈ sparks. Besides, we also proposed a simple parallelization strategy to increase computational speed for this chanllenging problem in cardiac modelling. © 2009 Springer Berlin Heidelberg.
Abstract.
Baddeley D, Jayasinghe I, Cremer C, Cannell MB, Soeller C (2009). Fluorescence Imaging of Ryanodine Receptor and Caveolin Distribution in Cardiac Myocytes at 30 nm Resolution.
Author URL.
Soeller C (2009). LOCAL CONTROL OF CARDIAC Ca2+ SIGNALING: MODELS, NANO-STRUCTURE, AND CHALLENGES.
JOURNAL OF APPLIED PHYSIOLOGY,
107(6), 1986-1987.
Author URL.
Baddeley D, Jayasinghe ID, Cremer C, Cannell MB, Soeller C (2009). Light-induced dark states of organic fluochromes enable 30 nm resolution imaging in standard media.
Biophys J,
96(2), L22-L24.
Abstract:
Light-induced dark states of organic fluochromes enable 30 nm resolution imaging in standard media.
We show that high quantum efficiency fluorophores can exhibit reversible photobleaching. This observation provides the basis for an imaging technique we call reversible photobleaching microscopy. We demonstrate applicability of this technique using antibody labeled biological samples in standard aqueous (or glycerol based) media to produce far-field images at approximately 30 nm resolution. Our novel method relies on intense illumination to reversibly induce a very long-lived (>10 s) dark state from which single fluorochromes slowly return stochastically. As in other localization microscopy methods, reversible photobleaching microscopy localizes single fluorochromes, but has the advantage that specialized photoactivatible and photoswitchable molecules or special immersion/embedding media are not required.
Abstract.
Author URL.
Baddeley D, Jayasinghe ID, Lam L, Rossberger S, Cannell MB, Soeller C (2009). Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes.
Proc Natl Acad Sci U S A,
106(52), 22275-22280.
Abstract:
Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes.
We have applied an optical super-resolution technique based on single-molecule localization to examine the peripheral distribution of a cardiac signaling protein, the ryanodine receptor (RyR), in rat ventricular myocytes. RyRs form clusters with a mean size of approximately 14 RyRs per cluster, which is almost an order of magnitude smaller than previously estimated. Clusters were typically not circular (as previously assumed) but elongated with an average aspect ratio of 1.9. Edge-to-edge distances between adjacent RyR clusters were often
Abstract.
Author URL.
Jayasinghe ID, Cannell MB, Soeller C (2009). Organization of ryanodine receptors, transverse tubules, and sodium-calcium exchanger in rat myocytes.
Biophys J,
97(10), 2664-2673.
Abstract:
Organization of ryanodine receptors, transverse tubules, and sodium-calcium exchanger in rat myocytes.
Confocal and total internal reflection fluorescence imaging was used to examine the distribution of caveolin-3, sodium-calcium exchange (NCX) and ryanodine receptors (RyRs) in rat ventricular myocytes. Transverse and longitudinal optical sectioning shows that NCX is distributed widely along the transverse and longitudinal tubular system (t-system). The NCX labeling consisted of both punctate and distributed components, which partially colocalize with RyRs (27%). Surface membrane labeling showed a similar pattern but the fraction of RyR clusters containing NCX label was decreased and no nonpunctate labeling was observed. Sixteen percent of RyRs were not colocalized with the t-system and 1.6% of RyRs were found on longitudinal elements of the t-system. The surface distribution of RyR labeling was not generally consistent with circular patches of RyRs. This suggests that previous estimates for the number of RyRs in a junction (based on circular close-packed arrays) need to be revised. The observed distribution of caveolin-3 labeling was consistent with its exclusion from RyR clusters. Distance maps for all colocalization pairs were calculated to give the distance between centroids of punctate labeling and edges for distributed components. The possible roles for punctate NCX labeling are discussed.
Abstract.
Author URL.
Peng H, Zhang L, Kilmartin PA, Zujovic Z, Soeller C, Travas-Sejdic J (2009). Quantum dots and nanostructured conducting polymers for biosensing applications.
International Journal of Nanotechnology,
6(3-4), 418-430.
Abstract:
Quantum dots and nanostructured conducting polymers for biosensing applications
Numerous methodologies are currently being employed for the preparation of nanomaterials in customised morphologies. In this paper, we outline recent progress in our laboratory to develop nanostructured inorganic semiconductors - quantum dots (QDs), and nanostructured organic semiconductors based on polyaniline (PANI), for biosensing applications. Blue luminescent CdTe QDs were prepared directly from an aqueous solution via photo-irradiation and used to demonstrate a simple homogenous DNA sensing platform. Highly luminescent CdTe/CdS core/shell QDs with enhanced photostability were also prepared in an aqueous phase using thioacetamide as a sulphur source. PANI nanostructures have been prepared using a template-free method and the mechanism of their formation is currently being investigated. The effect of having amino acids in the polymerisation solution and using polymeric acids as dopants on the morphology of the nanotubes has been examined. An electrochemical DNA sensor was constructed using PANI nanotubes doped with poly(methyl vinyl ether-alt-maleic acid). Copyright © 2009 Inderscience Enterprises Ltd.
Abstract.
Savio-Galimberti E, Sachse FB, Goldhaber JI, Soeller C, Bridge JHB (2009). Relationship of Ryanodine Receptors to the Sarcolemma in Rabbit Ventricular Myocytes.
Author URL.
Zhang L, Peng H, Sui J, Soeller C, Kilmartin PA, Travas-Sejdic J (2009). Self-assembly of poly(o-methoxyaniline) hollow microspheres.
Journal of Physical Chemistry C,
113(21), 9128-9134.
Abstract:
Self-assembly of poly(o-methoxyaniline) hollow microspheres
Hollow microspheres of poly(o-methoxyaniline) (POMA) were prepared in a solution of p-toluenesulfonic acid (p-TSA) using ammonium persulfate (APS) as oxidant. The morphology of the final polymers was characterized by scanning electron microscopy and transmission electron microscopy techniques. The molecular structure and electrical properties of the products were investigated using a range of spectroscopic methods. The mechanism of nanostructure formation was studied in the light of the chemical nature of the starting monomer and other synthesis conditions, such as the reaction temperature and time and the concentrations of the oxidant and of the dopant molecule. It was established that the mechanism of microsphere formation is based on polymer growth on preformed micelles, which could be seen in freeze-fracture experiments. The presence of p-TSA led to microspheres with a very regular size distribution. The CH3O-group on the aniline side chain also increased the electrocatalytic activity of nanostructured POMA films cast on a glassy carbon electrode for ascorbic acid (AA) oxidation, in comparison with polyaniline nanotubes. © 2009 American Chemical Society.
Abstract.
Li P, Soeller C, Cannell M, Wei W, Cai X, Holden AV (2009). THE ROLE OF STRUCTURAL BIFURCATION OF Z-DISKS ON REGULATING SPONTANEOUS CALCIUM ACTIVITIES IN CARDIAC MYOCYTES: a THEORETICAL INVESTIGATION.
Author URL.
Soeller C, Jayasinghe ID, Li P, Holden AV, Cannell MB (2009). Three-dimensional high-resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes.
Exp Physiol,
94(5), 496-508.
Abstract:
Three-dimensional high-resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes.
Quantitative understanding of the Ca(2+) handling in cardiac ventricular myocytes requires accurate knowledge of cardiac ultrastructure and protein distribution. We have therefore developed high-resolution imaging and analysis approaches to measure the three-dimensional distribution of immunolabelled proteins with confocal microscopy. Labelling of single rat cardiac myocytes with an antibody to the Z-line marker alpha-actinin revealed a complex architecture of sarcomere misalignment across single cells. Double immunolabelling was used to relate the Z-line structure to the distribution of ryanodine receptors (RyRs, the intracellular Ca(2+) release channels) and the transverse tubular system. Both RyR and transverse tubular system distributions exhibited frequent dislocations from the simple planar geometry generally assumed in existing mathematical models. To investigate potential effects of these irregularities on Ca(2+) dynamics, we determined the three-dimensional distribution of RyR clusters within an extended section of a single rat ventricular myocyte to construct a model of stochastic Ca(2+) dynamics with a measured Ca(2+) release unit (CRU) distribution. Calculations with this model were compared with a second model in which all CRUs were placed on flat planes. The model with a realistic CRU distribution supported Ca(2+) waves that spread axially along the cell at velocities of approximately 50 mum s(-1). By contrast, in the model with planar CRU distribution the axial wave spread was slowed roughly twofold and wave propagation often nearly faltered. These results demonstrate that spatial features of the CRU distribution on multiple length scales may significantly affect intracellular Ca(2+) dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.
Abstract.
Author URL.
2008
Travas-Sejdic J, Peng H, Spires J, Soeller C (2008). Conjugated polymers as novel electrochemical and optical DNA sensors.
2008 2nd IEEE International Nanoelectronics Conference, INEC 2008, 1074-1079.
Abstract:
Conjugated polymers as novel electrochemical and optical DNA sensors
A range of novel gene sensing methodologies based on intrinsically conducting polymers composed of modified polypyrroles and polythlophenes that were synthesized in the authors' laboratory are outlined and discussed. These sensors are based on the electrochemical transduction of the hybridization event by conducting polymer thin films. Optical gene detection schemes have also been developed based on cationic conjugated polymer and quantum dots homogeneous assays. The electrochemical and optical readout modalities are compared and discussed. © 2008 IEEE.
Abstract.
Kjällman THM, Peng H, Soeller C, Travas-Sejdic J (2008). Effect of probe density and hybridization temperature on the response of an electrochemical hairpin-DNA sensor.
Anal Chem,
80(24), 9460-9466.
Abstract:
Effect of probe density and hybridization temperature on the response of an electrochemical hairpin-DNA sensor.
Detection of specific sequences of target DNA is of high importance in many fields, especially in medicinal diagnostics. This study focuses on the response of an electrochemical, label-free DNA sensor at two different hybridization temperatures (37 and 44 degrees C). The stem-loop structured probes and the blocking polyethylene glycol molecules were self-assembled on the electrode through S-Au bonding, to form a mixed monolayer employed as the sensing platform. Impedance spectroscopy was used for investigation of the electron transfer processes at a modified gold electrode before and after hybridization with the target DNA. The sensor showed sensitive and selective detection of the target DNA at the lower temperature, whereas the higher temperature affected the dynamics of the hairpin significantly, reflected in an increased sensitivity of the sensor.
Abstract.
Author URL.
Kong CHT, Soeller C, Cannell MB (2008). Increasing sensitivity of Ca2+ spark detection in noisy images by application of a matched-filter object detection algorithm.
Biophys J,
95(12), 6016-6024.
Abstract:
Increasing sensitivity of Ca2+ spark detection in noisy images by application of a matched-filter object detection algorithm.
Microscopic calcium (Ca2+) events (such as Ca2+ sparks) are an important area for study, as they help clarify the mechanism(s) underlying intracellular signaling. In the heart, Ca2+ sparks occur as a result of Ca2+ release from the sarcoendoplasmic reticulum, via ryanodine receptor channels. Measurement of Ca2+ spark properties can provide valuable information about the control of ryanodine receptor channel gating in situ, but requires high spatiotemporal resolution imaging, which produces noisy datasets that are problematic for spark detection. Automated detection algorithms may overcome visual detection bias, but missed and false-positive events can distort the distribution of measured Ca2+ spark properties. We present a sensitive and reliable method for the automated detection of Ca2+ sparks in datasets obtained using confocal line-scanning or total internal reflection fluorescence microscopy. This matched-filter detection algorithm(MFDA) employs a user-defined object, chosen to mimic Ca2+ spark properties, and the experimental dataset is searched for instances of the object. Detection certainty is provided by nonparametric statistical testing. The supplied codes can also refine the search object on the basis of those detected to further increase detection sensitivity. In comparison to a commonly used, intensity-thresholding algorithm, the MFDA is more sensitive and reliable, particularly at low signal/noise ratios. The MFDA can also be easily adapted to other signal-detection problems in noisy datasets.
Abstract.
Author URL.
Zhang L, Peng H, Kilmartin PA, Soeller C, Travas-Sejdic J (2008). Poly(3,4-ethylenedioxythiophene) and polyaniline bilayer nanostructures with high conductivity and electrocatalytic activity.
Macromolecules,
41(20), 7671-7678.
Abstract:
Poly(3,4-ethylenedioxythiophene) and polyaniline bilayer nanostructures with high conductivity and electrocatalytic activity
Bilayer nanostructured poly(3,4-ethylenedioxythiophene) (PEDOT) and polyaniline (PANI) conducting polymer composites have been successfully prepared by oxidative polymerization of the parent monomers, aniline (An) and 3,4-ethylenedioxythiophene (EDOT), in aqueous solutions of p-toluenesulfonic acid (p-TSA). As the first step, PANI nanofibers were obtained in the p-TSA solution using ammonium persulfate (APS) as the oxidant. Subsequently, PEDOT was coated onto the PANI nanofibers by the oxidative polymerization of EDOT to form PEDOT/PANI bilayer nanofibers. The resulting nanostructured material was characterized by SEM and a range of spectroscopic methods, which confirmed that the surface layer of the synthesized materials had features typical of chemically synthesized PEDOT. The presence of the PEDOT layer increased the room temperature electrical conductivity of the PEDOT/PANI nanocomposites by 2 orders of magnitude in comparison with the parent PANI nanofibers. Moreover, PEDOT/PANI nanocomposites on a glassy carbon electrode showed stronger electrocatalytic activity for the oxidation of ascorbic acid than PANI nanofibers. © 2008 American Chemical Society.
Abstract.
Kjällman T, Peng H, Soeller C, Travas-Sejdic J (2008). Quenching of an indocarbocyanine dye and functionalized CdSe/ZnS quantum dots by gold surfaces.
Current Applied Physics,
8(3-4), 308-311.
Abstract:
Quenching of an indocarbocyanine dye and functionalized CdSe/ZnS quantum dots by gold surfaces
Quantum dots (QDs) have unique optical properties and several advantages over conventional organic dyes. The ability to make QDs water soluble allows for use in various biological applications. Smooth metallic surfaces are known to quench the fluorescence of both organic dyes and of certain QDs. This paper provides a comparative study of the quenching ability of an unmodified and a poly(ethyleneglycol) (PEG)-modified Au surface, of the emission from an organic dye and from CdSe/ZnS QDs. PEG is a biocompatible molecule widely used for modification of different surfaces when protein resistance is required. Fluorescence measurements show that both an unmodified and a PEG-modified Au surface are able to quench the emission from the dye and the QDs. Some interactions between the QDs and the unmodified Au surface were also observed, although the interactions are significantly suppressed by the presence of PEG. The PEG-modified Au surface is suitable for further applications, involving biomolecules, where its efficient quenching ability and protein resistivity can be utilized. © 2007 Elsevier B.V. All rights reserved.
Abstract.
Zhang L, Peng H, Kilmartin PA, Soeller C, Tilley R, Travas-Sejdic J (2008). Self-assembled hollow polyaniline/Au nanospheres obtained by a one-step synthesis.
Macromolecular Rapid Communications,
29(7), 598-603.
Abstract:
Self-assembled hollow polyaniline/Au nanospheres obtained by a one-step synthesis
Self-assembled hollow nanosphere composites of polyaniline and Au nanoparticles (PANI-p-TSA/Au) were chemically synthesized from solutions containing p-toluenesulfonic acid (p-TSA) with the addition of gold chloride trihydrate as the oxidant. The composite materials were characterized by SEM, TEM, and a range of spectroscopic methods. Spectroscopic characterizations confirmed that the polymeric product is a form of doped PANI, while electron diffraction and X-ray diffraction showed that elemental Au was present in the PANI-p-TSA/Au nanocomposites. The room temperature electrical conductivity of the PANI-p-TSA/Au nanocomposites was two orders of magnitude greater than a PANI-p-TSA obtained in the presence of ammonium persulfate as the oxidant under the same conditions. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA.
Abstract.
Spires JB, Peng H, Williams DE, Wright BE, Soeller C, Travas-Sejdic J (2008). The effect of the oxidation state of a terthiophene-conducting polymer and of the presence of a redox probe on its gene-sensing properties.
Biosensors and Bioelectronics,
24(4), 928-933.
Abstract:
The effect of the oxidation state of a terthiophene-conducting polymer and of the presence of a redox probe on its gene-sensing properties
The gene-sensing properties of sensor films made of a terthiophene-conducting polymer, poly(3-((2′:2″, 5″:2″′-terthiophene)-3″-yl)acrylic acid) (PTAA), were evaluated using electrochemical impedance spectroscopy for films in their reduced and oxidised states with and without the Fe(CN)63-/4- redox probe (RP) in dilute tris-EDTA buffer. Porous films of PTAA were prepared and attached to an oligonucleotide sequence specific to the Salmonella virulence gene InvA. These films could be described with a dual transmission line model in which the polymer conductivity was increased as a consequence of surface binding of complementary DNA. The effect is analogous to that reported for silicon nanowires and field-effect transistors in dilute electrolyte modified by charge exchange across the polymer-electrolyte interface. As a result, gene sensing could be conveniently observed as a change in the impedance phase angle at a fixed frequency. © 2008 Elsevier B.V. All rights reserved.
Abstract.
Spires JB, Peng H, Williams DE, Wright BE, Soeller C, Travas-Sejdic J (2008). The effect of the oxidation state of a terthiophene-conducting polymer and of the presence of a redox probe on its gene-sensing properties.
Biosens Bioelectron,
24(4), 934-939.
Abstract:
The effect of the oxidation state of a terthiophene-conducting polymer and of the presence of a redox probe on its gene-sensing properties.
The gene-sensing properties of sensor films made of a terthiophene-conducting polymer, poly(3-((2':2'', 5'':2'''-terthiophene)-3''-yl)acrylic acid) (PTAA), were evaluated using electrochemical impedance spectroscopy for films in their reduced and oxidised states with and without the Fe(CN)(6)(3-/4-) redox probe (RP) in dilute tris-EDTA buffer. Porous films of PTAA were prepared and attached to an oligonucleotide sequence specific to the Salmonella virulence gene InvA. These films could be described with a dual transmission line model in which the polymer conductivity was increased as a consequence of surface binding of complementary DNA. The effect is analogous to that reported for silicon nanowires and field-effect transistors in dilute electrolyte modified by charge exchange across the polymer-electrolyte interface. As a result, gene sensing could be conveniently observed as a change in the impedance phase angle at a fixed frequency.
Abstract.
Author URL.
2007
Vlajkovic SM, Wang CJH, Soeller C, Zimmermann H, Thorne PR, Housley GD (2007). Activation-dependent trafficking of NTPDase2 in Chinese hamster ovary cells.
Int J Biochem Cell Biol,
39(4), 810-817.
Abstract:
Activation-dependent trafficking of NTPDase2 in Chinese hamster ovary cells.
Membrane-bound NTPDase2 is a member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family involved in the regulation of P2 receptor signaling. NTPDase2 has broad substrate specificity for extracellular nucleotides, but hydrolyses nucleoside 5'-triphosphates with high preference over nucleoside 5'-diphosphates. In this study, we have sought to determine how enzyme substrates acting on P2 receptors affect intracellular NTPDase2 trafficking. To achieve this, Chinese hamster ovary (CHO) cells were transiently transfected with rat-specific NTPDase2 cDNA tagged with green fluorescent protein (GFP), to allow direct visualisation of subcellular localisation and trafficking of NTPDase2. Cells were superfused with NTPDase2 substrates (ATP and UTP) and synthetic nucleotide analogues (ATPgammaS and ADPbetaS), and confocal image stacks were acquired at regular time intervals. NTPDase2 incorporation into the plasma membrane was determined by comparative analysis of fluorescence intensity in the cytosolic and membrane compartments. GFP-tagged NTPDase2 was fully functional and ATP and ATPgammaS induced membrane incorporation of GFP-NTPDase2 from putative intracellular stores, whilst UTP and ADPbetaS were ineffective. The increased ATP hydrolysis rate correlated with increased NTPDase2 trafficking to the plasma membrane. ATP-induced NTPDase2 trafficking was mediated by activation of endogenous P2X receptors involving Ca2+ entry rather than by P2Y receptor-induced release of Ca2+ from intracellular stores. Our results suggest that P2X receptor activation stimulates insertion of latent NTPDase2 into the plasma membrane. The increase in surface-located NTPDase2 may reflect a regulatory mechanism counteracting excessive stimulation and desensitisation of P2 receptors.
Abstract.
Author URL.
Soeller C, Crossman D, Gilbert R, Cannell MB (2007). Analysis of ryanodine receptor clusters in rat and human cardiac myocytes.
Proc Natl Acad Sci U S A,
104(38), 14958-14963.
Abstract:
Analysis of ryanodine receptor clusters in rat and human cardiac myocytes.
Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and alpha-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and deconvolution. The RyR label formed discrete puncta representing clusters of RyRs or "couplons" around the edges of the myofilaments with a nearest-neighbor spacing of 0.66 +/- 0.06 microm in rat and 0.78 +/- 0.07 microm in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of approximately 0.6 microm(2) in rat and approximately 0.8 microm(2) in human. Although the couplons were in reasonable registration with z-lines, there were discontinuities in the longitudinal position of sarcomeres so that dislocations in the order of RyR clusters occurred. There was approximately 53% longitudinal registration of RyR clusters, suggesting a nonrandom placement of couplons around the sarcomere. These data can explain the spherical propagation of Ca(2+) waves and provide quantitative 3D data sets needed for accurate modeling of cardiac Ca(2+)-induced Ca(2+) release. By quantifying labeling intensity in rat ventricular myocytes, a lower limit of 78 RyRs per cluster (on average) was obtained. By modeling the couplon as a disk wrapping around a t-tubule and fitting cluster images, 95% of couplons contained between 120 and 260 RyRs (assuming that RyRs are tight packed with a spacing of 29 nm). Assuming similar labeling efficiency in human, from the fluorescence intensity alone we estimate that human ventricular myocytes contain approximately 30% fewer RyRs per couplon than rat.
Abstract.
Author URL.
Cannell MB, Kilmartin P, Jacobs M, Valiavalappil S, Travas-Sejdic J, Soeller C (2007). Chapter 14 Nanoscale fluid motion via molecular pores and polymer actuators.
,
3, 207-224.
Abstract:
Chapter 14 Nanoscale fluid motion via molecular pores and polymer actuators
To develop "lab-on-a-chip" devices, it would be useful to develop microscopic pumping systems as well as ways of separating reactants. We have been developing methods to study a protein based molecular filter based on connexin proteins found in cells. The connexins form gap-junctions with a molecular weight cut-off of 1kDa. To probe the porosity of a native gap-junction system we have combined two-photon excited flash photolysis and confocal microscopy to study diffusion through gap-junctions. To move sub-picolitre solution volumes, we have been examining volume changes within conducting polymers with atomic force microscopy (AFM). Rat lenses (a cell system with extensive gap-junction mediated cell-cell permeability) were loaded with CMNB-caged fluorescein and positioned on the stage of a confocal microscope. By focusing the light from a Ti:Sapphire laser into a selected fiber cell a point source of free fluorescein was created. The movement of released fluorescein within and between fiber cells was monitored using an Argon ion laser. Parameter fits to experimental data gave estimates for both intracellular and intercellular diffusion coefficients. From this analysis, the gap junctions in eye lens fiber cells permit exchange of low molecular weight compounds between cells at about 0.4% of the rate of free diffusion. Out-of-plane volume changes of microscopic polypyrrole strips were monitored with a Nanoscope II AFM. The actuator polymerization was carried out on gold strip electrodes patterned onto a glass slide. After polymerisation, alternate strips were oxidized and reduced to measure swelling and shrinkage as voltage was changed (+/- 1V). With suitable electrolytes, 20% (or more) volume change occurred. Since the volume change is reversible by reversing the electrical polarity, there is net movement of electrolyte into the conducting polymer matrix. The resulting movement could be used to propel solute and provide a pumping action. Additionally, the swelling of the polymer could be used as a valve to direct different solute streams to a reaction chamber. © 2007 Elsevier B.V. All rights reserved.
Abstract.
Peng H, Zhang L, Kjällman THM, Soeller C, Travas-Sejdic J (2007). DNA hybridization detection with blue luminescent quantum dots and dye-labeled single-stranded DNA.
J Am Chem Soc,
129(11), 3048-3049.
Author URL.
Peng H, Soeller C, Travas-Sejdic J (2007). DNA sensors based on conducting polymers functionalized with conjugated side chain.
Proceedings of IEEE Sensors, 1124-1127.
Abstract:
DNA sensors based on conducting polymers functionalized with conjugated side chain
In this work, label-free DNA sensors were prepared based on polypyrrole and polythiophene functionalized with conjugated side chain. Their sensing properties were investigated by electrochemical impedance spectroscopy. The relative performance of sensors based on functionalized polypyrrole and polythiophene was compared and discussed. © 2007 IEEE.
Abstract.
Kjällman T, Peng H, Travas-Sejdic J, Soeller C (2007). DNA-sensors based on functionalized conducting polymers and quantum dots.
Proceedings of SPIE - the International Society for Optical Engineering,
6416Abstract:
DNA-sensors based on functionalized conducting polymers and quantum dots
The availability of rapid and specific biosensors is of great importance for many areas of biomedical research and modern biotechnology. This includes a need for DNA sensors where the progress of molecular biology demands routine detection of minute concentrations of specific gene fragments. A promising alternative approach to traditional DNA essays utilizes novel smart materials, including conducting polymers and nanostructured materials such as quantum dots. We have constructed a number of DNA sensors based on smart materials that allow rapid one-step detection of unlabeled DNA fragments with high specificity. These sensors are based on functionalized conducting polymers derived from polypyrrole (PPy) and poly(p-phenylenevinylene) (PPV). PPy based sensors provide intrinsic electrical readout via cyclic voltammetry and electrochemical impedance spectroscopy. The performance of these sensors is compared to a novel self-assembled monolayer-PNA construct on a gold electrode. Characterization of the novel PNA based sensor shows that it has comparable performance to the PPy based sensors and can also be read out effectively using AC cyclic voltammetry. Complementary to such solid substrate sensors we have developed a novel optical DNA essay based on a new PPV derived cationic conducting polymer. DNA detection in this essay results from sample dependent fluorescence resonance energy transfer changes between the cationic conducting polymer and Cy3 labeled probe oligonucleotides. As an alternative to such fluorochrome based sensors we discuss the use of inorganic nanocrystals ('quantum dots') and present data from water soluble CdTe quantum dots synthesized in an aqueous environment.
Abstract.
Tai DCS, Hooks DA, Harvey JD, Smaill BH, Soeller C (2007). Illumination and fluorescence collection volumes for fiber optic probes in tissue.
J Biomed Opt,
12(3).
Abstract:
Illumination and fluorescence collection volumes for fiber optic probes in tissue.
Optical fibers can deliver light to, and collect it from, regions deep in tissue. However, reported illumination and fluorescence collection volumes adjacent to the fiber tip have been inconsistent, and systematic data on this topic are not available. Illumination and fluorescence collection profiles were characterized with high spatial resolution for different optical fibers in tissue and various fluids using two-photon flash photolysis and excitation. We confirm that illumination and fluorescence collection volumes for optical fibers are near identical. Collection volume is determined by the core dimensions and numerical aperture (NA) of the fiber and the scattering properties of the medium. For a multimode optical fiber with 100 microm core diam and NA=0.22, 80% of the total fluorescence is collected from a depth of 170 microm in tissue and 465 microm in nonscattering fluid. A semiempirical mathematical description of photon flux adjacent to the fiber tip was also developed and validated. This was used to quantify the extent of temporal blurring associated with propagation of a wavefront of altered fluorescence emission across the region addressed by fiber optic probes. We provide information that will facilitate the design of optical probes for tissue imaging or therapeutic applications.
Abstract.
Author URL.
Peng H, Soeller C, Vigar NA, Caprio V, Travas-Sejdic J (2007). Label-free detection of DNA hybridization based on a novel functionalized conducting polymer.
Biosens Bioelectron,
22(9-10), 1868-1873.
Abstract:
Label-free detection of DNA hybridization based on a novel functionalized conducting polymer.
A new functionalized pyrrole monomer, 3-pyrrolylacrylic acid (PAA) was synthesized. It was used to prepare a copolymer with pyrrole, poly(Py-co-PAA), which was investigated by reflective FT-IR, UV-vis spectroscopy and cyclic voltammetry. A label-free DNA sensor was prepared based on a poly(Py-co-PAA) film. Hybridization with complementary and non-complementary DNA targets was studied by electrochemical impedance spectroscopy. Results show a significant increase in the charge-transfer resistance upon addition of complementary target. The impedance spectra were analyzed by using a modified Randles and Ershler equivalent circuit model. The change in charge-transfer resistance that was used as an index of sensor response was found to be linear with logarithmic target concentration in the range of 2 x 10(-9) to 2 x 10(-7)M. The detection limit was 0.98 nM.
Abstract.
Author URL.
Peng H, Soeller C, Travas-Sejdic J (2007). Novel conducting polymers for DNA sensing.
Macromolecules,
40(4), 909-914.
Abstract:
Novel conducting polymers for DNA sensing
Novel functionalized pyrroles, 3-pyrrolylacrylic acid (PAA), 5-(3-pyrrolyl) 2,4-pentadienoic acid (PPDA), and 3-pyrrolylpentanoic acid (PPA) were synthesized and used to construct label-free gene sensors based on pyrrole copolymer films. Electrochemical impedance spectroscopy (EIS) was used to obtain electrical readout from these gene sensors. Comparison of the performance of PPDA-containing sensors to that containing 3-pyrrolylpentanoic acid (PPA), a similar functionalized pyrrole but with a saturated side chain, showed that functionalized polymers with unsaturated side chains have superior properties for use in biosensor applications. Sensors based on copolymers of both PAA and PPDA were evaluated across a wide range of oligonucleotide concentrations. The highest sensitivity was exhibited by a poly(Py-co-PPDA) sensor whose EIS spectra were well resolved, and the changes in charge-transfer resistance, taken as the index of sensor response, were largest among the materials studied. This sensor had a detection limit of 0.5 nM and a good selectivity. © 2007 American Chemical Society.
Abstract.
Zhang L, Peng H, Kilmartin PA, Soeller C, Travas-Sejdic J (2007). Polymeric acid doped polyaniline nanotubes for oligonucleotide sensors.
Electroanalysis,
19(7-8), 870-875.
Abstract:
Polymeric acid doped polyaniline nanotubes for oligonucleotide sensors
Template-free, self-assembled polyaniline (PANI) nanotubes were prepared in a solution of poly(methyl vinyl ether-alt-maleic acid) (PMVEA) by oxidative polymerization using ammonium persulfate as the oxidant. The weight ratio of the polymeric acid to aniline in the solution had a significant effect on the size of the polyaniline nanotubes as determined by SEM. The outer diameter of the nanotubes increased from 65 nm to 160 nm with increased levels of PMVEA in the reaction solution from 1 to 4 wt/v%. The PANI/PMVEA nanotubes were characterized by FTIR and cyclic voltammetry. The nanotubes were used to construct a simple electrochemical oligonucleotide (ODN) sensor where ODN probes were covalently grafted onto the residual carboxylic acid functionalities on the nanotubes. The potential pulse amperometry technique was used to obtain a direct and fast electrochemical readout. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.
Abstract.
Peng H, Zhang L, Soeller C, Travas-Sejdic J (2007). Preparation of water-soluble CdTe/CdS core/shell quantum dots with enhanced photostability.
Journal of Luminescence,
127(2), 721-726.
Abstract:
Preparation of water-soluble CdTe/CdS core/shell quantum dots with enhanced photostability
CdTe/CdS core/shell quantum dots (QDs) have been synthesized in an aqueous phase using thioacetamide as a sulfur source. The quantum yield was greatly enhanced by the epitaxial growth of a CdS shell, which was confirmed by X-ray photoelectron spectroscopy (XPS) results. The quantum yield of as-prepared CdTe/CdS core/shell QDs without any post-preparative processing reached 58%. The experimental results illustrate that the QDs with core/shell structure show better photostability than thioglycolic acid (TGA)-capped CdTe QDs. The cyclic voltammograms reveal higher oxidation potentials for CdTe/CdS core/shell QDs than for TGA-capped CdTe QDs, which explains the superior photostability of QDs with a core/shell structure. This enhanced photostability makes these QDs with core/shell structure more suitable for bio-labeling and imaging. © 2007 Elsevier B.V. All rights reserved.
Abstract.
Peng H, Zhang L, Spires J, Soeller C, Travas-Sejdic J (2007). Synthesis of a functionalized polythiophene as an active substrate for a label-free electrochemical genosensor.
Polymer,
48(12), 3413-3419.
Abstract:
Synthesis of a functionalized polythiophene as an active substrate for a label-free electrochemical genosensor
The synthesis of a new functionalized terthiophene monomer with an unsaturated side chain, 3-((2′:2″,5″:2‴-terthiophene)-3″-yl) acrylic acid, is described. A conducting polymer was obtained by electropolymerization of the functionalized monomer. The properties of the polymer were investigated using FT-IR, Raman and UV-vis spectroscopy. The application of the polymer as an active substrate for a genosensor is demonstrated by the covalent attachment of amino-end modified oligonucleotide probes to the carboxylic acid group of the polymer. The hybridization of the complementary ODNs can be clearly detected by an increase in the admittance without the need for an indicator or any sample modifications. © 2007 Elsevier Ltd. All rights reserved.
Abstract.
Raybould NP, Jagger DJ, Kanjhan R, Greenwood D, Laslo P, Hoya N, Soeller C, Cannell MB, Housley GD (2007). TRPC-like conductance mediates restoration of intracellular Ca2+ in cochlear outer hair cells in the guinea pig and rat.
J Physiol,
579(Pt 1), 101-113.
Abstract:
TRPC-like conductance mediates restoration of intracellular Ca2+ in cochlear outer hair cells in the guinea pig and rat.
Ca2+ signalling is central to cochlear sensory hair cell physiology through its influence on sound transduction, membrane filter properties and neurotransmission. However, the mechanism for establishing Ca2+ homeostasis in these cells remains unresolved. Canonical transient receptor potential (TRPC) Ca2+ entry channels provide an important pathway for maintaining intracellular Ca2+ levels. TRPC3 subunit expression was detected in guinea pig and rat organ of Corti by RT-PCR, and localized to the sensory and neural poles of the inner and outer hair cells (OHCs) by confocal immunofluorescence imaging. A cation entry current with a TRPC-like phenotype was identified in guinea pig and rat OHCs by whole-cell voltage clamp. This slowly activating current was induced by the lowering of cytosolic Ca2+ levels ([Ca2+]i) following a period in nominally Ca2+-free solution. Activation was dependent upon the [Ca2+]o and was sustained until [Ca(2+)]i was restored. Ca2+ entry was confirmed by confocal fluorescence imaging, and rapidly recruited secondary charybdotoxin- and apamin-sensitive K(Ca) currents. Dual activation by the G protein-coupled receptor (GPCR)-phospholipase C-diacylglycerol (DAG) second messenger pathway was confirmed using the analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Ion substitution experiments showed that the putative TRPC Ca2+ entry current was selective for Na+ > K+ with a ratio of 1: 0.6. The Ca2+ entry current was inhibited by the TRPC channel blocker 2-aminoethyl diphenylborate (2APB) and the tyrosine kinase inhibitor, erbstatin analogue. We conclude that TRPC Ca2+ entry channels, most likely incorporating TRPC3 subunits, support cochlear hair cell Ca2+ homeostasis and GPCR signalling.
Abstract.
Author URL.
Soeller C, Gilbert R, Cannell MB (2007). The distribution of ryanodine receptor clusters and its relationship to the contractile apparatus in rat ventricular myocytes.
Author URL.
2006
Peng H, Soeller C, Travas-Sejdic J (2006). A novel cationic conjugated polymer for homogeneous fluorescence-based DNA detection.
Chem Commun (Camb)(35), 3735-3737.
Abstract:
A novel cationic conjugated polymer for homogeneous fluorescence-based DNA detection.
A novel water-soluble cationic conjugated polymer, poly({2,5-bis[3-(N,N-diethylamino)-1-oxapropyl]-para-phenylenevinylene}-alt-para-phenylenevinylene) dibromide, was synthesized and used to develop a simple label-free DNA detection essay.
Abstract.
Author URL.
Travas-Sejdic J, Peng H, Cooney RP, Bowmaker GA, Cannell MB, Soeller C (2006). Amplification of a conducting polymer-based DNA sensor signal by CdS nanoparticles.
Current Applied Physics,
6(3), 562-566.
Abstract:
Amplification of a conducting polymer-based DNA sensor signal by CdS nanoparticles
A novel DNA sensor based on a polypyrrole substrate and nanoparticle labeled ODN probes was prepared and characterized. In this sensor type the DNA sample to be analyzed was physically entrapped into the polypyrrole film during electropolymerization. The resulting sensor film was then exposed to ODN probes in solution. Sensitive electrical readout was achieved by electrochemical impedance spectroscopy. A further amplification in sensor response (as compared to using unlabeled ODN probes) was achieved by tagging ODN probes with CdS nanoparticles. We suggest that the sensor response is caused by an increase in charge transfer resistance upon binding of complementary CdS-ODN nanoparticle probes. The selectivity of the sensor was tested with various match and mismatch sequences. By appropriate choice of hybridization conditions the sensor was able to robustly discriminate between the exact match and a two-point mismatch sequence. © 2005 Elsevier B.V. All rights reserved.
Abstract.
Cannell MB, Crossman DJ, Soeller C (2006). Effect of changes in action potential spike configuration, junctional sarcoplasmic reticulum micro-architecture and altered t-tubule structure in human heart failure.
J Muscle Res Cell Motil,
27(5-7), 297-306.
Abstract:
Effect of changes in action potential spike configuration, junctional sarcoplasmic reticulum micro-architecture and altered t-tubule structure in human heart failure.
Using a Monte-Carlo model of L-type Ca2+ channel (DHPR) gating, we have examined the effect of changes in the early time course of the action potential as seen in human heart failure on excitation contraction coupling. The time course of DHPR Ca2+ influx was coupled into a simple model of sarcoplasmic reticulum Ca2+ release. Our model shows that the loss of the initial spike in human heart failure should reduce the synchrony of Ca2+ spark production and lead to the appearance of late Ca2+ sparks and greater non-uniformity of intracellular Ca2+. Within the junctional space of the cardiac dyad, a small increase in the mean distance of a DHPR from a RyR results in a marked decrease in the ability of the DHPR-mediated increase in local [Ca2+] concentration to activate RyRs. This suggests that the efficiency of EC coupling may be reduced if changes in micro-architecture develop and such effects have been noted in experimental models of heart failure. High resolution imaging of t-tubules in tachycardia-induced heart failure show deranged t-tubule structure. While in normal human hearts t-tubules run mainly in a radial direction, t-tubules in the heart failure samples were oriented more toward the long axis of the cell. In addition, t-tubules may become dilated and bifurcated. Our data suggest that changes in the micro-architecture of the cell and membrane structures associated with excitation-contraction coupling, combined with changes in early action potential configuration can reduce the efficiency by which Ca2+ influx via DHPRs can activate SR calcium release and cardiac contraction. While the underlying cause of these effects is unclear, our data suggest that geometric factors can play an important role in the pathophysilogy of the human heart in failure.
Abstract.
Author URL.
Peng H, Soeller C, Cannell MB, Bowmaker GA, Cooney RP, Travas-Sejdic J (2006). Electrochemical detection of DNA hybridization amplified by nanoparticles.
Biosens Bioelectron,
21(9), 1727-1736.
Abstract:
Electrochemical detection of DNA hybridization amplified by nanoparticles.
Detection of specific oligonucleotide (ODN) fragments has become an important field in many areas of biomedicine. We describe a novel ODN sensor based on electropolymerization of a conducting polymer (polypyrrole) in the presence of a sample containing ODN(s). The resulting trapped ODN(s) are then probed by addition of complimentary sequence ODN. By incorporating CdS nanoparticles with the probe, a significant improvement in sensor sensitivity was observed. Impedance spectroscopy suggested that optimal detection of hybridization occurred at frequencies>or=3000 Hz (for a 0.07 cm2 85 nm thick film). At these frequencies, the impedance signal was almost linear with the logarithm of ODN concentration in the range 3.7-370 nM with a detection limit of approximately 1 nM ODN (for the sensor fabricated). Importantly, the sensor could be regenerated by removing hybridized ODN with NaOH suggesting possibility of the sensor re-use.
Abstract.
Author URL.
Tai DCS, Soeller C, Hooks DA, Harvey JD, Smaill BH (2006). Illumination and effective collection volumes for fiber optic probes in tissue.
Optics InfoBase Conference PapersAbstract:
Illumination and effective collection volumes for fiber optic probes in tissue
Illumination and fluorescence collection volumes of different optical fibres in tissue were characterized using 2-photon techniques. A 3D illumination model was also developed and validated. This study provides important information for optical probe design in biomedical applications. © 2006 Optical Society of America.
Abstract.
Tai DCS, Soeller C, Hooks DA, Harvey JD, Smaill BH (2006). Illumination and effective collection volumes for fiber optic probes in tissue.
Conference on Lasers and Electro-Optics and 2006 Quantum Electronics and Laser Science Conference, CLEO/QELS 2006Abstract:
Illumination and effective collection volumes for fiber optic probes in tissue
Illumination and fluorescence collection volumes of different optical fibres in tissue were characterized using 2-photon techniques. A 3D illumination model was also developed and validated. This study provides important information for optical probe design in biomedical applications. © 2006 Optical Society of America.
Abstract.
Cannell MB, McMorland A, Soeller C (2006). Image enhancement by deconvolution.
, 488-500.
Abstract:
Image enhancement by deconvolution
In this chapter we will try to provide a more intuitive (and less mathematical) insight into image formation and practical image restoration by deconvolution methods. The mathematics of image formation and deconvolution microscopy have been described in greater detail elsewhere (see Chapters 11, 22, 23, and 24), so we will limit our discussion to fundamental issues and gloss over most of the mathematics of image restoration. We will also focus on practical ways of assessing microscope performance and getting the best possible data before applying more sophisticated image processing methods than are usually seen in the literature. © 2006, 1995, 1989 Springer Science+Business Media, LLC. All rights reserved.
Abstract.
Chen-Izu Y, McCulle SL, Ward CW, Soeller C, Allen BM, Rabang C, Cannell MB, Balke CW, Izu LT (2006). Three-dimensional distribution of ryanodine receptor clusters in cardiac myocytes.
Biophys J,
91(1), 1-13.
Abstract:
Three-dimensional distribution of ryanodine receptor clusters in cardiac myocytes.
The clustering of ryanodine receptors (RyR2) into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling. Using immunolabeling and confocal microscopy, we have analyzed the distribution of RyR2 clusters in rat and ventricular atrial myocytes. The resolution of the three-dimensional structure was improved by a novel transverse sectioning method as well as digital deconvolution. In contrast to earlier reports, the mean RyR2 cluster transverse spacing was measured 1.05 microm in ventricular myocytes and estimated 0.97 microm in atrial myocytes. Intercalated RyR2 clusters were found interspersed between the Z-disks on the cell periphery but absent in the interior, forming double rows flanking the local Z-disks on the surface. The longitudinal spacing between the adjacent rows of RyR2 clusters on the Z-disks was measured to have a mean value of 1.87 microm in ventricular and 1.69 microm in atrial myocytes. The measured RyR2 cluster distribution is compatible with models of Ca2+ wave generation. The size of the typical RyR2 cluster was close to 250 nm, and this suggests that approximately 100 RyR2s might be present in a cluster. The importance of cluster size and three-dimensional spacing for current E-C coupling models is discussed.
Abstract.
Author URL.
2005
Cannell MB, Jacobs MD, Donaldson PJ, Soeller C (2005). Application of two-photon flash photolysis to measure microscopic diffusion and calcium fluxes.
Proceedings of SPIE - the International Society for Optical Engineering,
5714, 116-125.
Abstract:
Application of two-photon flash photolysis to measure microscopic diffusion and calcium fluxes
Two-photon excitation (TPE) via a microscope objective lens poduces a spatially confined excitation volume where UV-excited caged molecules may be broken (uncaged) to release active products. We describe an optical system that creates a stationary parfocal TPE uncaging spot on the stage of a conventional confocal microscope. With this system, we have examined the ability of two dyes to track microscopic calcium changes produced by TPE photolysis of DM-nitrophen. We find that, even when EGTA is used with a low affinity indicator, the dye signals are complicated by diffusion of both indicator-Ca complex and CaEGTA to produce a signal that does not simply report the spatial dimensions of the calcium release site. In addition, the time course of calcium release is poorly reported. This suggests that considerable caution must be applied to the interpretation of spatially resolved calcium signals inside cells. We have also used TPE of CMND-caged fluorescein to measure the rate of fluorescein production in test solution (2500 s-1) as well as the diffusion of fluorescein in drops of solution and within and between between eye lens fiber cells. While diffusion of uncaged fluorescein was about an order of magnitude slower inside fiber cells than in aequeous solution, slower diffusion between cells could also be detected and could be explained by the gap junctions joining the cells behaving as a barrier to diffusion. By using a computer model, parameter fits to experimental data gave estimates for both intracellular and intercellular diffusion coefficients. From this anaysis, the gap junctions in eye lens fiber cells permit exchange of low molecular weight compounds between cells at about 0.4% of the rate of free diffusion.
Abstract.
Wang CJH, Vlajkovic SM, Housley GD, Braun N, Zimmermann H, Robson SC, Sévigny J, Soeller C, Thorne PR (2005). C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation.
Biochem J,
385(Pt 3), 729-736.
Abstract:
C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation.
The present study provides functional characterization of alternative splicing of the NTPDase2 (ecto-nucleoside triphosphate diphosphohydrolase-2) involved in the regulation of extracellular nucleotide concentrations in a range of organ systems. A novel NTPDase2beta isoform produced by alternative splicing of the rat NTPDase2 gene provides an extended intracellular C-terminus and distinguishes itself from NTPDase2alpha isoform in gaining several intracellular protein kinase CK2 (casein kinase 2) phosphorylation sites and losing the intracellular protein kinase C motif. The plasmids containing NTPDase2alpha or NTPDase2beta cDNA were used to stably transfect Chinese-hamster ovary-S cells. Imaging studies showed that NTPDase2alpha was predominantly membrane-bound, whereas NTPDase2beta had combined cell surface and intracellular localization. alpha and beta isoforms showed variations in divalent cation dependence and substrate specificity for nucleoside-5'-triphosphates and nucleoside-5'-diphosphates. NTPDase2beta exhibited reduced ATPase activity and no apparent ADPase activity. NTPDase2 isoforms demonstrated similar sensitivity to inhibitors such as suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid, and differential regulation by protein kinases. NTPDase2beta was up-regulated by intracellular protein kinase CK2 phosphorylation, whereas NTPDase2alpha activity was down-regulated by protein kinase C phosphorylation. The results demonstrate that alternative coding of the intracellular C-terminal domain contributes distinctive phenotypic variation with respect to extracellular nucleotide specificity, hydrolysis kinetics, protein kinase-dependent intracellular regulation and protein trafficking. These findings advance the molecular physiology of this enzyme system by characterizing the contribution of the C-terminal domain to many of the enzyme's signature properties.
Abstract.
Author URL.
Soeller C, Song X, Cannell MB (2005). Colocalization of proteins involved in cardiac excitation-contraction coupling studied at high spatial resolution.
Author URL.
Travas-Sejdic J, Peng H, Kilmaitin PA, Cannell MB, Bowmaker GA, Cooney RP, Soeller C (2005). DNA sensor based on functionalized polypyrrole.
Synthetic Metals,
152(1-3), 37-40.
Abstract:
DNA sensor based on functionalized polypyrrole
A novel DNA sensor based on a functionalized polypyrrole was prepared. The sensor film was obtained by electrocopolymerizing poly(pyrrole-co-4-(3-pyrrolyl) butanoic acid) onto which an NH2-ODN probe was grafted. The obtained sensor is a simple, label-free DNA sensor with direct electrical read-out. Hybridisation with complementary ODN was investigated by observing changes in die cyclic voltammogram and the AC impedance spectrum. Significant modifications of both the voltammogram and AC impedance spectrum were observed when the sensor was incubated in the presence of complementary ODN. The AC impedance spectra show an increased charge transfer resistance of the film after hybridisation. © 2005 Elsevier B.V. All rights reserved.
Abstract.
Peng H, Soeller C, Vigar N, Kilmartin PA, Cannell MB, Bowmaker GA, Cooney RP, Travas-Sejdic J (2005). Label-free electrochemical DNA sensor based on functionalised conducting copolymer.
Biosens Bioelectron,
20(9), 1821-1828.
Abstract:
Label-free electrochemical DNA sensor based on functionalised conducting copolymer.
A simple and label-free electrochemical sensor for recognition of the DNA hybridization event was prepared based on a new functionalised conducting copolymer, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid]. This precursor copolymer can be easily electrodeposited on the electrode surface and shows high electroactivity in an aqueous medium. An amino-substituted oligonucleotide (ODN) probe was covalently grafted onto the surface of the copolymer in a one step procedure and tested on hybridization with complementary ODN segments. The cyclic voltammogram of ODN probe-modified copolymer showed very little change when incubated in presence of non-complementary ODN, while a significant, and reproducible, modification of the voltammogram was observed after addition of complementary ODN. The AC impedance spectrum showed an increased charge transfer resistance (Rct) and double layer capacitance of the sensor film after hybridisation. Sensors with thinner films showed higher sensitivity than thicker films, suggesting that hybridisation at or near the surface of the film produces a larger change in electrical properties than that within the body of the film.
Abstract.
Author URL.
Soeller C, Peng H, Cannell MB, Travas-Sejdic J (2005). Novel DNA sensors with electrical read-out based on conducting polymers and quantum dots.
Author URL.
2004
Soeller C, Cannell MB (2004). Analysing cardiac excitation-contraction coupling with mathematical models of local control.
Prog Biophys Mol Biol,
85(2-3), 141-162.
Abstract:
Analysing cardiac excitation-contraction coupling with mathematical models of local control.
Cardiac excitation-contraction (E-C) coupling describes the process that links sarcolemmal Ca2+ influx via L-type Ca2+ channels to Ca2+ release from the sarcoplasmic reticulum via ryanodine receptors (RyRs). This process has proven difficult to study experimentally, and complete descriptions of how the cell couples surface membrane and intracellular signal transduction proteins to achieve both stable and sensitive intracellular calcium release are still lacking. Mathematical models provide a framework to test our understanding of how this is achieved. While no single model is yet capable of describing all features of cardiac E-C coupling, models of increasing complexity are revealing unexpected subtlety in the process. In particular, modelling has established a general failure of 'common-pool' models and has emphasized the requirement for 'local control' so that microscopic sub-cellular domains can separate local behaviour from the whole-cell average (common-pool) behaviour. The micro-architecture of the narrow diadic cleft in which the local control takes place is a key factor in determining local Ca2+ dynamics. There is still considerable uncertainty about the number of Ca2+ ions required to open RyRs within the cleft and various gating models have been proposed, many of which are in reasonable agreement with available experimental data. However, not all models exhibit a realistic voltage dependence of E-C coupling gain. Furthermore, it is unclear which model features are essential to producing reasonable gain properties. Thus, despite the success of local-control models in explaining many features of cardiac E-C coupling, more work will be needed to provide a sound theoretical basis of cardiac E-C coupling.
Abstract.
Author URL.
Cannell MB, Soeller C (2004). Control of the Ca spark during E-C coupling.
Author URL.
Donaldson PJ, Grey AC, Merriman-Smith BR, Sisley AMG, Soeller C, Cannell MB, Jacobs MD (2004). Functional imaging: new views on lens structure and function.
Clin Exp Pharmacol Physiol,
31(12), 890-895.
Abstract:
Functional imaging: new views on lens structure and function.
1. We have developed an experimental imaging approach that allows the distribution of lens membrane proteins to be mapped with subcellular resolution over large distances as a function of fibre cell differentiation. 2. Using this approach in the rat lens, we have localized precisely histological sites of connexin 46 cleavage, quantitatively mapped changes in gap junction distribution and fibre cell morphology and correlated these changes to differences in intercellular dye transfer. 3. Profiling of glucose transporter isoform expression showed that lens epithelial cells express GLUT1, whereas deeper cortical fibre cells express the higher-affinity GLUT3 isoform. Near the lens periphery, GLUT3 was located in the cytoplasm of fibre cells, but it underwent a differentiation-dependent membrane insertion. 4. Similarly, the putative adhesion protein membrane protein 20 is inserted into fibre cell membranes at the stage when the cells lose their nuclei. This redistribution is strikingly rapid in terms of fibre cell differentiation and correlates with a barrier to extracellular diffusion. 5. Our imaging-orientated approach has facilitated new insights into the relationships between fibre cell differentiation and lens function. Taken together, our results indicate that a number of strategies are used by the lens during the course of normal differentiation to change the subcellular distribution, gross spatial location and functional properties of key membrane transport proteins.
Abstract.
Author URL.
Jacobs MD, Soeller C, Sisley AMG, Cannell MB, Donaldson PJ (2004). Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens.
Invest Ophthalmol Vis Sci,
45(1), 191-199.
Abstract:
Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens.
PURPOSE: to map changes in the structure and function of fiber cell gap junctions that occur with lens differentiation. METHODS: Equatorial lens sections were fluorescently labeled with antibodies to the gap junction protein connexin (Cx)46, the membrane marker wheat germ agglutinin, and the nuclear stain propidium iodide. Two-photon microscopy and digital image analysis were used to quantify label and cell morphology as a function of radial distance (r/a) across the lens. Loop- and tail-specific Cx46 antibodies were used to identify regions of posttranslational modification. Local fiber cell coupling was imaged in situ using two-photon flash photolysis of caged fluorescein. RESULTS: Antibody labeling showed that the cytoplasmic tail of Cx46 was removed in two zones (r/a approximately 0.9 and r/a approximately 0.7). In addition, with increasing depth, the large radially aligned plaques of peripheral fiber cells became fragmented and dispersed around the cell membrane, and cells became more circular in cross section. Fluorescein transfer between peripheral fiber cells was highly anisotropic and occurred predominantly within a column of fiber cells, resulting in radially directed transport. In regions beyond the zone of nuclear loss, transport was more isotropic and occurred across columns of fiber cells. CONCLUSIONS: the cleavage of Cx46 is associated with a spatial redistribution of gap junction plaques. The distribution of gap junction plaques around the cell membrane can explain the observed directionality of intercellular solute transfer. The findings suggest that the processing and redistribution of gap junction proteins is central to controlling radial and circumferential solute gradients in different regions within the lens.
Abstract.
Author URL.
Chen-Izu Y, McCulle SL, Allen BM, Wehrens XH, Soeller C, Cannell MB, Robinson SW, Ward CW, Reiken SR, Marks AR, et al (2004). Geometry of ryanodine receptor localization in ventricular and atrial myocytes.
Author URL.
Cannell MB, Jacobs MD, Donaldson PJ, Soeller C (2004). Probing microscopic diffusion by 2-photon flash photolysis: measurement of isotropic and anisotropic diffusion in lens fiber cells.
Microsc Res Tech,
63(1), 50-57.
Abstract:
Probing microscopic diffusion by 2-photon flash photolysis: measurement of isotropic and anisotropic diffusion in lens fiber cells.
Two-photon excited flash photolysis (TPEFP) was used to photorelease caged fluorescein in test solutions and inside fiber cells of the eye lens. Accurate alignment between the focus of the IR beam and the probe beam from the confocal microscope was achieved with an accessory focussing lens and computer models of diffusion were fit to experimental data to extract apparent diffusion coefficients. Inside a fiber cell, the diffusion coefficient for fluorescein was 4 x 10(-7) cm(2)/s at 21 degrees C, a value an order of magnitude lower than observed in free solution. Fluorescence also diffused between fiber cells via gap junctions. In the periphery, diffusion between cells occurred mainly in a radial direction while deep in the lens the diffusion between cells appeared more isotropic. Diffusion between cells was slower than inside cells and corresponded to less than approximately 1% of the area between cells being available for diffusion. This value is in good agreement with that expected from measurements of gap junction structure and packing density if a 1-1.5-nm aqueous gap junction pore is nearly always open.
Abstract.
Author URL.
2003
Soeller C, Cannell MB (2003). A numerical study of ryanodine receptor gating in the cardiac diad and comparison with experimental data.
Author URL.
Soeller C, Jacobs MD, Jones KT, Ellis-Davies GCR, Donaldson PJ, Cannell MB (2003). Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements (vol 8, pg 418, 2003).
JOURNAL OF BIOMEDICAL OPTICS,
8(4), 668-668.
Author URL.
Soeller C, Jacobs MD, Jones KT, Ellis-Davies GCR, Donaldson PJ, Cannell MB (2003). Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements.
J Biomed Opt,
8(3), 418-427.
Abstract:
Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements.
Two-photon excitation makes it possible to excite molecules in volumes of much less than 1 fl. In two-photon flash photolysis (TPFP) this property is used to release effector molecules from caged precursors with high three-dimensional resolution. We describe and examine the benefits of using TPFP in model solutions and in a number of cell systems to study their spatial and temporal properties. Using TPFP of caged fluorescein, we determined the free diffusion coefficient of fluorescein (D=4 x 0(-6) cm(2)/s at 20 degrees C, which is in close agreement with published values). TPFP of caged fluorescein in lens tissue in situ revealed spatial nonuniformities in intercellular fiber cell coupling by gap junctions. At the lens periphery, intercellular transport was predominantly directed along rows of cells, but was nearly isotropic further from the periphery. To test an algorithm aiming to reconstruct the Ca(2+) release flux underlying physiological Ca(2+) signals in heart muscle cells, TPFP of DM-Nitrophen was utilized to generate artificial microscopic Ca(2+) signals with known underlying Ca(2+) release flux. In an experiment with mouse oocytes, the recently developed Ca(2+) cage dimethoxynitrophenyl-ethyleneglycol-bis-(beta-aminoethylether)-N,N,N('),N(') tetraacetic acid-4 (DMNPE-4) was released in the oocyte cytosol and inside a nucleolus. Analysis of the resulting fluorescence changes suggested that the effective diffusion coefficient within the nucleolus was half of that in the cytosol. These experiments demonstrate the utility of TPFP as a novel tool for the optical study of biomedical systems.
Abstract.
Author URL.
Soeller C, Jacobs MD, Jones KT, Ellis-Davies GCR, Donaldson PJ, Cannell MB (2003). Erratum: Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca<sup>2+</sup> movements (Journal of Biomedical Optics (July 2000) 8:3 (418-427)). Journal of Biomedical Optics, 8(4).
Soeller C, Jacobs M, Donaldson P, Cannell MB (2003). Fiber cell coupling in the mammalian lens probed by two-photon flash photolysis of caged fluorescein.
Author URL.
Jacobs MD, Soeller C, Cannell MB, Donaldson PJ (2003). Quantitative real-time imaging of local fiber cell gap junction coupling in the rat lens.
Author URL.
Jacobs MD, Donaldson PJ, Cannell MB, Soeller C (2003). Resolving morphology and antibody labeling over large distances in tissue sections.
Microsc Res Tech,
62(1), 83-91.
Abstract:
Resolving morphology and antibody labeling over large distances in tissue sections.
Protein expression patterns are a primary determinant of tissue function and in this study we developed methods to study protein expression over macroscopic distances at subcellular levels of detail. Using the mammalian lens as a model tissue system, we show that by combining two-photon microscopy with novel image montage methods (fast beam blanking coupled with mathematical alignment tools) we have extended the limited field of view of laser scanning microscopes. To illustrate the utility of our approach, the distribution of connexin-46 was visualized across equatorial sections of the rat mammalian lens. By optimizing fixation protocols, good morphological preservation could be achieved over the thickness of the lens (approximately 4 mm) while preserving antigenicity of lens proteins. Using the same image data, changes in lens fiber cell morphology were mapped quantitatively by automatic image analysis routines. The methods presented should be generally applicable to any tissue system where changes in antibody labeling and tissue structure occur over large and small distances.
Abstract.
Author URL.
2002
Soeller C, Cannell MB (2002). A Monte Carlo model of ryanodine receptor gating in the diadic cleft of cardiac muscle.
Author URL.
Cannell MB, Soeller C (2002). A mode of thought in excitation-contraction coupling.
Biophys J,
83(1), 1-2.
Author URL.
Donaldson PJ, Grey AC, Merriman-Smith BR, Kistler J, Cannell MB, Soeller C, Jacobs MD (2002). Chances in the distribution of lens membrane proteins as a function of fiber cell differentiation: Functional implications for lens transparency.
Author URL.
Soeller C, Cannell MB (2002). Estimation of the sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks.
Biophys J,
82(5), 2396-2414.
Abstract:
Estimation of the sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks.
Using a combination of experimental and numerical approaches, we have tested two different approaches to calculating the sarcoplasmic reticulum (SR) Ca2+ release flux, which gives rise to cardiac muscle Ca2+ sparks. By using two-photon excited spot photolysis of DM-Nitrophen, known Ca2+ release flux time courses were generated to provide the first experimental validation of spark flux reconstruction algorithms. These artificial Ca2+ sparks show that it is possible to calculate the SR Ca2+ release waveform with reasonable accuracy, provided the flux equations reasonably reflect the properties of the experimental system. Within cardiac muscle cells, we show that Ca2+ flux reconstruction is complicated by the substantial dye binding to proteins, a factor that has not been adequately addressed in previous flux reconstruction algorithms. Furthermore, our numerical experiments suggest that the calculated time course of release flux inactivation based on conventional flux reconstruction algorithms is likely to be in error. We therefore developed novel algorithms based on an explicit dye binding scheme. When these algorithm were applied to evoked Ca2+ sparks in rat cardiac ventricular myocytes, the reconstructed Ca2+ release waveform peaked in ~5 ms and decayed with a halftime of approximately 5 ms. The peak flux magnitude was 7-12 pA, suggesting that sparks must arise from clusters of >15 ryanodine receptors.
Abstract.
Author URL.
2001
McMorland AJC, Robinson DM, Soeller C, Cannell MB, Funk GD (2001). Respiratory Research, 2(Suppl 1), P27-P27.
Wong C, Soeller C, Burton L, Cannell MB (2001). Changes in transverse-tubular system architecture in myocytes from diseased human ventricles.
Author URL.
Soeller C, Cannell MB (2001). Modeling the Ca2+ spark and estimation of the SR Ca2+ release flux.
Author URL.
Jacobs MD, Soeller C, Cannell MB, Donaldson PJ (2001). Quantifying changes in gap junction structure as a function of lens fiber cell differentiation.
Cell Commun Adhes,
8(4-6), 349-353.
Abstract:
Quantifying changes in gap junction structure as a function of lens fiber cell differentiation.
The ocular lens is an ideal model system for studying gap junction structure-function relationships. Here we apply novel methods to quantitatively compare connexin expression over macroscopic distances while simultaneously resolving the intracellular distribution of gap junctions in sub-micron detail. Our approach has identified three distinct zones of connexin density and allowed changes in gap junction plaque size, number and dispersion to be quantified. Our analysis is the first to precisely correlate changes in gap junction plaque structure with the reported changes in gap junction function that occur as a consequence of fiber cell differentiation.
Abstract.
Author URL.
Wan H, Winton HL, Soeller C, Taylor GW, Gruenert DC, Thompson PJ, Cannell MB, Stewart GA, Garrod DR, Robinson C, et al (2001). The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus.
Clin Exp Allergy,
31(2), 279-294.
Abstract:
The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus.
There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells. In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions. Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry. HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs. HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens.
Abstract.
Author URL.
2000
Hollingworth S, Soeller C, Baylor SM, Cannell MB (2000). Erratum: (Journal of Physiology (2000) vol. 526 (551-560)). Journal of Physiology, 527(3).
Jacobs MD, Soeller C, Cannell MB, Donaldson PJ (2000). Quantifying changes in gap junction structure as a function of lens fiber cell differentiation.
Cell Adhesion and Communication,
8(4-6), 349-353.
Abstract:
Quantifying changes in gap junction structure as a function of lens fiber cell differentiation
The ocular lens is an ideal model system for studying gap junction structure-function relationships. Here we apply novel methods to quantitatively compare connexin expression over macroscopic distances while simultaneously resolving the intracellular distribution of gap junctions in sub-micron detail. Our approach has identified three distinet zones of connexin density and allowed changes in gap junction plaque size, number and dispersion to be quantified. Our analysis is the first to precisely correlate changes in gap junction plaque structure with the reported changes in gap junction function that occur as a consequence of fiber cell differentiation. Copyright © 2001 Taylor and Francis.
Abstract.
Wan H, Winton HL, Soeller C, Gruenert DC, Thompson PJ, Cannell MB, Stewart GA, Garrod DR, Robinson C (2000). Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1.
Clin Exp Allergy,
30(5), 685-698.
Abstract:
Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1.
BACKGROUND: House dust mite allergen Der p 1 is a cysteine peptidase. Previously, we have suggested that the proteolytic activity of this allergen may contribute to asthma by damaging the barrier formed by the airways epithelium. OBJECTIVE: the present study applied novel techniques to compare changes in permeability with quantitative events in tight junctions (TJs) and desmosomes (DMs) of epithelial cells exposed to Der p 1. METHODS: Confluent monolayers of Madin-Darby canine kidney (MDCK) and 16HBE14o-human bronchial epithelial cells were used as experimental models. Permeability was estimated from mannitol clearance. Digital imaging with quantification of TJs and DMs was achieved by fluorescent antibody staining and 2-photon molecular excitation microscopy (2PMEM). Biochemical changes in TJs were studied by immunoblotting, radiolabelling and immunoprecipitation. RESULTS: Der p 1 caused a time-dependent breakage of TJs and reduction in their content of the protein ZO-1. Reduction in ZO-1 immunofluorescence at TJs occurred with a small increase in the amount of diffuse, cytoplasmic immunoreactive ZO-1 staining. Morpho-logical changes in TJs occurred in synchrony with increases in epithelial permeability. DM puncta increased both in size and intensity of staining. Immunoblotting demonstrated that the disruption of TJ morphology was associated with cleavage of ZO-1 and occludin. Cells recovered from allergen exposure by de novo synthesis of occludin. CONCLUSION: Der p 1 could contribute to sensitization and allergic responses by degrading the function of the airway epithelial barrier.
Abstract.
Author URL.
Hollingworth S, Soeller C, Baylor SM, Cannell MB (2000). Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscoping (vol 526, pg 551, 2000).
JOURNAL OF PHYSIOLOGY-LONDON,
527(3), 654-654.
Author URL.
Hollingworth S, Soeller C, Baylor SM, Cannell MB (2000). Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy.
J Physiol,
526 Pt 3, 551-560.
Abstract:
Sarcomeric Ca2+ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy.
Intra-sarcomeric gradients of [Ca2+] during activation of action potential stimulated frog single fibres were investigated with the Ca2+ indicator fluo-3 and confocal and two-photon microscopy. The object of these experiments was to look for evidence of extra-junctional Ca2+ release and examine the microscopic diffusion of Ca2+ within the sarcomere. By exploiting the spatial periodicity of sarcomeres within the fibre, we could achieve a high effective line-scanning rate ( approximately 8000 lines s-1), although the laser scanning microscope was limited to < 1000 lines s-1. At this high time resolution, the time course of fluorescence changes was very different at the z- and m-lines, with a significant delay ( approximately 1 ms; 22 C) between the rise of fluorescence at the z-line and the m-line. To calculate the expected fluorescence changes, we used a multi-compartment model of Ca2+ movements in the half-sarcomere in which Ca2+ release was restricted to triadic junctions (located at z-lines). Optical blurring by the microscope was simulated to generate fluorescence signals which could be compared directly to experimental data. The model which reproduced our experimental findings most accurately included Ca2+ binding by ATP, as well as indicator binding to immobile sarcomeric proteins. After taking sarcomeric misregistration within the fibre into account, there was very good agreement between the model and experimental results. We conclude that there is no experimental evidence for Ca2+ release at locations other than at z-lines. In addition, our calculations support the conclusion that rapidly diffusing Ca2+ buffers (such as ATP) are important in shaping the Ca2+ transient and that the details of intracellular indicator binding need to be considered to explain correctly the time course of fluorescence change in the fibre.
Abstract.
Author URL.
Wan H, Winton HL, Soeller C, Stewart GA, Thompson PJ, Gruenert DC, Cannell MB, Garrod DR, Robinson C (2000). Tight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o-.
Eur Respir J,
15(6), 1058-1068.
Abstract:
Tight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o-.
Tight junctions (TJs) make a vital contribution to the barrier properties of the airway lining. Opening of TJs, or their frank cleavage, is suspected as a pathophysiological event in the lung, but research into the cellular and molecular mechanisms involved has been impeded by technical limitations of available experimental models. The authors have compared the properties of two epithelial cell lines derived from bronchial epithelium to explore whether these cell lines could constitute appropriate tools for the study of TJ regulation in bronchial epithelium. Investigations of TJs in 16HBE14o- cells and Calu-3 cells were made by fluorescent antibody labelling in conjunction with wide-field, confocal or 2-photon molecular excitation microscopy (2PMEM). The presence of TJ proteins was confirmed by immunoblotting and functional properties of the monolayers were studied by measurements of transepithelial electrical resistance and mannitol permeability. Cells of both lines formed confluent monolayers in which the cells expressed the TJ proteins occludin and ZO-1 in continuous circumferential patterns suggestive of functional TJs. This interpretation was supported by the development of transepithelial electrical resistances and of low paracellular permeability to solutes. Within the limits of resolution offered by 2PMEM, occludin and ZO-1 appeared to colocalize at TJs. These studies suggest that the 16HBE14o- cells and Calu-3 cell lines are potentially useful in vitro models to study how tight junction opening or cleavage changes the functional barrier properties of bronchial epithelium.
Abstract.
Author URL.
1999
Robinson C, Wan H, Winton HL, Soeller C, Cannell MB, Gruenert DC, Thompson PJ, Stewart GA, Taylor GW, Garrod DR, et al (1999). Cleavage of occludin by Der P 1 facilitates transepithelial permeation of allergen.
Author URL.
Cannell MB, Soeller C (1999). Correction. Journal of General Physiology, 113(5).
Wan H, Winton HL, Soeller C, Tovey ER, Gruenert DC, Thompson PJ, Stewart GA, Taylor GW, Garrod DR, Cannell MB, et al (1999). Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.
J Clin Invest,
104(1), 123-133.
Abstract:
Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.
House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.
Abstract.
Author URL.
Cannell MB, Soeller C (1999). Erratum: (The Journal of General Physiology (March 1999) 113:3 (373- 376)). Journal of General Physiology, 113(5).
Soeller C, Cannell MB (1999). Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques.
Circ Res,
84(3), 266-275.
Abstract:
Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques.
The transverse tubular system (t-system) of cardiac muscle is a structure that allows rapid propagation of excitation into the cell interior. Using 2-photon molecular excitation microscopy and digital image-processing methods, we have obtained a comprehensive overview of the t-system of rat ventricular myocytes in living cells. We show that it is possible to quantify the morphology of the t-system in terms of average local tubule diameter, branching pattern, and local abundance of the t-system by immersing living myocytes in a dextran-linked fluorescein solution. Our data suggest that previous electron microscopic examinations of t-system structure have underestimated both the geometric complexity of the t-system morphology and the fraction of cell volume occupied by the t-system (3.6% in this species). About 40% of tubules occur between Z-lines, and the t-tubule diameter is 255+/-0.85 nm (mean+/-SEM). The t-tubules leave the outer surface of the cell in an approximately rectangular array; however, at some points junctions between the t-tubules and the surface membrane are missing. In view of the complexity of the t-system apparent from our images, we propose that the t-system be renamed the "sarcolemmal Z rete." the methods presented here are generally applicable to the quantification of the sarcolemmal Z rete and other structures within cells by fluorescence microscopy in a variety of cell types.
Abstract.
Author URL.
Housley GD, Kanjhan R, Raybould NP, Greenwood D, Salih SG, Järlebark L, Burton LD, Setz VC, Cannell MB, Soeller C, et al (1999). Expression of the P2X(2) receptor subunit of the ATP-gated ion channel in the cochlea: implications for sound transduction and auditory neurotransmission.
J Neurosci,
19(19), 8377-8388.
Abstract:
Expression of the P2X(2) receptor subunit of the ATP-gated ion channel in the cochlea: implications for sound transduction and auditory neurotransmission.
Extracellular ATP has multimodal actions in the cochlea affecting hearing sensitivity. ATP-gated ion channels involved in this process were characterized in the guinea pig cochlea. Voltage-clamped hair cells exhibited a P2 receptor pharmacology compatible with the assembly of ATP-gated ion channels from P2X(2) receptor subunits. Reverse transcription-PCR experiments confirmed expression of the P2X(2-1) receptor subunit mRNA isoform in the sensory epithelium (organ of Corti); a splice variant that confers desensitization, P2X(2-2), was the predominant subunit isoform expressed by primary auditory neurons. Expression of the ATP-gated ion channel protein was localized using a P2X(2) receptor subunit-specific antiserum. The highest density of P2X(2) subunit-like immunoreactivity in the cochlea occurred on the hair cell stereocilia, which faces the endolymph. Tissues lining this compartment exhibited significant P2X(2) receptor subunit expression, with the exception of the stria vascularis. Expression of ATP-gated ion channels at these sites provides a pathway for the observed ATP-induced reduction in endocochlear potential and likely serves a protective role, decoupling the "cochlear amplifier" in response to stressors, such as noise and ischemia. Within the perilymphatic compartment, immunolabeling on Deiters' cells is compatible with purinergic modulation of cochlear micromechanics. P2X(2) receptor subunit expression was also detected in spiral ganglion primary afferent neurons, and immunoelectron microscopy localized these subunits to postsynaptic junctions at both inner and outer hair cells. The former supports a cotransmitter role for ATP in a subset of type I spiral ganglion neurons, and latter represents the first characterization of a receptor for a fast neurotransmitter associated with the type II spiral ganglion neurons.
Abstract.
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Hollingworth S, Soeller C, Baylor SM, Cannell MB (1999). In skeletal muscle fibers, a rise in myoplasmic calcium appears to speed the diffusion of fluo-3 by perturbing the equilibrium between protein-free and protein-bound indicator.
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Cannell MB, Soeller C (1999). Mechanism underlying calcium sparks in cardiac muscle (vol 113, pg 373, 1999).
JOURNAL OF GENERAL PHYSIOLOGY,
113(5), 761-761.
Author URL.
Cannell MB, Soeller C (1999). Mechanisms underlying calcium sparks in cardiac muscle.
J Gen Physiol,
113(3), 373-376.
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Full text.
Soeller C, Cannell MB (1999). Two-photon excited flash photolysis for the study of model cell systems.
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Soeller C, Cannell MB (1999). Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis.
Microsc Res Tech,
47(3), 182-195.
Abstract:
Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis.
Two-photon molecular excitation microscopy has several advantages over conventional confocal fluorescence microscopy, including the ability to section deeper into scattering samples and to allow spatially resolved flash photolysis. We describe and examine the benefit of incorporating non-descanned fluorescence detection in our microscope system. In a scattering sample where almost no signal could be obtained at a depth of 50 microm with confocal detection, non-descanned detection resulted in an improvement of signal strength by more than an order of magnitude at depths >40 microm. The spatio-temporal properties of stationary spot two-photon excited flash photolysis (TPEFP) in drops of test solutions and cardiac myocytes were also examined. At input powers that produce >10% of the maximum rate of DM-nitrophen photolysis, serious photodestruction of the reporter fluorochrome (Fluo-3) at the photolysis spot occurred. At power levels of approximately 4 mW for periods
Abstract.
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1998
Cannell MB, Bridge JH, Soeller C (1998). Dissecting the basis of the spark in cardiac muscle.
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Dillmann A, Wetzel T, Soeller C (1998). Interferometric measurement and tomography of the density field of supersonic jets.
Experiments in Fluids,
25(5-6), 375-387.
Abstract:
Interferometric measurement and tomography of the density field of supersonic jets
The density field of cylindrical supersonic jets is investigated by Mach-Zehnder interferometry. The optical phase shift is extracted from the interferograms by digital image processing. Disturbing turbulence effects in the free shear layer are eliminated by sequential averaging of the phase shifts of several experimental images. The resulting steady state phase shift is used to calculate time-averaged interferograms and to reconstruct the density field by computerized tomography. The obtained results are compared with theoretical predictions and good quantitative agreement is found. © Springer-Verlag 1998.
Abstract.
Soeller C, Cannell MB (1998). Limits of accuracy in reconstructing local release fluxes from fluorescence records of calcium sparks.
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Cannell MB, Soeller C (1998). Local Control in Calcium Signalling during Cardiac E-C Coupling. In (Ed) Integrative Aspects of Calcium Signalling, Springer US, 381-398.
Hollingworth S, Soeller C, Baylor SM, Cannell MB (1998). Local calcium gradients during activation of frog skeletal muscle measured with Mg-green and fluo-3.
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Cannell MB, Soeller C (1998). Sparks of interest in cardiac excitation-contraction coupling.
Trends Pharmacol Sci,
19(1), 16-20.
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Jones KT, Soeller C, Cannell MB (1998). The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse.
Development,
125(23), 4627-4635.
Abstract:
The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse.
Mouse sperm-egg fusion was examined using two-photon and confocal microscopy. A delay of several minutes occurred between the first observable event of fusion (which was the diffusion of Ca2+-sensitive dyes from egg into sperm) and any change in egg cytoplasmic Ca2+. When indo-1 dextran was used to obtain ratiometric two-photon images, there was no detectable local increase in egg cytoplasmic Ca2+ near the site of sperm fusion. However, the sperm underwent a Ca2+ transient which appeared to be coincident with the egg cytoplasm Ca2+ transient, which suggested that there was a high permeability pathway for Ca2+ between egg and sperm. To exclude this pathway from providing trigger Ca2+ for the egg transient, we reduced bathing [Ca2+] to approx. 18 microM and 13nM (with EGTA). In these conditions the first egg Ca2+ transient was not prevented, which makes an obligatory role for extracellular Ca2+ in the initiation of the egg Ca2+ transient unlikely. Both FITC-albumin (70 kDa) and 10 kDa dextran-linked Ca2+ indicators were able to diffuse into the sperm from the egg. In addition, phycoerythrin (240 kDa) rapidly diffused into the sperm shortly after fusion (but before any changes in Ca2+ occurred). This suggests that the 'pore(s)' that form during sperm-egg fusion must be at least 8 nm in diameter. These data are compatible with the idea that a diffusible sperm protein could trigger the observed changes in intracellular Ca2+ in the egg, but do not exclude the possibility that other second messengers are generated during sperm-egg fusion.
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1997
Soeller C, Cannell MB (1997). Investigation into the morphology of the t-tubular system in living cardiac myocytes by two photon excitation microscopy.
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Cannell MB, Soeller C (1997). Numerical analysis of ryanodine receptor activation by L-type channel activity in the cardiac muscle diad.
Biophys J,
73(1), 112-122.
Abstract:
Numerical analysis of ryanodine receptor activation by L-type channel activity in the cardiac muscle diad.
Computer simulations were used to examine the response of ryanodine receptors (RyRs) to the sarcolemmal calcium influx via L-type calcium channels (DHPRs). The effects of ryanodine receptor organization, diad geometry, DHPR single-channel current, and DHPR gating were examined. In agreement with experimental findings, the simulations showed that RyRs can respond rapidly (approximately 0.4 ms) to calcium influx via DHPRs. The responsiveness of the RyR depends on the geometrical arrangement between the RyRs and the DHPR in the diad, with wider diads being generally less responsive. When the DHPR single-channel current is small (approximately 25 fA), the organization of RyRs into small clusters results in an improved responsiveness. With experimentally observed DHPR mean open and closed times (0.17 ms and 4 ms, respectively) it is the first opening of the DHPR that is most likely to activate the RyR. A measure of the efficiency (Q) by which DHPR gating evokes sarcoplasmic reticulum release is defined. Q is at maximum for tau approximately 0.3 ms, and we interpret this finding in terms of the "tuning" of DHPR gating to RyR response. If certain cardiac myopathies are associated with a mismatch in the "tuning," then modification of DHPR gating with drugs to "retune" calcium-induced calcium release should be possible.
Abstract.
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Soeller C, Cannell MB (1997). Numerical simulation of local calcium movements during L-type calcium channel gating in the cardiac diad.
Biophys J,
73(1), 97-111.
Abstract:
Numerical simulation of local calcium movements during L-type calcium channel gating in the cardiac diad.
Computer simulation was used to investigate the calcium levels after sarcolemmal calcium influx through L-type calcium channels (DHPRs) into the narrow diadic space of cardiac muscle. The effect of various cytosolic and membranebound buffers, diad geometry, DHPR properties (open time and current), and surface charge were examined. The simulations showed that phospholipid binding sites on the sarcolemmal membrane are the major buffer affecting free calcium ([Ca2+]) levels in the diad. The inclusion of surface charge effects calculated from Gouy-Chapman theory resulted in a marked decrease in [Ca2+] levels at all times and a faster decay of [Ca2+] after termination of DHPR influx. For a DHPR current of 200 fA, [Ca2+] at the center of the diad reached peak levels of approximately 73 microM. In larger diads (> or = 400 nm diameter), [Ca2+] decayed more slowly than in smaller diads (100-200 nm diameter), although peak [Ca2+] levels reached during typical DHPR open times were similar. For a wide range of DHPR single-channel current magnitudes (Ica = 25-200 fA), [Ca2+] levels in the diad were approximately proportional to ICa. The decrease in calculated [Ca2+] levels due to the effects of surface charge can be interpreted as resulting from an effective "volume expansion" of the diad space. Furthermore, the layer of increased [Ca2+] close to the sarcolemmal membrane can act as a fast buffer.
Abstract.
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1996
Soeller C, Cannell MB (1996). Construction of a two-photon microscope and optimisation of illumination pulse duration.
Pflugers Arch,
432(3), 555-561.
Abstract:
Construction of a two-photon microscope and optimisation of illumination pulse duration.
The construction of a two-photon/confocal microscope system is described in detail. For two-photon illumination, a Ti:sapphire modelocked laser generating 62-fs pulses at 715 nm was used. The effect of the optical train on illumination pulse width was examined and the observed increase in pulse duration was almost completely removed by the addition/adjustment of a prism compressor system. The imaging capabilities of the two-photon microscope are demonstrated and it is shown that the imaging performance of the two-photon microscope is similar to that of a conventional confocal microscope. With two-photon illumination, the resolution (full width at half-maximum intensity) was 0.42 microM (x-y) and 0.81 microM axially, while with single-photon illumination (at 488 nm in the same instrument with a confocal pinhole detector) the resolution was 0.3 microM (x-y) and 0.75 microM axially. The results are discussed with regard to the general problem of femtosecond pulse distortion in an optical system and a simple procedure for optimal pulse restoration is described.
Abstract.
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Soeller C, Cannell MB (1996). Numerical analysis of cardiac EC coupling.
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