Current developments in optical microscopy aim to visualise complex dynamic
biomolecular processes close to their native state. To capture transient phenomena,
rapid three-dimensional stacks are acquired by translating the objective or sample
stage to refocus into different depths of the specimen. Such conventional refocusing
strategies introduce vibrational artefacts when imaging specimens that are in direct
contact with the immersion media of the objective.
Remote focusing is a methodology in which agitation-free refocusing can be
performed using high numerical aperture (NA) objectives without compromising
on resolution or imaging speed. It compensates for aberrations from the imaging
objective by introducing equal and opposite aberration with a second microscope
placed in reverse to the first. As the NA of the imaging objective increases, there
are significant constraints placed on the tolerance in optical design to reach perfect
phase-matching condition.
In the first part of the thesis, the computational model developed to predict
the performance of remote focusing microscopes is presented. From the model,
the increased sensitivity of high-NA systems to magnification mismatch is inferred
where the diffraction limited volume reduces by half for a 1% error.
Informed by the sensitivity analysis, the decrease in resolution across depth for
a remote focusing microscope with a 4% magnification mismatch is demonstrated.
A protocol for magnification and resolution characterisation is presented and is
applied to a novel Spinning Disk Remote Focusing microscope. The microscope
is then applied to perform live volumetric imaging to study the normal neural
activity of Platynereis dumerilii larvae. The studies presented here paves way
for a standardised characterisation of remote focusing systems allowing for wider
implementation.
In the final part of the thesis, the spherical aberration generated by the correction
collar on an immersion objective is exploited to compensate for residual
spherical aberration in an ideal remote focusing system. The wavefront aberrations
are measured using a Shack-Hartmann sensor and sub-resolution beads are imaged
for point spread function measurements. Results from the Shack-Hartmann measurements
show a 60% increase in axial range compensated for spherical aberration.
In addition, the contribution of off-axis aberrations to the overall image quality at
defocussed positions is explored further.

We introduce a random-access parallel (RAP) imaging modality that uses a novel design inspired by a Newtonian telescope to image multiple spatially separated samples without moving parts or robotics. This scheme enables near-simultaneous image capture of multiple petri dishes and random-access imaging with sub-millisecond switching times at the full resolution of the camera. This enables the RAP system to capture long-duration records from different samples in parallel, which is not possible using conventional automated microscopes. The system is demonstrated by continuously imaging multiple cardiac monolayer and Caenorhabditis elegans preparations.

Remote focusing (RF) is a technique that greatly extends the aberration-free axial scan range of an optical microscope. To maximise the diffraction limited depth range in an RF system, the magnification of the relay lenses should be such that the pupil planes of the objectives are accurately mapped on to each other. In this paper we study the tolerance of the RF system to magnification mismatch and quantify the amount of residual spherical aberration present at different focusing depths. We observe that small deviations from ideal magnification results in increased amounts of residual spherical aberration terms leading to a reduction in the diffracted limited range. For high-numerical aperture objectives, the simulation predicts a 50% decrease in the diffracted limited range for 1% magnification mismatch. The simulation has been verified against an experimental RF system with ideal and nonideal magnifications. Experimentally confirmed predictions also provide a valuable empirical method of determining when a system is close to the ideal phase matching condition, based on the sign of the spherical aberration on either side of focus.

Fast confocal imaging was achieved by combining remote focusing with differential spinning disk optical sectioning to rapidly acquire images of live samples at cellular resolution. Axial and lateral full width half maxima less than 5 μm and 490 nm respectively are demonstrated over 130 μm axial range with a 256 × 128 μm field of view. A water-index calibration slide was used to achieve an alignment that minimises image volume distortion. Application to live biological samples was demonstrated by acquiring image volumes over a 24 μm axial range at 1 volume/s, allowing for the detection of calcium-based neuronal activity in Platynereis dumerilii larvae.

The present work is concerned with the development and application of a novel fringe analysis technique based on the principles of the windowed-Fourier-transform (WFT) for the determination of temperature and concentration fields from interferometric images for a range of heat and mass transfer applications. Based on the extent of the noise level associated with the experimental data, the technique has been coupled with two different phase unwrapping methods: the Itoh algorithm and the quality guided phase unwrapping technique for phase extraction. In order to generate the experimental data, a range of experiments have been carried out which include cooling of a vertical flat plate in free convection conditions, combustion of mono-propellant flames, and growth of organic as well as inorganic crystals from their aqueous solutions. The flat plate and combustion experiments are modeled as heat transfer applications wherein the interest is to determine the whole-field temperature distribution. Aqueous-solution-based crystal growth experiments are performed to simulate the mass transfer phenomena and the interest is to determine the two-dimensional solute concentration field around the growing crystal. A Mach-Zehnder interferometer has been employed to record the path-integrated quantity of interest (temperature and/or concentration) in the form of interferometric images in the experiments. The potential of the WFT method has also been demonstrated on numerically simulated phase data for varying noise levels, and the accuracy in phase extraction have been quantified in terms of the root mean square errors. Three levels of noise, i.e. 0%, 10%, and 20% have been considered. Results of the present study show that the WFT technique allows an accurate extraction of phase values that can subsequently be converted into two-dimensional temperature and/or concentration distribution fields. Moreover, since WFT is a local processing technique, speckle patterns and the inherent noise in the interferometric data do not affect the resultant phase values. Brief comparisons of the accuracy of the WFT with other standard techniques such as conventional Fourier-filtering methods are also presented.

The present work is concerned with exploring the potential of refractive index-based imaging techniques for investigating the heat transfer characteristics of impinging turbulent synthetic jets. The line-of-sight images of the convective field have been recorded using a Mach Zehnder interferometer. Heat transfer experiments have been conducted in infinite fringe setting mode of the interferometer with air as the working fluid. The effect of the excitation frequency of the synthetic jet on the resultant temperature distribution and local heat transfer characteristics has been studied. The fringe patterns recorded in the form of interferograms have first been qualitatively discussed and thereafter, quantitatively analyzed to determine the two-dimensional temperature field. Local heat transfer coefficients along the width of the heated copper block have been determined from the temperature field distribution thus obtained from the interferograms. The results have been presented in the form of interferometric images recorded as a function of frequency of the synthetic jet, corresponding two-dimensional temperature distributions and local variation of heat transfer coefficients. Interferometric measurements predicted maxima of the heat transfer coefficient at the resonance frequency of the synthetic jet and at a jet-to-plate surface spacing (z/d) of 3. These observations correlate well with the thermocouple-based measurements of temperature and heat transfer coefficient performed simultaneously during the experiments. The interferometry-based study, as reported in the present work for the first time in the context of synthetic jets, highlights the importance of refractive index-based imaging techniques as a potential tool for understanding the local heat transfer characteristics of synthetic jets.