Equipment & Technologies
Narrow band spectrally focused Coherent Raman system
This custom-built system provides fast hyperspectral CRS imaging using chirped femtosecond laser pulses to achieve rapid Raman spectra acquisition while retaining the full speed and image quality of narrowband SRS imaging.
This system is powered by an InSightX3 fs laser, which provides a tuneable output between 680-1300nm and a fixed wavelength output at 1045nm. The 2 beams are combined in the SF-TRU spectral focusing unit. This unit controls the laser powers applied to the sample, the time delay between the beams. It also provides the intensity modulations in the Stokes beam necessary for SRS and chirps the beams from fs to ps pulses. The imaging is carried out on a modified confocal inverted microscope (Olympus Flouview3000 and IX81). The light is focused on the sample with a 1.2NA water objective and transmitted light is collected with either an oil or water condenser depending on the sample type. For transparent samples SRS and CARS are measured in the forwards direction, however for thicker samples there is also an option to collect backscattered CARS in an epi channel.
Our Exeter lab offers users the ability to combine Coherent Raman imaging with complementary techniques including Second Harmonic Generation (SHG) and Two Photon fluorescence (TPF), This highly powerful combination of techniques provides complementary information about the collagen structures and endogenous fluorophores or added fluorescent labels within a sample. This flexible system allows 3D imaging and large area scans in addition to rapid hyperspectral imaging.
Further reading:
Module for multiphoton high-resolution hyperspectral imaging and spectroscopy (Aram Zeytunyan; Tommaso Baldacchini; Ruben Zadoyan) - February 2018
Broadband CARS Microscope
This custom-built micro-spectrometer provides two- and three-dimensional mapping of biomolecules based on their unique Raman fingerprint. Based on broadband coherent anti-Stokes Raman scattering this system provides a combination of speed, sensitivity and spectral breadth. The system utilizes a configuration of laser sources that probes the entire biologically relevant Raman window (500–3500 cm–1). By utilising intra-pulse three-colour excitation and the non-resonant background to heterodyne-amplify weak Raman signals it is possible to obtain efficient acquisition of the typically weak ‘fingerprint’ region.
At the heart of the system are two co-seeded fibre lasers (Toptica, FemtoPro), custom designed specifically for broadband CARS. The system provides synchronization of narrowband (∼3.4 ps) flat-top pulses at 770 nm synchronised with supercontinuum pulses (∼16 fs) spanning ∼900–1400 nm. The first fibre laser, a FemtoFiber pro NIR is a frequency doubled ultrafast Erbium fibre laser coupled to a FF 1PS NIR FemtoFiber pro to increase the pulse length to ~3.4 ps. The second fibre laser is an ultrashort-pulsed Erbium fibre amplifier (FF PRO UCP AMP) with motorised control for optimisation of the supercontinuum power and pulse duration. The FF PRO UCP AMP is seeded by the FF PRO NIR so that the two pulse trains are intrinsically rep-rate locked.
The beams are temporally and spatially overlapped and delivered onto the sample via a 1.2NA objective and the CARS signal collected in the forwards direction by and inverted microscope (Olympus IX73) with a 0.7NA objective. The anti-Stokes signal is spectrally isolated from the excitation wavelengths using band-pass filters and focused onto the entrance slits of a spectrograph (Princeton Instruments Isoplane 160) equipped with a fast CCD camera (Princeton Instruments Pixis).
Further reading:
- High-speed coherent Raman fingerprint imaging of biological tissues (Charles H. Camp Jr, Young Jong Lee, John M. Heddleston, Christopher M. Hartshorn, Angela R. Hight Walker, Jeremy N. Rich, Justin D. Lathia & Marcus T. Cicerone) - July 2014
Spontaneous Raman Systems
In additions to the coherent Raman systems there are 2 spontaneous Raman systems which are available to for booking, these are not currently TRAC managed but can be booked by contacting the Contrast team.
Renishaw InVia Quantor
- Simple user-friendly system
- Choice between 532nm and 785nm laser
- Hyperspectral mapping capabilities
- Confocal imaging
- Wide range of objectives
Cell Culture Facility
Contrast facility users will have access to a shared cell culture laboratory. We have a class II biosafety facilities however do not routinely handle pathogenic, infectious or genetically modified organisms therefore work involving such agents would require discussion with the team.
We can image both fixed and living cells on the coherent Raman microscopes. Living cells are best imaged in 1inch diameter well plates with a coverslip bottom.