Coherent Raman Scattering Microscopy: Technology Developments and Biological Applications

Optical microscopy is an indispensable tool that is driving progress in cell biology, and is still the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Coherent Raman Scattering (CRS) microscopy has emerged in the last decade as a powerful multiphoton microscopy technique which overcomes the need for fluorescent labelling and yet retains biomolecular specificity and intrinsic 3D resolution. We have developed several home-built CRS microscopes featuring innovative excitation/detection schemes and quantitative image analysis. Our second-generation multimodal CRS instrument is based on a single 5fs Ti:Sa laser source with 350nm bandwidth capable of exciting a wide vibrational range from 1200 cm(-1) to 3800 cm(-1), thus enabling hyperspectral microscopy. Alongside CRS, it also features simultaneous detection of two-photon fluorescence and second harmonic generation for correlative studies. Our latest technology developments include high-content high-throughput label-free quantitative chemical imaging via Bessel beam illumination and sparse sampling. Our latest biological applications feature quantitative imaging of i) intra-cellular lipid droplets in live mouse oocytes and early embryos, ii) cancer organoids and their responses to drugs, iii) freshwater microalgae as potential food supplements for omega-3 fatty acids.