Paola Borri (Cardiff University) is visiting today and giving the AMOPP/BioP seminar on Novel Multiphoton Microscopy Techniques for Cell Imaging: CARS Microscopy and Resonant Four-Wave Mixing.
Abstract: 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. Much effort is being devoted recently to achieve intrinsic three-dimensional (3D) spatial resolution by exploiting optical nonlinear effects which can only take place in the small focal volume where high photon densities are reached. One of the most utilised multiphoton (ie nonlinear) microscopy techniques is two-photon fluorescence where the biomolecules of interest are labelled with fluorophores, which are optically excited via simultaneous absorption of two photons. However, these modified biomolecules raise questions if their behaviour is real or artefactual. Furthermore, all organic fluorophores are prone to photo-bleaching which severely limits time-course observations and is accompanied by toxicity effects and consequent cell damage.
Coherent Antistokes Raman Scattering (CARS) microscopy has recently emerged as a new multiphoton microscopy technique which overcomes the need of fluorescent labelling and yet retains biomolecular specificity and intrinsic 3D resolution. We have developed in our laboratory a fully home-built CARS microscope featuring innovative CARS excitation/detection schemes. In particular, we have demonstrated differential-CARS (D-CARS) and single-laser CARS utilising femtosecond laser pulses linearly chirped by glass dispersion. Furthermore we have invented and demonstrated a novel imaging modality, based on the resonant Four-Wave Mixing (FWM) of colloidal nanoparticles. Results on this work showed that nanoparticles, both semiconductor and metallic, can be used as alternative labels beyond fluorescence by exploiting their resonant FWM, to achieve a novel coherent multiphoton microscopy modality free from background and with a spatial resolution significantly surpassing the one-photon diffraction limit. I will present our latest progress with both techniques and their applications to cell imaging.