Coherent Anti-Stokes Raman multiplex microscopy for non-invasive imaging of living cells

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Our objective is to develop and demonstrate a new microscope generation with improved sensitivity and chemical specificity for real-time studies on living cells. Such an instrument will allow non-invasive, microscopic examination of cells and subcellular structures under physiological conditions with chemical contrast without the need to stain or express tagged proteins, together with three-dimensional imaging capability of high penetration depth in intact tissues. 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. However, most cellular constituents have no colour and they are hard to distinguish under a light microscope unless they are stained. Fluorescence microscopy, using antibodies labelled with dyes or fusion of proteins with fluorescent tags has provided a highly sensitive and specific method of visualizing biomolecules. However, when used for real time observations in living cells, these modified biomolecules raise questions if their behaviour is real or artefactual. Furthermore, all fluorescent probes are prone to photo-bleaching that is an irreversible degradation of the fluorescence intensity after excitation with light. Another major difficulty in light microscopy occurs when attempting to image living cells within intact tissues with high axial resolution and long penetration depth. The key idea of this new microscope is to obtain the image contrast via scattering of light with the specific resonances of vibration in chemical bonds (Raman scattering). This phenomenon can be coherently enhanced when using two short laser pulses to resonantly excite the vibrations and generate the so-called Coherent Antistokes Raman Scattering (CARS). Since CARS depends nonlinearly on the exciting light intensity, sufficient intensities for CARS generation are achieved only in the small focal volume where the exciting photons are concentrated resulting in an intrinsic high resolution and sectioning capability similar to multi-photon fluorescence microscopy. The application of CARS microscopy for imaging in cell biology has been recently explored by a few groups but is still at a rather early stage. Significant development effort is needed to improve this technique to a mature state. We propose the construction of a new multiplex CARS microscope able to detect several chemical species in parallel with improved chemical selectivity and sensitivity as compared to what reported in literature up to now. To develop the instrument for the biological context we will collaborate with biologists within the School of Biosciences, who will provide biological samples and interesting problems that are difficult to approach even with state-of-the-art fluorescence microscopy. These problems are: a) the change in structure of mitochondria in the pathogenic yeast Candida albicans during programmed cell death (apoptosis) and in the model yeast Saccharomyces cerevisiae during respiratory oscillations, b) the end-bud growth, development and apoptosis of cells in mouse mammary glands, c) the dynamic changes in cell water content and membrane dynamics during development of the slime mold Dictyostelium discoideum. Besides the collaborators within the School of Biosciences, researchers from both physics and biological disciplines worldwide might benefit from the outcome of this work. The usage of this novel microscopy technique is also likely to be of relevance in medical applications, to improve the diagnostic and treatment of diseases. Additionally, the proposed research contains the realization of an economic design of the multiplex CARS microscope for its possible widespread application, so that microscope manufacturers are likely to be interested in this project.