Functional imaging of cells by CARS microscopy

The intention of the present project is to apply "Coherent anti-Stokes Raman Scattering (CARS)" in a novel way of nonlinear microscopy: The goal is to obtain images of biological samples, such as unstained living cells or cell organelles with functional "color-contrast" for arbitrary substances of interest within the sample. The resulting images are similar to those obtained in fluorescence microscopy, however, with the great advantage that no preceding cell preparation is necessary, such as staining with fluorescent dyes, which might affect the living cells in an unknown and uncontrollable way. The method is based on nonlinear mixing of two incoming laser beams in the microscope sample, generating a strong signal with a much shorter wavelength in sample regions which contain the selected Raman-active substances. Theses substances can be chosen by adjusting the difference in laser frequency of the two incoming beams. Only recently, this new type of microscopic imaging was demonstrated in a confocal setup, where the sample area was scanned pixel by pixel to obtain an image. In our project we intend to develop a modification of this method, which allows a whole sample to be imaged at once, i.e. without scanning. This promises higher speed of imaging. For this purpose we constructed a novel CARS excitation geometry where the so-called wave-matching condition (necessary for obtaining a CARS signal) is satisfied at the whole sample area simultaneously. Furthermore, we use a nanosecond laser system with a smaller spectral bandwidth which provides better spectroscopic resolution for different biochemical substances of interest. The emphasis of the present project lies on the development of the novel method and the demonstration of fast imaging of biological samples and finally of living cells with a high chemical selectivity. We expect that our novel method of CARS microscopy has the potential to strongly promote biomedical, physiological and pharmaceutical research.

The intention of the present project is to apply "Coherent anti-Stokes Raman Scattering (CARS)" in a novel way of nonlinear microscopy: The goal is to obtain images of biological samples, such as unstained living cells or cell organelles with functional "color-contrast" for arbitrary substances of interest within the sample. The resulting images are similar to those obtained in fluorescence microscopy, however, with the great advantage that no preceding cell preparation is necessary, such as staining with fluorescent dyes, which might affect the living cells in an unknown and uncontrollable way. The method is based on nonlinear mixing of two incoming laser beams in the microscope sample, generating a strong signal with a much shorter wavelength in sample regions which contain the selected Raman-active substances. Theses substances can be chosen by adjusting the difference in laser frequency of the two incoming beams. Only recently, this new type of microscopic imaging was demonstrated in a confocal setup, where the sample area was scanned pixel by pixel to obtain an image. In our project we intend to develop a modification of this method, which allows a whole sample to be imaged at once, i.e. without scanning. This promises higher speed of imaging. For this purpose we constructed a novel CARS excitation geometry where the so-called wave-matching condition (necessary for obtaining a CARS signal) is satisfied at the whole sample area simultaneously. Furthermore, we use a nanosecond laser system with a smaller spectral bandwidth which provides better spectroscopic resolution for different biochemical substances of interest. The emphasis of the present project lies on the development of the novel method and the demonstration of fast imaging of biological samples and finally of living cells with a high chemical selectivity. We expect that our novel method of CARS microscopy has the potential to strongly promote biomedical, physiological and pharmaceutical research.