Development of molecular imaging microscope for biological specimen and its application for tissue examination

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Aim of this research project is to develop an effective method that can sense objective biomolecles without staining and to apply this method to identification of the situation of the molecles. For ths purpose, we have focused on non-linear optical effect in the tissue that is induced by the irradiation with high-intensity very short duration pulsed laser light such as a femtosecond laser. In the actual research, we have developed three microscopes ; fluorescence lifetime imaging microscope in nanosecond range, a collagen sensitive microscope using second harmonic generation light (SCG light) and a coherent anti Stokes coherent Roman scattering (CARS) microscope.The most successful one is the SHG microscope. Based on the reflection-type microscope with a probe light spot of 10 micrometers in diameter and the polarization measurement of SHG light induced by collagen molecules, we have determined distribution of collagen fiber orientation in various human tissues. The resulting SHG data in human skin imply that the reticular dermis posses a tangled structure of collagen fibers which is consistent with the result of the anatomical examination of the skin. We have improved the SHG microscope to obtain 3D dissection image using the con-focal optics and the 3D sample scanning stage. With this microscope, tomograph of the collage fiber orientation in the human skin and bone have been measured.Performance of the fluorescence lifetime imaging microscope was demonstrated by the measurement of the autofluorescence in liposome. Also we have measured the distribution of hyaluronic acid around an erythrocyte with CARS microscope.