This work was supported by grants from The National Natural Science Foundation of China through the Outstanding Young Overseas Scientists Program (19928408), Major Program (60138010) and Free Application Program (10274039).
A new method is developed for two-photon fluorescence anisotropy imaging. Its biological applications are tested. This system is based on a two-photon laser scanning fluorescence microscope. A polarization beam splitter was inserted into the optical path to separated fluorescence into components of orthogonal polarization. By rotating the polarization of the excitation beam by 90 ° , four images were collected for each sample. These images were then processed pixel-by-pixel to generate a new fluorescence anisotropy image. The capability of this method is tested for different samples, including FITC, FITC-CD44 in solution and FITC-CD44 attached to the membranes of tumor cells. The results show that fluorescence images can distinguish fluorescence molecules of different molecular mass, or detect changes in microenvironment.
WANG Xue-Feng, WANG Yi, JIANG Yan, MA Hui. Two-photon Fluorescence Anisotropy Imaging[J]. Progress in Biochemistry and Biophysics,2005,32(2):161-167
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