Two-photon fluorescence anisotropy imaging

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 90degrees, 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.