We are working on developing a new technique of microscopy which will combine ISM (Image Scanning Microscopy) with STED. ISM will help us to achieve higher resolution in xy plane without photobleaching the sample with high-intensity excitation beam and simultaneous STED will only be used for fluorescence limitation in z-direction. The real-time image is generated by direct representation from the central pixel; after the acquisition, the higher resolution image is calculated from all measured ISM signals using photon reassignment. With ISM all photons can be used, especially for the fluorescence lifetime calculation (FLIM). STED is used to suppress fluorescence from areas above and below the image plane, but the FPs in the image plane are never quenched by the STED lasers.
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