Lifetime‐selective fluorescence imaging using an rf phase‐sensitive camera

We report the creation of two‐dimensional fluorescence lifetime images, based on a sinusoidally modulated image intensifier that is operated as a radio‐frequency phase‐sensitive camera, synchronized to a mode‐locked and cavity dumped picosecond dye laser. By combining the image intensifier with a CCD camera and applying digital image processing, lifetime‐selective signal suppression can be realized even for fluorophores with comparable lifetimes. This phase‐sensitive technique can be used to create fluorescence lifetime images, that is, images in which the contrast is based upon the fluorescence lifetimes rather than upon local probe concentration and/or intensity. Because the lifetimes of many dyes are sensitive to the chemical environments surrounding the fluorophore, fluorescence lifetime imaging (FLIM) can reveal the local chemical composition and properties of the molecular environment that surrounds the fluorophore. As an example we created images of rhodamine 6G (4 ns) and rhodamine B (1.5 ns) solu...

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