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

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Abstract
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) solutions in which the difference in lifetimes results in 100% contrast, whereas the total fluorescence intensity is similar. In order to estimate the time resolution obtainable with our phase‐sensitive imaging setup, we also performed measurements using distance‐selective suppression of optical signals backscattered from laser‐illuminated targets. In these studies, we have created distance‐selective images with a resolution of 3.75 cm, which corresponds to a lifetime resolution of 0.25 ns for fast‐decaying fluorophores. Since we observed nearly 100% contrast for this 0.25 ns difference, still smaller distances and/or lifetime differences could be observed, which seems to imply some advantages of phase fluorometric lifetime imaging. Using these same intensifiers in the pulse‐gating mode one expects a typical 5 ns gating time, which result in a distance resolution of only 75 cm. The distance‐selective imaging principle has the potential for manifold applications related to robotics, construction, and even to spacecraft maneuvering. Fluorescence lifetime imaging, in combination with fluorescence microscopy, can have numerous applications to biochemistry, biophysics, and cell physiology.