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Fluorescence Recovery after Photobleaching with the Leica TCS SP2

FRAP Introduction Among all photobleaching experiments which have been described, fluorescence recovery after photo-bleaching (FRAP) is the most popular. It employs irradiation of a fluorophore in a living sample with a short laser pulse to degrade it and thereby abolish fluores-cence followed by time-resolved image recording of the sample. If the fluorophore (or a so-called mobile fraction of it, see below) is able to freely diffuse through the sample a recovery of fluorescence can be observed. Photobleaching experiments can be conducted with modern laser scanning microscopes, where the laser is used at high intensity for bleaching and low intensity for image recording. The LCS software provided by Leica contains a FRAP application wizard that guides the user through the steps of a FRAP experiment. This wizard will be used in the examples below. FRAP experiments have been applied to study the dynamics within and between subcellular compartments (Phair and Misteli, 2000) as well as changes in their morphology (Beaudouin et al. 2002). In this way qualitative information about intracellular transport processes can be rendered in live cells. If a time series is recorded immediately after the bleaching pulse at high rate (> 5 s-1) a quantitative evaluation becomes possible. Parameters that can be obtained are the diffusion coefficient D, and the mobile fraction M f. Alternatively, instead of D the characteristic half-time of the recovery t 1/2 , can be retrieved from the data. In fact, t 1/2 is reciprocally proportional to D with the assumptions made by Axelrod et al. (1976). See also " data processing " in this paper. Preparation The most common way to fluorescently tag proteins in living cells is the GFP technology (green fluorescent protein cloned from the jelly fish A. victoria). Of the different spectral GFP mutants EGFP is the best, because of its high quantum yield, its low tendency of photobleaching as well as its relative photostability during post-bleach image acquisition. Cells both stably or transiently expressing an EGFP-tagged version of your protein of interest (ypi) are suitable for FRAP experiments. Transiently transfected cells are often used 16 – 48 hours post transfection. Here the optimal time for the experiment has to be determined empirically depending on the construct and the cell type or culture conditions. To maintain appropriate culture conditions for most cells it is necessary to have at least a heatable stage, ideally contained within a moisturized and CO 2-controlled box. …