Photobleaching

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Abstract

Thanks to the wide variety of applications, fluorescence microscopy is now one of the most popular imaging techniques in biology (Weber, 1960; Lakowicz, 1999; Periasamy, 2001; Michalet et al., 2003; Tsien, 2003; Bastiens and Hell, 2004; Taroni and Valentini, 2004; Diaspro et al., 2005). Fluorescence microscopy utilizes fluorescently labeled probes of high biochemical affinity to image the molecular composition and dynamics of biological structures. Moreover, the use of probes that change their fluorescence properties in response to specific physiological parameters enables one to analyze the physiological state of cells or tissues (Birks, 1970; Emptage, 2001; Zhang et al., 2002; Lippincott-Schwartz and Patterson, 2003; Stephens and Allan, 2003). Fluorescence is highly specific either as an exogenous label (e.g., 4’,6-diamidino-2-phenylindole (DAPI) bound to DNA) or an endogenous tracker [(e.g., autofluorescence of NADH, or visible fluorescent proteins such as green fluorescent protein (GFP)] providing spatial and functional information through precise photophysical properties such as absorption, emission, lifetime, and anisotropy. Furthermore, sample preparation is relatively simple, allowing non-invasive imaging and three-dimensional (3D) mapping within cells and tissues to be achieved by means of computational optical sectioning, confocal laser-scanning microscopy (CLSM), and two-photon excitation microscopy (TPEM) (Periasamy, 2001). In particular, CLSM and TPEM are two comparatively recent fluorescence microscopy techniques that have improved the quality of biological images (Wilson and Sheppard, 1984; Denk et al., 1990; Pawley, 1995a; Diaspro, 2002, 2004; Matsumoto, 2002; Amos and White, 2003).