Abstract
Fluorescence microscopy is one of the most important tools for the visualization and examination of biological cells and tissues, both fixed and live. Fluorescent probes can target specific low concentrations molecules with high selectivity and specificity. Modern imaging techniques and instrumentation, along with new fluorescent tags and fluorophores, and advanced computer and informatics technology have compounded its uses in studying the temporal and spatial dynamics of cellular components and activity. This chapter covers the principles of fluorescence imaging, solutions to problems inherent in fluorescence microscopy and modern image processing techniques for enhancing and analyzing fluorescence images. These techniques include calibration and background subtraction, immunofluorescence, fluorescence in situ hybridization (FISH), quantitative co-localization, ratio imaging, Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), Time Correlated Single Photon Counting (TCSPC), Fluorescence Recovery After Photobleaching (FRAP), and Fluorescence Correlation Spectroscopy (FCS).
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