Abstract
Time-resolved fluorescence microscopy (TRFM) of objects stained with luminescent lanthanides chelate-based reagents requires adaptation of microscope equipment to distinguish the long-lived fluorescence of the lanthanide from scattered excitation light and relatively short-lived autofluorescence of biological objects and optical components. How this is accomplished depends on the choice of image scanning, e.g., wide-field microscopy (image plane scanning) or object plane scanning [(confocal) laser microscopy]. Typically, excitation light is presented as pulses by applying mechanically chopping of a continuous light beam, or by using flash lamps or pulse lasers. The detector (CCD camera, photomultiplier tube) is time-gated to record the delayed signal in a defined time interval, either electronically or using phase-locked choppers in the emission pathway. This chapter discusses strategies for optimal TRFM and reviews the main existing configurations used for lanthanide imaging. In addition, special techniques such are Fluorescence Lifetime Imaging Microscopy (FLIM) and Time-Correlated Single Photon Counting (TCSPC) are presented and discussed as well.
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