Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach
to study fixed and living cells because of its versatility,
specificity, and high sensitivity. Fluorescence microscopes can
both detect the fluorescence emitted from labeled molecules in
biological samples as images or photometric data from which
intensities and emission spectra can be deduced. By exploiting the
characteristics of fluorescence, various techniques have been
developed that enable the visualization and analysis of complex
dynamic events in cells, organelles, and sub-organelle components
within the biological specimen. The techniques described here are
fluorescence recovery after photobleaching (FRAP), the related
fluorescence loss in photobleaching (FLIP), fluorescence
localization after photobleaching (FLAP), Forster or fluorescence
resonance energy transfer (FRET) and the different ways how to
measure FRET, such as acceptor bleaching, sensitized emission,
polarization anisotropy, and fluorescence lifetime imaging
microscopy (FLIM). First, a brief introduction into the mechanisms
underlying fluorescence as a physical phenomenon and fluorescence,
confocal, and multiphoton microscopy is given. Subsequently, these
advanced microscopy techniques are introduced in more detail, with
a description of how these techniques are performed, what needs to
be considered, and what practical advantages they can bring to cell
biological research.