Abstract
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), Förster 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.
Highlights
fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), fluorescence resonance energy transfer (FRET), and fluorescence lifetime imaging microscopy (FLIM) are fluorescence microscopy techniques that in some way take advantage of particular aspects of the fluorescence process by which fluorochromes are excited and emit fluorescent light, are damaged during repetitive excitation, or undergo non-radiative decay prior to light emission
Quantum dots are characterized by a number of additional unique properties [30,31]: (i) QDs are about 10–100 times brighter than organic fluorogenic dyes; (ii) are 100–1,000 times more resistant to photobleaching, because the shell and various coatings form physical barriers that separate the excited state from surrounding biomolecules and molecular oxygen; and (iii) show narrower and more symmetric emission spectra compared with other fluorochromes (typical full-width at half max (FWHM) of ~25–40 nm [36])
Green fluorescent protein is the most widely used fluorescent probe for cellular studies because of its stability, low cytotoxicity, because it does not bleach significantly at low light intensities, does not seem to be damaging to the cell after undergoing irreversible photobleaching, it can be readily expressed in several cell types where it is fused to a particular protein, and in many cases tagging a protein of interest with green fluorescent protein (GFP) has no significant influence on the function and localization of the protein under investigation [85,121,122,123,124]
Summary
FRAP, FLIP, FLAP, FRET, and FLIM are fluorescence microscopy techniques that in some way take advantage of particular aspects of the fluorescence process by which fluorochromes are excited and emit fluorescent light, are damaged during repetitive excitation, or undergo non-radiative decay prior to light emission. In order to understand the basic principles underpinning these advanced fluorescence techniques, first some general aspects of fluorescence and fluorescence microscopy are introduced before going into the technical details and practicalities of FRAP, FLIP, FLAP, FRET and FLIM. This article is not meant to be a comprehensive report on the aforementioned techniques, but rather to introduce these advanced fluorescence imaging techniques to a broad biological and bio(medical) research audience and give the reader some feeling for the field. The reader is referred to more specialized and comprehensive books and manuscripts for further reading throughout the text
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