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Molecules 2012, 17(4), 4047-4132; doi:10.3390/molecules17044047

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

1,†,* , 2 and 3,†,*
1 Ludwig Maximilian University of Munich, Institute of Anatomy and Cell Biology, Schillerstr. 42, 80336 München, Germany 2 Carl Zeiss Microimaging GmbH, Kistlerhofstr. 75, 81379 München, Germany 3 Bionanoscience and Bio-Imaging Program, Cellular Stress and Ageing Program, Bio&Nano-Solutions, Helmutstr. 3A, 40472 Düsseldorf, Germany These authors contributed equally to this work.
* Authors to whom correspondence should be addressed.
Received: 14 March 2012 / Revised: 21 March 2012 / Accepted: 21 March 2012 / Published: 2 April 2012


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.
Keywords: fluorescence microscopy; fluorescence; fluorochrome; techniques; confocal; multiphoton; anisotropy; FRET; homo-FRET; FRAP; FLIP; FLIM; FLAP fluorescence microscopy; fluorescence; fluorochrome; techniques; confocal; multiphoton; anisotropy; FRET; homo-FRET; FRAP; FLIP; FLIM; FLAP
This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Ishikawa-Ankerhold, H.C.; Ankerhold, R.; Drummen, G.P.C. Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM. Molecules 2012, 17, 4047-4132.

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