Fluorescence lifetime imaging energy transfer

The lifetime of the excited state of fluorophores may be altered by physical and biochemical properties of its environment. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful analytical tool for the quantitative mapping of fluorescent molecules that reports, for instance, on local ion concentration, pH, and viscosity, the fluorescence lifetime of a donor fluorophore, Forster resonance energy transfer can be also imaged by FLIM. This provides a robust method for mapping protein-protein interactions and for probing the complexity of molecular interaction networks. 

In previous chapters it was shown that FRET can be reliably detected by donor fluorescence lifetime imaging. Here, we will focus on what is perhaps the most intuitive and straightforward way to record FRET imaging of sensitized emission (s.e., that is, the amount of acceptor emission that results from energy transferred by the donor through resonance) by filterFRET. While simple in principle, determinations of s.e. are complicated by overlap of excitation and emission spectra of the donors and acceptors, and by several imperfections of the recording optics, light sources and detectors. 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Elangovan, M., Day, R. N. and Periasamy, A. (2002). Nanosecond fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy to localize the protein interactions in a single living cell. J. Microsc. 205, 3-14. 

B. J. Bacskai, J. Skoch, G.A. Hickey, R. Allen, B.T. Hyman, Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques, J. Biomed. Opt 8, 368-375 (2003)... 

Fluorescence lifetime imaging microscopy (FLIM)-based guantitative fluorescence resonance energy transfer (FRET). Direct detection of biomolecular interactions is possible with FRET measurements, where a donor fluorophore transfers the energy to an acceptor fluorophore in case they are close in space. The combination of this technique together with FLIM, based on the decrease in donor fluorophore lifetime that is induced by FRET, has recently enabled the quantitative assessment of the protein-interacting fractions [12]. 

Keywords Fluorescence lifetime imaging microscopy Frequency domain Time-correlated single photon counting Fluorescence resonance energy transfer Protein-protein interactions... 

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