Fluorescent imaging FRET

Rizzo, M. A. and Piston, D. W. (2005). High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy. Biophys. J. 88, L14—6. 

Domingo, B., Sabariegos, R., Picazo, F. and Llopis, J. (2007). Imaging FRET standards by steady-state fluorescence and lifetime methods. Microsc. Res. Tech. 70, 1010-21. 

Fig. 10. (Top) Schematic of the experimental arrangement for carrying out NSOM/ FRET measurements. An acceptor dye of a FRET pair is attached to the NSOM probe while the sample contains the donor dye in the bottom and top layers of a multi-layer film. The left fluorescence image shows the donor fluorescence and the right the fluorescence from the tip-bound acceptor dye. Reproduced with permission from Ref. [26]. Copyright 1999 The Biophysical Society.

TCSPC with two-dimensional position-sensitive detection can be used to acquire time-resolved images with wide-field illumination. The complete sample is illuminated by the laser and a fluorescence image of the sample is projected on the detector. For each photon, the coordinates in the image area and the time in the laser pulse sequence are determined. These values are used to build up the photon distribution over the image coordinates and the time (see Fig. 3.12, page 40). The technique dates back to the 70s [312] and is described in detail in [262]. Lifetime imaging with a TCSPC wide-field system and its application to GFP-DsRed FRET is described in [162]. A spatially one-dimensional lifetime system based on a delay-line MCP is described in [509]. 

Keywords Spectral imaging Linear unmixing Fluorescent proteins - FRET... 

Tramier M, Coppey-Moisan M (2008) Fluorescence anisotropy imaging microscopy for homo-FRET in living cells. Methods Cell Biol 85 395-414... 

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