Fluorescence Lifetime Imaging (FLIM) on a Spectral Confocal Microscope: : The Ultimate Tool for Fluorescence Resonance Energy Transfer (FRET) Analysis

✍ Scribed by Chay Keogh

• Publisher: Wiley (John Wiley & Sons)
• Year: 2007
• Weight: 253 KB
• Volume: 9
• Category: Article
• ISSN: 1439-4243
• DOI: 10.1002/imic.200790159

✦ Synopsis

Providing researchers with insights into the complex world of cellular signalling, protein interactions are an increasingly important aspect of live cell research. Advanced developments in fluorescence proteins have enabled live cell microscopy to become an important tool in these studies. Fluorescence resonance energy transfer (FRET)

, in particular, is a key technique for visualising protein interactions in living cells. In this article, FRET between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) is examined with particular reference to fast spectral detection and fluorescence lifetime imaging (FLIM).

measurement of FRET efficiencies, a problem that can be overcome by applying FLIM to FRET studies. By characterising donor lifetimes in the absence and presence of an acceptor, FRET efficiencies can be obtained using the equation:

FRET efficiency = 1 -(tDA / tD) where t = fluorescence lifetime, D = donor, and A = acceptor.

FRET can also be studied in living cells using spectral imaging. The Nikon C1si spectral confocal microscope is capable of recording full spectra (spectral range of 320 nm using 512 x 512 pixels) in less than two seconds and can record spectral changes due to FRET. The C1si, in addition, can be equipped with a high-speed lifetime detector (Limo) to record donor lifetime changes. The use of Limo in FRET applications is examined here using a CFP/YFP FRET pair.