FRET-FLIM techniques

Hence, for modern FRET and FLIM techniques in Molecular Biology and Biochemistry it is important to keep the enthusiasm for the in situ technique, yielding unprecedented rich information on molecular states in live cells, and to keep the advantages of easy labeling techniques, modern microscopes and automated data processing. However, we need to educate the new generations of FRET scientists in the theoretical background of the technique, how it should be done correctly, and what the sources of errors are. Only then it will be clear that FRET-(FLIM) is a very direct, robust, extremely sensitive, and reliable technique. 

Laboratory Techniques in Biochemistry and Molecular Biology, Volume 33 FRET and FLIM Techniques T. W. J. Gadella (Editor)... 

Measuring FRET by fluorescence lifetime imaging microscopy (FRET-FLIM) offers the ability to see beyond the resolution of the optical system ( 10-100 times that of modern far field microscopes [5]). FRET efficiency can be used as a proxy for molecular distance, thereby allowing the easy detection and somewhat more challenging quantification of molecular interactions. Although many types of assay exist, FRET-FLIM is a highly suitable technique that is capable of in situ measurements of molecular interactions and conformation in living and fixed cells. 

The distance between two different fiuorophore molecules can be probed by fluorescence resonance energy transfer (FRET) [308]. The energy transfer rate from the donor to the aeeeptor depends on the sixth power of the distance. FRET becomes noticeable at distanees on the order of a few mn and therefore occurs only if the donor and aeeeptor are physically linked. With FLIM techniques, FRET results are obtained from a single lifetime image of the donor [15, 32, 38, 61, 62, 63, 73, 80, 93, 147, 209, 405, 508]. 

The local environment, the binding or aggregation state, the quenching rate, and the FRET efficiency of the fiuorophore moleeules in cells are normally inhomogeneous. Moreover, different fluorophores may overlap within the same pixel. Therefore, the fluorescenee deeay funetions found in cells are usually multiexponential. A FLIM technique should not only resolve lifetimes down to 50 ps, it should also be able to resolve multiexponential deeay funetions. 

Comparison of FLIM with Other FRET Microscopy Techniques. 165... 

With each method of FRET estimation there are advantages and disadvantages. Here, specifically the three most widely used other FRET-imaging techniques besides FRET-FLIM i) acceptor-bleaching, ii) filter-FRET and iii) spectral imaging are briefly discussed in relation to FLIM. Also elsewhere, reviews and experimental studies have appeared comparing FLIM with other FRET-microscopy techniques [61,63,108]. 

On September 13 2005 I received the invitation from professor P.C. van der Vliet (the editor of the Laboratory Techniques in Biochemistry and Molecular Biology Series) to become the editor of a new volume in the series on FRET and FLIM . In the letter it was mentioned that in view of the rapid developments in single cell technology, we feel that a book on imaging techniques in living cells, such as FRET and FFIM, is appropriate and timely . 

However, FLIM measurements by both frequency and time domain using commercially available software suffers from variation in the measured lifetime from region to region in cells [36], which can be confusing. In the light of the potential pitfalls associated with each of the above-mentioned FRET techniques, potential protein associations should ideally be tested independently by a combination of two or more FRET-based methods in addition to biochemical techniques such as co-immunoprecipitations. 

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