FRET-FLIM application

In Chapter 11, by Phill Jones et al., biomedical FRET-FLIM applications are reviewed and illustrated. The molecular background of a variety of diseases (e.g., Alzheimer s disease) can be uncovered by using FRET-FLIM. In this major funding area, the killer -applications of the technology are and will be found, leading to a further boost of the implementation and commercial availability of high-end microscopes with automated acquisition and standardized analysis features. 

While VFPs have boosted the applications of FRET-FLIM, chemical FRET probes should not be dismissed. The advantage of chemical probes is that they are much smaller in size and that they often have much better spectral readout than VFP probes. In Chapter 6, Amanda Cobos Correa and Carsten Schultz highlight the various small molecule-based FRET probes and their use in bioimaging. 

FRET-FLIM has been applied to numerous biological problems, centering on protein-protein interactions, protein conformation, posttranslational modifications, and activation state of enzymes, with lipid microdomains. Each of these applications takes advantage... 

In principle, any couple of fluorophores can be used for FRET, provided that the emission spectrum of the donor overlaps with the absorption of the acceptor. For a review of FRET-couples (and RO values) of chemical dyes see [62]. Furthermore, donors with a high fluorescence quantum-yield and acceptors with a high molar absorbance will display increased FRET. For FLIM it will be important to tune the instrument-performance to ensure maximal sensitivity to small changes in lifetimes at the control donor lifetime. Usually this is easily achieved. Many FRET-pairs have been used for FRET-FLIM including chemical probes as Fluorescein-Rhodamine [54,93],calcein-sulforhodamine B [94], and Cy3-Cy5, [70]. Since 1996, the availability of genetic-encoded fluorophores such as CFP, GFP, YFP has boosted application of FRET-FLIM enormously [95]. Nowadays fluorescent-tagging of proteins no longer depends on laborious protein pu-... 

To demonstrate an application of TIRF-FLIM, a FRET study of annexin A4 translocation and self-aggregation near the plasma membrane is shown in Fig. 9.4. This is a particularly useful application of TIRF-FLIM, since TIRF provides the spatial contrast of detecting only molecules immediately adjacent to the plasma membrane and the lifetime contrast reports on the aggregation state of annexin A4. Annexins are calcium-dependent lipid-binding domains with a different type of lipid binding domain compared to the common C2 domains (e.g., found in protein kinase C). Annex-ins consist of an N-terminal domain and a core domain binding calcium and phospholipids. The core domain is conserved in the... 

An application of TCSPC FLIM to CFP-YFP FRET is shown in Fig. 5.87 and Fig. 5.88. The microscope was a Zeiss LSM 510 NLO two-photon laser scanning microscope in the Axiovert 200 version. An excitation wavelength of 860 nm was used. The nondescanned fluorescence signal from the sample was fed out of the... 

Hyman, B. T. Berezovska, O. Bacskai, B. Lleo, A. FRET- and FLIM-based assays for monitoring protein conformation and protein/protein proximity, and drug screening applications based on preseni-lin 1-APP interaction. U.S. Pat. Appl. Publ. US 2005048539, 2005 Chem. Abstr. 2005,142, 276400. 

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