Total internal reflectance fluorescence energy transfer

Khakh, B. S., Fisher, J. A., Nashmi, R., Bowser, D. N. and Lester, H. A. (2005). An angstrom scale interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy. J. Neurosci. 25, 6911-20. 

T. P. Burghardt and D. Axelrod, Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin, Biochemistry 22, 979-985 (1983). 

The application of total internal reflection fluorescence spectroscopy (TIRF) by this laboratory to the study of protein adsorption at solid-liquid interfaces is reviewed. TIRF has been used to determine adsorption isotherms and adsorption rates from single-and multi-component protein solutions. Initial adsorption rates of BSA can be explained qualitatively by the properties of the adsorbing surface. Most recently, a TIRF study using monoclonal antibodies to probe the conformation of adsorbed sperm whale myoglobin (Mb) elucidated two aspects of the Mb adsorption process 1) Mb adsorbs in a non-random manner. 2) Conformational changes of adsorbed Mb, if they occur, are minor and confined to local regions of the molecule. Fluorescence energy transfer and proteolytic enzyme techniques, when coupled with TIRF, can characterize, respectively, the conformation and orientation of adsorbed Mb. 

Key words Confocal fluorescence microscopy, Forster resonance energy transfer, Total internal reflection fluorescence microscopy, Single-molecule imaging, GPCR, Heterotrimeric G-proteins, Spatiotemporal dynamics... 

Several other modes of fluorescence microscopy have been developed, including automated fluorescence image c3dometry (AFIC), fluorescence resonance energy transfer microscopy (FRETM), two-photon fluorescence microscopy, total internal reflection fluorescence microscopy, and standing-wave fluorescence microscopy. 

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. 

FRET fluorescence resonance energy transfer PDMS polydimethylsiloxane TIR total internal reflection... 

FRET fluorescence resonance energy transfer FCS fluorescence correlation spectroscopy TIRF total internal reflection fiuorescence PCFI photon counting histogram ICCD intensified charge coupled device EMCCD eiectron muitipiying charge coupled device CMOS complimentary metal oxide semiconductor AFM atomic force microscope. 

---