Fluorescence Resonance Energy Transfers Measurements on Cell Surfaces via Fluorescence Polarization

Abstract

A method has been developed for the determination of the efficiency of fluorescence resonance energy transfer efficiency between moieties located on cell surfaces by performing individual cell fluorescence polarization (FP) measurements. The absolute value of energy transfer efficiency (E) is calculated on an individual cell basis. The examination of this methodology was carried out using model experiments on human T lymphocyte cells. The cells were labeled with fluorescein-conjugated Concanavalin A (ConA) as donor, or rhodamine-conjugated ConA as acceptor. The experiments and results clearly indicate that determination of E via FP measurements is possible, efficient, and more convenient than other methods.

Real-time monitoring of changes in plasma membrane potential via imaging of fluorescence resonance energy transfer at individual cell resolution in suspension

“…15 Very briefly, Perrin's equation teaches that 25 Next, since both options (emission and FRET) of evacuating the excited level of the donor are active, donor FLT-τ F (of the excited state in the absence of acceptor) is shortened, consequently increasing its FP.…”

“…15,28 For all intents and purposes, Here, M is the microscope correction factor that compensates for the distortion of FP measurement due to the microscope objective numerical aperture and optical pass.…”

Fluorescence resonance energy transfer imaging via fluorescence polarization measurement

“…1 Microscopic FRET imaging methods currently used to measure energy transfer are based on monitoring donor photobleaching, 2 variations of sensitized acceptor fluorescence, 3 donor quenching, 4 donor lifetime, 5 and fluorescence polarization ͑FP͒. 6 In principle, the measurement of FRET using a microscope is as informative as the current macroscopic FRET measurements; however, it enables the visualization of the spatial distribution of FRET efficiency over the entire image, rather than averaging the values over the whole object and/or object population. Since energy transfer is possible in the distance range of 1 to 10 nm between the donor and acceptor, an additional increase in spatial resolution is enabled.…”

“…In an earlier study, we proposed a methodology for a direct determination of the efficiency of energy transfer ͑E͒ via FP, and showed its correlation with other techniques. 6 Briefly, FP is defined as the ratio:…”

Visualization of Protein Interactions in Living Cells

“…95 Unlike other FRET imaging modalities, anisotropy imaging can detect FRET between fluorophores of the same type, obviating the need for double labeling with different donors and acceptors. 96,97 This way, anisotropy imaging is particularly convenient to visualize homotypic aggregation and multimerization of FP-labeled proteins.…”