Reliable and Global Measurement of Fluorescence Resonance Energy Transfer Using Fluorescence Microscopes

[13] Fluorescence Resonance Energy Transfer

of the extent of binding. This effect (known informally as "Chip dip") is illustrated in Figs. 6 and 7 which concern the ethidium bromide/tRNA system. 41 In this case the free ethidium bromide has a lifetime of 1.86 nsec and a rotational relaxation time of 0.54 nsec. On binding the PhetRNA Phe the lifetime increases to 26 nsec and the rotational relaxation increases to 136 nsec. A single rotati...

Fluorescence resonance energy transfer (FRET) measurement by gradual acceptor photobleaching.

Fluorescence resonance energy transfer (FRET) is an extremely effective tool to detect molecular interaction at suboptical resolutions. One of the techniques for measuring FRET is acceptor photobleaching: the increase in donor fluorescence after complete acceptor photobleaching is a measure of the FRET efficiency. However, in wide-field microscopy, complete acceptor photobleaching is difficult ...

Fluorescence Resonance Energy Transfer using Color Variants of GFP

Polarized fluorescence resonance energy transfer microscopy.

Current methods for fluorescence resonance energy transfer (FRET) microscopy of living cells involve taking a series of images with alternating excitation colors in separate camera exposures. Here we present a new FRET method based on polarization that requires only one camera exposure and thereby offers the possibility for better time resolution of dynamic associations among subcellular compon...

Single-molecule fluorescence resonance energy transfer.

Fluorescent resonance energy transfer (FRET) is a powerful technique for studying conformational distribution and dynamics of biological molecules. Some conformational changes are difficult to synchronize or too rare to detect using ensemble FRET. FRET, detected at the single-molecule level, opens up new opportunities to probe the detailed kinetics of structural changes without the need for syn...