Photo-bleaching

FRAP (fluorescence recovery after photo bleaching) analysis was originally developed in the mid-1970s to study the diffusion of biomolecules in living cells (Edidin et al, 1976). Flowever, due to the increased availability of GFP tags and advances in the bleaching capabilities of confocal microscopes, this... 

For this particular experiment, we also carried out additional photobleaching measurements. Here, the acceptor was photo bleached and it was found that the donor lifetime returned to the donor only lifetime of about 2.19 ns. This confirms that the observed lifetime reduction is indeed due to FRET [49],... 

Purify the labeled protein from excess dye using dialysis or gel filtration on a desalting resin. Protect the labeled protein from light to avoid photo bleaching the dye and losing fluorescence intensity. 

Two-photon excitation provides intrinsic 3-D resolution in laser scanning fluorescence microscopy. The 3-D sectioning effect is comparable to that of confocal microscopy, but it offers two advantages with respect to the latter because the illumination is concentrated in both time and space, there is no out-of-focus photo-bleaching, and the excitation beam is not attenuated by out-of-focus absorption, which results in increased penetration depth of the excitation light. 

Giloh, H. and Sadat, J. W. (1982) Fluorescence microscopy reduced photo-bleaching of rhodamine and fluorescein protein coniugates by n-propyl gallate. Science 217, 1252-1255. 

Szentesi G, Vereb G, Horvath G et al (2005) Computer program for analyzing donor photo-bleaching FRET image series. Cytometry A 67A 119-128... 

Tertiary amines with an a-hydrogen are among the most effective electron donors other electron donors include alcohols, amides, amino acids, and ethers. A third process, direct hydrogen atom transfer from RH to the ketone, is not common hut does occur with some photoinitiators. The overall result is the same as the electron-transfer process. Although two radicals are produced by photolysis of the photoinitiator, only one of the radicals is typically active in initiation—the aroyl and amine radicals in Eqs. 3-48 and 3-49, respectively. The other radical may or may not initiate polymerization, hut is active in termination. The decrease in photoinitiator concentration during polymerization is referred to as photo-bleaching. 

In discussing the use of photoconduction measurements for testing the photo-bleaching process, it is important to recognize that the photoconduction of dyes is due to the formation and migration of electronic charge carriers and not to impurities of photochemical decomposition. However, the following facts should not be overlooked 91>. 

Fig. 1. ESR spectra of pure 3-methylpentane glass irradiated by y-rays to a dose of 3 x 10s rad at 77° K. Solid line, 6 min after irradiation dotted line, after photo-bleaching by room lights (Ref. 11)...

The integrated intensity of the satellite spectrum is very close to that of the trapped electron spectrum. The former spectrum decays concomitantly with the latter either with photo-bleaching with the infrared... 

Fluorescein-labeled proteins are also used to measure the translational mobility of proteins and lipids by the Fluorescence Recovery After Photo-bleaching technique [54-59]. The uniformly labeled fluorescent sample is flashed with an intense light source to bleach a spot, thus producing a concentration gradient. The rate of recovery of fluorescence in that bleached area is measured and used to calculate the diffusion coefficient of the probe dye into the bleached zone. Such diffusion coefficient measurements have been used to determine the association constants of proteins in cells [60], to measure the exchange of tubulin between the cytoplasm and the microtubules [61,62], to study the polymerization-depolymerization process of actin [63-65] and to monitor the changes that occur upon cell maturation [66,67]. 

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