Al. Stout et D. Axelrod, SPONTANEOUS-RECOVERY OF FLUORESCENCE BY PHOTOBLEACHED SURFACE-ADSORBED PROTEINS, Photochemistry and photobiology, 62(2), 1995, pp. 239-244
Fluorescence photobleaching of a carboxyfluorescein-labeled protein (e
rythrocyte cytoskeletal protein 4.1) immobilized on bare glass is foun
d to be spontaneously reversible, provided that the sample is deoxygen
ated. After a short (hundredths of seconds) photobleaching laser flash
, the subsequent fluorescence excited by a dim probe beam partly recov
ers on a long (tenths of second) time scale, even in the absence of ch
emical exchange or diffusion processes. Neither the fraction of the fl
uorescence that bleaches reversibly nor its recovery rate is a strong
function of fluorophore surface concentration. At a fixed surface conc
entration, the reversibly photobleached fr action and its recovery rat
e decreases with increasing duration or intensify of the bleaching fla
sh. On the other hand, nondeoxygenated air-equilibrated samples exhibi
t almost total irreversible bleaching on this time scale. Quantitative
fluorescence microscopy experiments occasionally require deoxygenatio
n to avoid photochemical crosslinking or photobleaching or to enhance
the tripler state population. The observations presented here indicate
that fluorescence recovery after photobleaching (FRAP) experiments pe
r formed under deoxygenated conditions for measuring diffusion or chem
ical kinetics should be interpreted with caution: fluorescence recover
ies may be due to intrinsic photochemical processes rather than fluoro
phore mobility. The recovery effect appears too slow to be ascribed si
mply to, a relaxation of a triplet state; other possible explanations
are offered.