SPONTANEOUS-RECOVERY OF FLUORESCENCE BY PHOTOBLEACHED SURFACE-ADSORBED PROTEINS

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.