Reversible Photobleaching of Fluorescein Conjugates in Air‐Saturated Viscous Solutions: Singlet and Triplet State Quenching by Tryptophan

Abstract

Fluorescence recovery after photobleaching (FRAP) measurements on air-saturated aqueous solutions of fluorescein made viscous with glycerol or sucrose revealed a rapid component of fluorescence recovery with exponential time constants of 30-120 microseconds at viscosities of 15-300 cP. The rapid recovery process was not related to fluorophore translational diffusion and was insensitive to fluorophore concentration and the additive used to increase solution viscosity. At constant viscosity, the rate of reversible photobleaching recovery increased 2.5-fold in an O2- vs N2-saturated solution. The relative efficiency of reversible-to-irreversible photobleaching decreased with increasing photobleaching time and/or beam intensity. Reversible photobleaching was also detected for conjugates of fluorescein with dextrans and proteins in viscous media. In screening triplet state quenchers that might influence the reversible recovery, it was found that tryptophan enhanced the rate of reversible photobleaching recovery (two-fold increase at 8 mM) and quenched the fluorescein singlet state (Stern-Volmer constant, 12 M-1). Analysis of fluorescein lifetimes and photobleaching parameters for a series of fluorescein-labeled proteins with different numbers of tryptophans were also carried out. The results provide evidence for an oxygen-dependent, reversible photobleaching mechanism for the fluorescein chromophore involving triplet state relaxation. The identification of reversible fluorescein photobleaching has important implications for FRAP measurements of rapid solute diffusion in biological systems.

Photobleached-fluorescence imaging of microflows

“…However, in aqueous solutions of borate buffer, water, and fluorescein or fluorescein-dextran conjugates, reversible photobleaching is negligible (Axelrod et al 1976;Periasamy et al 1996;Saylor 1995). This observation has been confirmed in the current study by photobleaching an entire microchannel cell and monitoring the fluorescence over periods of several hours.…”

Mechanism and Cellular Applications of a Green Fluorescent Protein-based Halide Sensor

“…In addition, a fast reversible fluorescence recovery process was identified with exponential time constants in the range 3-5 ms (bottom, left curve). Similar recovery processes in GFP-S65T and fluorescein were shown to arise from triplet-state quenching processes (27,28). The efficiency (bleach depth) and recovery rates for reversible fluorescein photobleaching were sensitive to triplet state quenchers, including the small molecules oxygen, azide, and Mn 2ϩ , whereas the reversible photobleaching of GFP-S65T and YFP-H148Q (Fig.…”

On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

“…These findings prompted us to investigate whether natural aromatic amino-acids might have a similar effect, since they are known to quench both singletand triplet-states of xanthene and oxazine dyes, via a PET quenching mechanism. [42][43][44] Our hypothesis was that aromatic amino-acids in close proximity to the fluorophore might reduce photostabilization by intermolecular stabilizers in a similar manner to covalently-linked NPA or COT moieties. Such a mechanism might explain the large variations in the size of the photostabilization effects observed with identical buffer cocktails and dye pairs on different targets such as proteins and oligonucleotides.…”