Selective Fluorescence Recovery after Bleaching of Single E²GFP Proteins Induced by Two‐Photon Excitation

Abstract

We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.

Improving autofluorescent proteins: Comparative studies of the effective brightness of Green Fluorescent Protein (GFP) mutants

“…Such photoconvertable isomeric states were found by experiments on individual molecules (Chirico et al, 2005;Dickson et al, 1997). Noteworthy is the case of E222Q/S65G, where a fluorescence enhancement of $400% by simultaneous two-color excitation was monitored (Fig.…”

“…A corresponding contrast in FCS experiments was not detected. Alternatively, one could imagine that these bulk experiments bring out the reversible photoconversion, which is investigated by other researchers in related mutants (Chirico et al, 2005;Dickson et al, 1997). In the evaluation of F eff , however, we neglect this small population.…”

Single-molecule spectroscopy of fluorescent proteins

“…In addition, many GFPs exhibit another, long lived, dark state that can be depopulated by illumination with light around 400 nm [46,97,107] or by two-photon excitation between 780 and 870 nm [108]. This switching was attributed to spontaneous protonation of the chromophore.…”

“…Illumination with light of short wavelengths into the absorbance band of the protonated chromophore favours the deprotonation of the chromophore in the excited state. Deprotonation results in the reconstitution of the red-shifted absorption and an efficiently emitting chromophore so that seemingly bleached chromophores were reactivated and regained their fluorescence [46,97,107,108]. The light-induced switching of fluorescent proteins between different emitting or non-emitting states bears great potential for cellular time / s Fig.…”

3D localized photoactivation of pa-GFP in living cells using two-photon interactions

“…Examination of the activated spatial volume as a function of the excitation energy showed that intensity modulation can be efficiently used to induce spatially controlled protein photoconversion along the optical axis providing a unique possibility to dynamically identify single 3D structures. Two-photon activation can be efficiently used to track the fate of proteins and other biological macromolecules in living cells following cell cycle steps, and applications in terms of 3D memories could be pursued [10]. .…”