Photostability of green and yellow fluorescent proteins with fluorinated chromophores, investigated by fluorescence correlation spectroscopy

Abstract

Abstract:The photostability of the widely used autofluorescent proteins EGFP and EYFP and their fluorinated counterparts were compared by means of fluorescence correlation spectroscopy.We analyzed the reduction of the apparent diffusional time in analogy to fluorescence quenching in which the 'photon concentration' is treated as the quencher concentration.The quantum yields of photobleaching Φ bl of EYFP (6.1×10 -5 ) and EGFP (8.2×10 -5 ) are in agreement with the previously published values. Among the investigated mutants, EYFP has the highest photostability and there is an enhanced photobleaching in (2-F) Tyr-EYFP. It turns out that the chromophore fluorination has no significant influence on the photostability.

Fluorescence in Nanobiotechnology: Sophisticated Fluorophores for Novel Applications

“…FPs can provide relatively large fluorescence QYs ( Φ ) and extinction coefficients ( ϵ ), e.g., Φ = 0.79 and ϵ = 30 000 M −1 cm −1 for the absorption maximum at 395 nm and ϵ = 7000 M −1 cm −1 for the one at 470 nm for wild type GFP (wtGFP). Popular FPs also have a high QY before photobleaching (6.1 × 10 −5 for enhanced GFP (eGFP) and 8.2 × 10 −5 for eYFP), describing the inverse number of photo cycles before the molecules are bleached 99. Another important photophysical property is the fluorescence lifetime.…”

“…Popular FPs also have a high QY before photobleaching (6.1 × 10 − 5 for enhanced GFP (eGFP) and 8.2 × 10 − 5 for eYFP), describing the inverse number of photo cycles before the molecules are bleached. [ 99 ] Another important photophysical property is the fl uorescence lifetime. wtGFP © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim reviews consists of two species one with a shorter (2.7-2.8 ns) and one with a longer lifetime component (3.3-3.4 ns).…”

Replacement of Highly Conserved E222 by the Photostable Non‐photoconvertible Histidine in GFP

“…[26] On the larger timescale, however, some photobleaching was detected, [49,50] and the reduction of the diffusion time at higher intensities (Figure 3 B) is comparable in extent to that for eYFP. [49] An explanation for the lack of photochemistry beyond photobleaching must be found in the structural difference ( Figure 1). Histidine is slightly larger than glutamic acid; moreover, histidine cannot undergo decarboxylation (as for glutamic acid).…”

“…Figure 3 B shows intensity-dependent FCS traces. The bright fraction [B] is constant (0.8) for GFP-E222H above 8 kW cm À2 , and thus slightly higher than for eGFP (0.7) and eYFP (0.65), [49] but significant higher than for GFP-E222Q (0.35). [26] A contrast essentially independent of excitation intensity excludes a significant triplet population.…”

Monofluorination and Trifluoromethylation of BODIPY Dyes for Prolonged Single‐Molecule Detection

“…[11] AS tern-Volmerlike plot allows for the assessment of the intensity-dependent rate constant k bl fora ll bleaching processes, as long as saturation due to an exhaustive triplet population can be neglected (see the Supporting Information for saturation curves) [Eq. (2)]: [11,40]…”