Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy - Archive ouverte HAL Access content directly
Journal Articles Journal of Physical Chemistry Letters Year : 2017

Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy

Abstract

Green-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2 and its derivatives are widely used in PhotoActivated Localization Microscopy (PALM). However, the complex photophysics of these genetically encoded markers complicates the quantitative analysis of PALM data. Here, we show that intense 561 nm light (∼1 kW/cm(2)) typically used to localize single red molecules considerably affects the green-state photophysics of mEos2 by populating at least two reversible dark states. These dark states retard green-to-red photoconversion through a shelving effect, although one of them is rapidly depopulated by 405 nm light illumination. Multiple mEos2 switching and irreversible photobleaching is thus induced by yellow/green and violet photons before green-to-red photoconversion occurs, contributing to explain the apparent limited signaling efficiency of this PCFP. Our data reveals that the photophysics of PCFPs of anthozoan origin is substantially more complex than previously thought, and suggests that intense 561 nm laser light should be used with care, notably for quantitative or fast PALM approaches.
Not file

Dates and versions

hal-01592521 , version 1 (25-09-2017)

Identifiers

Cite

Daniel Thédié, Romain Berardozzi, Virgile Adam, Dominique Bourgeois. Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy. Journal of Physical Chemistry Letters, 2017, 8 (18), pp.4424 - 4430. ⟨10.1021/acs.jpclett.7b01701⟩. ⟨hal-01592521⟩
311 View
0 Download

Altmetric

Share

Gmail Facebook Twitter LinkedIn More