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Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy.
J Phys Chem Lett 2017; 8(18):4424-4430JP

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/cm2) 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.

Authors+Show Affiliations

Institut de Biologie Structurale, CNRS, Université Grenoble Alpes, CEA, IBS, 38044 Grenoble, France.Institut de Biologie Structurale, CNRS, Université Grenoble Alpes, CEA, IBS, 38044 Grenoble, France.Institut de Biologie Structurale, CNRS, Université Grenoble Alpes, CEA, IBS, 38044 Grenoble, France.Institut de Biologie Structurale, CNRS, Université Grenoble Alpes, CEA, IBS, 38044 Grenoble, France.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28850784

Citation

Thédié, Daniel, et al. "Photoswitching of Green mEos2 By Intense 561 Nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy." The Journal of Physical Chemistry Letters, vol. 8, no. 18, 2017, pp. 4424-4430.
Thédié D, Berardozzi R, Adam V, et al. Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy. J Phys Chem Lett. 2017;8(18):4424-4430.
Thédié, D., Berardozzi, R., Adam, V., & Bourgeois, D. (2017). Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy. The Journal of Physical Chemistry Letters, 8(18), pp. 4424-4430. doi:10.1021/acs.jpclett.7b01701.
Thédié D, et al. Photoswitching of Green mEos2 By Intense 561 Nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy. J Phys Chem Lett. 2017 Sep 21;8(18):4424-4430. PubMed PMID: 28850784.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy. AU - Thédié,Daniel, AU - Berardozzi,Romain, AU - Adam,Virgile, AU - Bourgeois,Dominique, Y1 - 2017/09/05/ PY - 2017/8/30/pubmed PY - 2017/8/30/medline PY - 2017/8/30/entrez SP - 4424 EP - 4430 JF - The journal of physical chemistry letters JO - J Phys Chem Lett VL - 8 IS - 18 N2 - 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/cm2) 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. SN - 1948-7185 UR - https://www.unboundmedicine.com/medline/citation/28850784/Photoswitching_of_Green_mEos2_by_Intense_561_nm_Light_Perturbs_Efficient_Green_to_Red_Photoconversion_in_Localization_Microscopy_ L2 - https://dx.doi.org/10.1021/acs.jpclett.7b01701 DB - PRIME DP - Unbound Medicine ER -