New light on fluorescent proteins dark states
Photoconvertible fluorescent proteins (PCFPs) are the most commonly used markers in super-resolution "PALM" microscopy. However, in addition to their green (native) and red (photo-converted) forms, these proteins have an unfortunate tendency to stochastically enter multiple non-fluorescent dark states, ie to “blink”. The present papers investigate dark states that are reached from the green form of PCFPs and reveal their importance for PALM microscopy. The first article shows that these dark states greatly limit the efficiency of green-to-red photoconversion. The second, in collaboration with a German team, shows that PCFPs can be divided into two categories. PCFPs of the first category blink less in their green form, and it is shown that they are amenable to “primed” green-to-red photoconversion. This is not the case for members of the second category, which blink more. It is shown that primed photoconversion, a mechanism recently discovered by a Swiss team, substantially reduces the phototoxicity of PALM experiments.
Photoswitching of Green mEos2 by Intense 561-nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy. D. Thédié, R. Berardozzi, V. Adam, D. Bourgeois. J. Phys. Chem. Lett., 2017, 8, 4424−4430 DOI : 10.1021/acs.jpclett.7b01701
A General Mechanism of Photoconversion of Green-to-Red Fluorescent Proteins Based on Blue and Infrared Light Reduces Phototoxicity in Live-Cell Single-Molecule Imaging. B. Turkowyd, A. Balinovic, D. Virant, H. G. Gölz Carnero, F. Caldana, M. Endesfelder, D. Bourgeois, and U. Endesfelder. Angewandte Chemie International Edition England ; 56, 1-7 DOI: 10.1002/anie.201702870