Fluorescent proteins don’t like sunburns !
Super resolution microscopy continues to reveal new insights into biology at the nanoscale. Quantitative measurements using super resolution microscopy, however, remain limited due to the highly complex photophysical behavior of the used fluorescent labels. In the case of green-to-red photoconvertible fluorescent proteins (PCFPs), their limited green-to-red conversion efficiency lowers the achievable spatial resolution and causes undercounting errors in quantitative experiments. Researchers of the PIXEL team have shown that the photoconversion efficiency of several popular PCFPs, including mEos4b, Dendra2 and pcStar, strongly depends on the applied illumination conditions. In particular, they revealed that the photoconversion efficiency depends in a non-linear manner on the used 405-nm light intensity, due to a non-linear photobleaching mechanism. Their findings contribute to our understanding of FP photophysics and inform the design of optimized illumination schemes for photoactivated localization microscopy.
mEos4b Photoconversion Efficiency Depends on Laser Illumination Conditions Used in PALM. Wulffele J, Thédié D, Glushonkov O, Bourgeois D. J. Phys. Chem. Lett. 13:5075–5080. DOI : 10.1021/acs.jpclett.2c00933