mEos4b photoconversion efficiency depends on laser illumination conditions used in PALM

Abstract

Green-to-red photoconvertible fluorescent proteins (PCFPs) are widely employed as markers in photoactivated localization microscopy (PALM). However, their highly complex photophysical behavior complicates their usage. The fact that only a limited fraction of a PCFP ensemble can form the photoconverted state upon near-UV light illumination, termed photoconversion efficiency (PCE), lowers the achievable spatial resolution in PALM and creates undercounting errors in quantitative counting applications. Here, we show that the PCE of mEos4b is not a fixed property of this PCFP, but strongly depends on illumination conditions. Attempts to reduce long-lived blinking in red mEos4b by application of 488 nm light leads to a reduction of the PCE. Furthermore, the PCE of mEos4b strongly depends on the applied 405-nm power density. A refined photophysical model of mEos4b accounts for the observed effects, involving nonlinear green-state photobleaching upon violet light illumination favored by photon absorption by a putative radical dark state.

Photophysical studies at cryogenic temperature reveal a novel photoswitching mechanism of rsEGFP2

“…Such a low recovery level is problematic for cryo-PALM applications, as only a minor fraction of the fluorescently labeled biological targets would then be detectable, giving rise to a low effective labeling efficiency. In comparison, in standard RT PALM using greento-red photoconvertible FPs, typically 60 to 70% of the labels can be imaged under favorable illumination conditions 39,40 .…”

Single molecule imaging simulations with advanced fluorophore photophysics

“…Furthermore, SMIS users may modify existing fluorophores as new experimental knowledge is gathered on them. For example, photoblueing mechanisms in organic dyes 39 (e.g., photoconversion of Cy5 to Cy3) or nonlinear green-state photobleaching in EosFP variants 40 could be readily implemented.…”

Single Molecule Imaging Simulations with Advanced Fluorophore Photophysics

“…For example photoblueing mechanisms in organic dyes 39 (e.g. photoconversion of Cy5 to Cy3) or nonlinear green-state photobleaching in EosFP variants 40 could be readily implemented…”

“…For example photoblueing mechanisms in organic dyes 39 (e.g. photoconversion of Cy5 to Cy3) or nonlinear green-state photobleaching in EosFP variants 40 could be readily implemented As of today, limited photophysical knowledge is available for a majority of fluorophores, including many popular ones. Moreover, the photophysical behavior of organic dyes, and -to a lesser extent-of fluorescent proteins, is heavily dependent on environmental conditions.…”