RT Journal Article
SR Electronic
T1 Rapid Ensemble Measurement of Protein Diffusion, Probe Blinking and Photobleaching Dynamics in the Complex Cellular Space
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.06.22.449491
DO 10.1101/2021.06.22.449491
A1 Simon Sehayek
A1 Xiyu Yi
A1 Shimon Weiss
A1 Paul W. Wiseman
YR 2021
UL http://biorxiv.org/content/early/2021/06/26/2021.06.22.449491.abstract
AB We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a non-uniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12 labeled beta-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.Competing Interest StatementThe authors have declared no competing interest.