Photobleaching step analysis for robust determination of protein complex stoichiometries.

ABSTRACT: The counting of discrete photobleaching steps in fluorescence microscopy is ideally suited to study protein complex stoichiometry in situ. The counting range of photobleaching step analysis has been significantly improved with more-sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high-quality data. Here, we address concerns regarding robustness, automation, and experimental validation, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labeling strategies with respect to their molecular brightness, photostability, and photoblinking. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we validate the d

SUBMITTER: Hummert J 

PROVIDER: S-EPMC8693960 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature