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 significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high data quality. 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 labelling strategies with respect to their molecular brightness, photostability, and photoblinking. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we validate the developed methods with experimental data acquired on DNA origami labeled with defined fluorophore numbers, demonstrating counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis by counting labeled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists

“…Fluorescence intensity traces associated with individual spots isolated from background were extracted and the number of photobleaching steps in each trace was determined (Fig. 8b, c ) 36 . Photobleaching step histograms show that majority (~71% of spots) of CD86-eGFP spots bleached in a single step consistent with a monomeric stoichiometry (Fig.…”

Single‐molecule localization microscopy goes quantitative

“…If the labeling stoichiometry is not controllable, fluorescence calibration methods can help to strengthen the accuracy of the counting. Both synthetic nanostructures and intracellular proteins can be used as calibration systems to optimize the quantitative analysis, reducing miscounting errors (Hummert et al, 2021) and automatizing the analysis (Danial et al, 2022). However, stepwise photobleaching presents some intrinsic limitations.…”

A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy

“…were included. Photobleaching steps were analyzed with use of quickPBSA package (Hummert et al, 2021).…”

“…Previous work along these lines showed that the size distribution of the vesicles could be determined by correlating the fluorescence intensity of a lipid marker in single vesicles with DLS data (Hatzakis et al, 2009;Malle et al, 2021;Lohr et al, 2009;Olsson et al, 2015), and that tightness and lamellarity could be assessed by bleaching of the marker (Heider et al, 2011). Other studies used step-wise photobleaching assays to determine the copy number of fluorescent proteins per vesicle (Hummert et al, 2021). Here, we present a broadly applicable proteoliposome characterization assay at the single vesicle level that permits parallel analysis of multiple parameters (physical size, tightness, unilamellarity, membrane protein content and orientation) of individual proteoliposomes to provide a detailed picture of the reconstituted membrane system.…”