Determination of the oligomerization state of plasma membrane proteins and lipids is key to understanding cellular signaling and function. TOCCSL (thinning out clusters while conserving stoichiometry of labeling)—an imaging modality based on single-molecule fluorescence microscopy—allows for measuring the degree of homo- or hetero-oligomerization of membrane constituents even at expression levels that are normally incompatible with single-molecule observations. In this study we determined the influence of different experimental parameters on the detected oligomerization state by utilizing Monte Carlo simulations. In silico TOCCSL experiments identified the diffraction-affected laser intensity profile and diffusion properties of the probe as major sources for influencing the determined oligomerization states. Furthermore, they allowed for optimizing experimental parameters and oligomerization data analysis.

Authors:

Clara Bodner ¹, Dominik Kiesenhofer ¹, Gerhard J Schütz ², Mario Brameshuber ³

Reference:

BioPhysical Journal. Published:April 22, 2023DOI:https://doi.org/10.1016/j.bpj.2023.04.021