Abstract

Protein oligomerization is a fundamental process in cell signaling and membrane organization, yet its precise quantification in native cellular environments remains challenging. Photoactivated Localization Microscopy (PALM) offers super-resolution capabilities, but its quantitative application (qPALM) for determining oligomer stoichiometry is highly sensitive to experimental parameters. This study investigates the applicability and limitations of qPALM for the quantitative analysis of membrane protein oligomerization under diverse physiological and environmental conditions. We employed a model system of fluorescently-tagged membrane proteins with known oligomeric states, systematically varying factors such as labeling density, photophysical properties, and sample preparation protocols. Our results demonstrate that while qPALM can accurately resolve oligomeric species, its quantitative reliability is significantly impacted by factors like protein mobility and the density of photoactivatable fluorophores. We establish a robust statistical framework and a set of critical parameters necessary to ensure the accurate determination of oligomer size and distribution. This work provides essential guidelines for the rigorous application of qPALM in cell and membrane biology, enabling more reliable insights into the dynamic organization and function of membrane protein complexes in living cells.