Quantitative Expansion Microscopy for In Situ Estimation of Endogenous Target Abundance

Abstract Spatially mapping protein abundance in situ can offer important insights into molecular mechanisms and the physiological functions of protein complexes. This is typically achieved by combining super-resolution microscopy to image fluorescently-labeled protein locations with statistical estimators to retrieve abundances, where accuracy is strongly impacted by labeling efficiency. We introduce quantitative expansion microscopy (qExM) as a method to estimate endogenous protein abundance on ExM data, which offers improved antibody targeting through molecular decrowding. Using cryo-fixation, we preserved ultrastructure and enhanced labeling efficiency to improve accuracy in abundance estimations. We benchmark the effectiveness of qExM by quantifying the stoichiometry of well-characterized nuclear pore complex subunits, and find a mean percent error of 9.4%. We further apply qExM to investigate the abundance of mitochondrial respiratory chain complexes in functionally distinct organelle subpopulations, and of mitochondrial respiratory chain super-complexes in differentially activated human T-cells. qExM provides a robust methodological framework for quantifying endogenous protein abundance in expanded samples in situ .