Quantitative Expansion Microscopy and its validation characterizing the spectrin membrane-associated periodic skeleton in axons

GAZAL N G; MARTÍNEZ G F; QUASSOLLO G; SZALAI A; DEL CID-PELLITERO E; DURCAN T M; FON EA; BISBAL M; STEFANI FD ; UNSAIN N

Congreso; XXXIV Congress of the Argentine Society for Research in Neuroscience; 2019

Argentine Society for Research in Neuroscience

Different fluorescent nanoscopy approaches have been usedto characterize a 1-D periodic organization of actin, spectrinand associated proteins in neuronal axons and dendrites.This membrane-associated periodic skeleton (MPS) hasbeen found in processes from all neuronal types examinedacross animals, suggesting that the structure is a conservedand fundamental component of these processes. The nanoscalearchitecture of the arrangement (periods of 190nm)lays bellow the resolution limit of conventional fluorescentmicroscopy. This finding has led to a small number of articlesand we believe it is because nanoscopy requires special analyzesand expensive equipment. In this report, we aimed atsolving this issue by using protein-retention expansionmicroscopy (ExM) to evidence the MPS of axons. We firstexplored means to estimate expansion factors for proteinstructures within the cell. We then describe the protocolthat produces an expanded specimen that can be examinedwith any conventional fluorescent microscopy (confocal, epifluorescenceo spinning disk) that allows quantitative nanoscalecharacterization of the MPS. We validate our characterizationby showing that the resolved details using prot-ExM rivals those obtained with commercially available stimulatedemission depletion microscope (STED). We concludethat ExM allows for three-dimensional, multicolor and quantitativecharacterization of the MPS using accessible reagentsand conventional fluorescent microscopes.