Quantitative expansion microscopy and its validation characterizing the spectrin membrane-associated periodic skeleton in axons

N. GUADALUPE GAZAL; GABY F. MARTINEZ; GONZALO QUASSOLLO; ALAN SZALAI; ESTHER DEL CID-PELLITERO; THOMAS M. DURCAN; EDWARD A. FON; MARIANO BISBAL; FERNANDO D. STEFANI; NICOLÁS UNSAÍN

Villa Carlos Paz

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

Sociedad Argentina de Investigación en Neurociencia

Different fluorescent nanoscopy approaches have been used to characterize a 1-D periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) has been found in processes from all neuronal types examined across animals, suggesting that the structure is a conserved and fundamental component of these processes. The nanoscale architecture of the arrangement (periods of ~190nm) lays bellow the resolution limit of conventional fluorescent microscopy. This finding has led to a small number of articles and we believe it is because nanoscopy requires special analyzes and expensive equipment. In this report, we aimed at solving this isuue by using protein-retention expansion microscopy (ExM) to evidence the MPS of axons. We first explored means to estimate expansion factors for protein structures within the cell. We then describe the protocol that produces an expanded specimen that can be examined with any conventional fluorescent microscopy (confocal, epifluorescence o spinning disk) that allows quantitative nanoscale characterization of the MPS. We validate our characterization by showing that the resolved details using prot-ExM rivals those obtained with commercially available stimulated emission depletion microscope (STED). We conclude that ExM allows for three-dimentional, multicolor and quantitative characterization of the MPS using accesible reagents and conventional fluorescent microscopes.