Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy.

Gaby F Martínez,Fernando D Stefani,Mariano Bisbal,Esther Del Cid-Pellitero,Nahir G Gazal,Alan M Szalai,Nicolas Unsain,Gonzalo Quassollo,Edward A Fon,Thomas M Durcan

doi:10.1038/s41598-020-59856-w

Abstract

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. This membrane-associated periodic skeleton (MPS) is conserved across animals, suggesting it is a fundamental component of neuronal extensions. The nanoscale architecture of the arrangement (190 nm) is below the resolution limit of conventional fluorescent microscopy. Fluorescent nanoscopy, on the other hand, requires costly equipment and special analysis routines, which remain inaccessible to most research groups. This report aims to resolve this issue by using protein-retention expansion microscopy (pro-ExM) to reveal the MPS of axons. ExM uses reagents and equipment that are readily accessible in most neurobiology laboratories. We first explore means to accurately estimate the expansion factors of protein structures within cells. We then describe the protocol that produces an expanded specimen that can be examined with any fluorescent microscopy allowing quantitative nanoscale characterization of the MPS. We validate ExM results by direct comparison to stimulated emission depletion (STED) nanoscopy. We conclude that ExM facilitates three-dimensional, multicolor and quantitative characterization of the MPS using accessible reagents and conventional fluorescent microscopes.

Highlights

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites
As a first test of our hypothesis, we compared the effective expansion of routine acrylamide gels used for expansion microscopy against the same recipe but containing yeast proteins that were dissolved in water and cross-linked before gelation
As a step we investigated whether this finding was relevant for expansion microscopy experiments

Summary

Introduction

Fluorescent nanoscopy approaches have been used to characterize the periodic organization of actin, spectrin and associated proteins in neuronal axons and dendrites. Its study has been governed by the use of a handful of high-end super-resolution microscopy approaches, often referred to as fluorescence nanoscopy, including stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED) and super-resolution structured illumination (SR-SIM) These techniques require specialized equipment, sophisticated data analysis and dedicated trained users[9,10], meaning their application is far www.nature.com/scientificreports from widespread in biomedical research laboratories. In 2015, Boyden and colleagues smartly used the swelling behavior of hydrogels to physically expand preserved biological specimens, which introduced the field of Expansion Microscopy[14] Their protein-retention approach (pro-ExM) version of the technique consisted of polymerizing a 2.5% acrylamide gel (ionized with sodium acrylate) within a cell and cross-linking endogenous proteins to the gel. We explored the possibility that the hydrogel built within the dense protein meshwork of the cell swells more than the hydrogel that lies outside the cells (the gel block)

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