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

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.

Visualizing subcellular structures in neuronal tissue with expansion microscopy

“…1F). Our average micro expansion factors are lower than the commonly reported 4-4.5X macro expansion factor for proExM, which is consistent with other reports using micro expansion factor measurements 14,15 (but see also ref 26 ), reinforcing the notion that each sample type needs to be independently optimized and validated for ExM.…”

“…Others, however, have reported minimal to no differences in micro vs macro expansion factors 6,18 . We did not measure macro expansion factors here, but our average micro expansion factors are lower than the commonly reported 4-4.5X macro expansion factor for proExM, which is consistent with other reports using micro expansion factor measurements 13,14 (but see also ref 25 ), reinforcing the notion that each sample type needs to be independently optimized and validated for ExM.…”

“…Others have used ExM to visualize subcellular structures, including mitochondria 1,2,23–25 and/or spines 2,20,23 in brain tissue, but few have systematically analyzed how fluorescence intensities and expansion factors compare across protocols or with unexpanded measurements. This is critically important as several groups have noted discrepancies in micro versus macro expansion factors in other sample types 7,14,17,26–28 , including dissimilarities in expansion factors of different subcellular organelles 14 or of subcellular organelles across neighboring cells and tissues 17 . Others, however, have reported minimal to no differences in micro vs macro expansion factors 6,19 .…”

Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics

“…However, these processes do not only depend on the properties of the motors and their assemblies, but also on the structural organization of the actin cytoskeleton. In the future, our approach should be complemented by imaging and modelling of the actin cytoskeleton using super-resolution microscopy (Martinez et al, 2020;Qi et al, 2019;Shelden et al, 2016;Wassie et al, 2019). In a next step, our insights into the distinct cellular roles of the different NM II isoforms should be transferred to the tissue context, e.g.…”

Direct Visualization of Actin Filaments and Actin-Binding Proteins in Neuronal Cells

“…Such a negative pattern reduces the size of the fluorescent region and scanning of this sharpened focal spot generates a super-resolution image. Through the use of STED, actin and ABP have been thoroughly investigated ( Urban et al, 2011 ; Lukinavičius et al, 2014 ; Leite et al, 2016 ; Sidenstein et al, 2016 ; Barabas et al, 2017 ; D’este et al, 2017 ; Krieg et al, 2017 ; Martínez et al, 2020 ).…”