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.
:C$\backslash$:/Users/Karoline/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Martínez et al. - 2020 - Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using flu.pdf:pdf
%0 Journal Article
%1 Martinez2020
%A Martínez, Gaby F.
%A Gazal, Nahir G.
%A Quassollo, Gonzalo
%A Szalai, Alan M.
%A Cid-Pellitero, Esther Del
%A Durcan, Thomas M.
%A Fon, Edward A.
%A Bisbal, Mariano
%A Stefani, Fernando D.
%A Unsain, Nicolas
%D 2020
%I Nature Research
%J Scientific Reports
%K axon cytoskeleton expansion microscopy neuro spectrin
%N 1
%P 1--11
%R 10.1038/s41598-020-59856-w
%T Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy
%V 10
%X 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.
@article{Martinez2020,
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.},
added-at = {2020-03-27T10:41:29.000+0100},
author = {Mart{\'{i}}nez, Gaby F. and Gazal, Nahir G. and Quassollo, Gonzalo and Szalai, Alan M. and Cid-Pellitero, Esther Del and Durcan, Thomas M. and Fon, Edward A. and Bisbal, Mariano and Stefani, Fernando D. and Unsain, Nicolas},
biburl = {https://www.bibsonomy.org/bibtex/2dae0f64977f727bdee8a4046463c41b7/kfriedl},
doi = {10.1038/s41598-020-59856-w},
file = {:C$\backslash$:/Users/Karoline/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Mart{\'{i}}nez et al. - 2020 - Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using flu.pdf:pdf},
interhash = {87191efb26c5a1d4892c1501a19c8a38},
intrahash = {dae0f64977f727bdee8a4046463c41b7},
issn = {20452322},
journal = {Scientific Reports},
keywords = {axon cytoskeleton expansion microscopy neuro spectrin},
month = dec,
number = 1,
pages = {1--11},
publisher = {Nature Research},
timestamp = {2020-04-06T15:15:43.000+0200},
title = {{Quantitative expansion microscopy for the characterization of the spectrin periodic skeleton of axons using fluorescence microscopy}},
volume = 10,
year = 2020
}