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Fluorescence Photobleaching as an Intrinsic Tool to Quantify the 3D Expansion Factor of Biological Samples in Expansion Microscopy
journal contribution
posted on 2020-03-17, 13:36 authored by Marisa Vanheusden, Raffaele Vitale, Rafael Camacho, Kris P. F. Janssen, Aline Acke, Susana Rocha, Johan HofkensFour years after
its first report, expansion microscopy (ExM) is
now being routinely applied in laboratories worldwide to achieve super-resolution
imaging on conventional fluorescence microscopes. By chemically anchoring
all molecules of interest to the polymer meshwork of an expandable
hydrogel, their physical distance is increased by a factor of ∼4–5×
upon dialysis in water, resulting in an imprint of the original sample
with a lateral resolution up to 50–70 nm. To ensure a correct
representation of the original spatial distribution of the molecules,
it is crucial to confirm that the expansion is isotropic, preferentially
in all three dimensions. To address this, we present an approach to
evaluate the local expansion factor within a biological sample and
in all three dimensions. We use photobleaching to introduce well-defined
three-dimensional (3D) features in the cell and, by comparing the
size and shape pre- and postexpansion, these features can be used
as an intrinsic ruler. In addition, our method is capable of pointing
out sample distortions and can be used as a quality control tool for
expansion microscopy experiments in biological samples.
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quality control tooloriginal spatial distributionconventional fluorescence microscopesexpansion microscopy experiments3d expansion factorexpansion microscopyoriginal sampleintrinsic toolfluorescence photobleachinguse photobleachingthree dimensionsshape presample distortionsroutinely appliedresolution imagingpolymer meshworkphysical distancelateral resolutionlaboratories worldwideintroduce wellintrinsic rulerfirst reportexpandable hydrogeldefined threecorrect representationchemically anchoringbiological samplesbiological sampleachieve super
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