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EVAnalyzer citation: Schürz, M., Danmayr, J., Jaritsch, M., Klinglmayr, E., Benirschke, H. M., Matea, C. -. T., Zimmerebner, P., Rauter, J., Wolf, M., Gomes, F. G., Kratochvil, Z., Heger, Z., Miller, A., Heuser, T., Stanojlovic, V., Kiefer, J., Plank, T., Johnson, L., Himly, M., … Meisner-Kober, N. (2022). EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin. Journal of Extracellular Vesicles, 11, e12282. https://doi.org/10.1002/jev2.12282
@ARTICLE{Schurz2022-kh,
title = "{EVAnalyzer}: High content imaging for rigorous characterisation
of single extracellular vesicles using standard laboratory
equipment and a new open-source {ImageJ/Fiji} plugin",
author = "Sch{\"u}rz, Melanie and Danmayr, Joachim and Jaritsch, Maria and
Klinglmayr, Eva and Benirschke, Heloisa Melo and Matea,
Cristian-Tudor and Zimmerebner, Patrick and Rauter, Jakob and
Wolf, Martin and Gomes, Fausto Gueths and Kratochvil, Zdenek and
Heger, Zbynek and Miller, Andrew and Heuser, Thomas and
Stanojlovic, Vesna and Kiefer, Jana and Plank, Tanja and Johnson,
Litty and Himly, Martin and Bl{\"o}chl, Constantin and Huber,
Christian G and Hintersteiner, Martin and Meisner-Kober, Nicole",
abstract = "Extracellular vesicle (EV) research increasingly demands for
quantitative characterisation at the single vesicle level to
address heterogeneity and complexity of EV subpopulations.
Emerging, commercialised technologies for single EV analysis
based on, for example, imaging flow cytometry or imaging after
capture on chips generally require dedicated instrumentation and
proprietary software not readily accessible to every lab. This
limits their implementation for routine EV characterisation in
the rapidly growing EV field. We and others have shown that
single vesicles can be detected as light diffraction limited
fluorescent spots using standard confocal and widefield
fluorescence microscopes. Advancing this simple strategy into a
process for routine EV quantitation, we developed 'EVAnalyzer',
an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated,
quantitative single vesicle analysis from imaging data. Using
EVAnalyzer, we established a robust protocol for capture,
(immuno-)labelling and fluorescent imaging of EVs. To exemplify
the application scope, the process was optimised and
systematically tested for (i) quantification of EV
subpopulations, (ii) validation of EV labelling reagents, (iii)
in situ determination of antibody specificity, sensitivity and
species cross-reactivity for EV markers and (iv) optimisation of
genetic EV engineering. Additionally, we show that the process
can be applied to synthetic nanoparticles, allowing to determine
siRNA encapsulation efficiencies of lipid-based nanoparticles
(LNPs) and protein loading of SiO(2) nanoparticles. EVAnalyzer
further provides a pipeline for automated quantification of cell
uptake at the single cell-single vesicle level, thereby enabling
high content EV cell uptake assays and plate-based screens.
Notably, the entire procedure from sample preparation to the
final data output is entirely based on standard reagents,
materials, laboratory equipment and open access software. In
summary, we show that EVAnalyzer enables rigorous
characterisation of EVs with generally accessible tools. Since we
further provide the plugin as open-source code, we expect
EVAnalyzer to not only be a resource of immediate impact, but an
open innovation platform for the EV and nanoparticle research
communities.",
journal = "J Extracell Vesicles",
volume = 11,
number = 12,
pages = "e12282",
month = dec,
year = 2022,
address = "United States",
keywords = "EV immunolabelling; cell uptake; exosomes; extracellular
vesicles; lipid nanoparticles; liposomes; open innovation; silica
nanoparticles; single particle imaging; single vesicle imaging",
language = "en"
}