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Perspectives of Microscopy Methods for Morphology Characterisation of Extracellular Vesicles from Human Biofluids

Extracellular vesicles (EVs) are nanometric membranous structures secreted from almostevery cell and present in biofluids. Because EV composition reflects the state of its parental tissue, EVs possess an enormous diagnostic/prognostic potential to reveal pathophysiological conditions.However, a prerequisite for such usage of EVs is their detailed characterisation, including visuali-sation which is mainly achieved by atomic force microscopy (AFM) and electron microscopy (EM).Here we summarise the EV preparation protocols for AFM and EM bringing out the main challengesin the imaging of EVs, both in their natural environment as biofluid constituents and in a salinesolution after EV isolation. In addition, we discuss approaches for EV imaging and identify thepotential benefits and disadvantages when different AFM and EM methods are applied, includingnumerous factors that influence the morphological characterisation, standardisation, or formationof artefacts. We also demonstrate the effects of some of these factors by using cerebrospinal fluidas an example of human biofluid with a simpler composition. Here presented comparison of ap-proaches to EV imaging should help to estimate the current state in morphology research of EVsfrom human biofluids and to identify the most efficient pathways towards the standardisation ofsample preparation and microscopy modes.

extracellular vesicles ; human biofluids ; nanotechnology ; atomic force microscopy ; electron microscopy ; morphology

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