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

Abstract

Extracellular vesicles (EVs) are nanometric membranous structures secreted from almost every 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 visualisation 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 challenges in the imaging of EVs, both in their natural environment as biofluid constituents and in a saline solution after EV isolation. In addition, we discuss approaches for EV imaging and identify the potential benefits and disadvantages when different AFM and EM methods are applied, including numerous factors that influence the morphological characterisation, standardisation, or formation of artefacts. We also demonstrate the effects of some of these factors by using cerebrospinal fluid as an example of human biofluid with a simpler composition. Here presented comparison of approaches to EV imaging should help to estimate the current state in morphology research of EVs from human biofluids and to identify the most efficient pathways towards the standardisation of sample preparation and microscopy modes.

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin

“…Generally, EVs detected by TEM are identified as particles with a cup‐shaped morphology, even though EVs are spherical structures. This cup‐shape is an artefact from TEM sample preparation, caused by sample dehydration (Jung & Mun, 2018 ; Malenica et al., 2021 ). Sample preparation can highly affect the structure and visualisation of particles by TEM (Malenica et al., 2021 ).…”

“…This cup‐shape is an artefact from TEM sample preparation, caused by sample dehydration (Jung & Mun, 2018 ; Malenica et al., 2021 ). Sample preparation can highly affect the structure and visualisation of particles by TEM (Malenica et al., 2021 ). For example, the different buffers in which the particles are suspended in, can affect the negative stain and make the particles appear more or less dark or have different morphology, leading to different visualisation of the same particles (Scarff et al., 2018 ).…”

“…This could be the cause for the dark appearance of the exoEasy samples, which has also been identified in other studies (Gutiérrez García et al., 2020 ; Lipps et al., 2019 ; Stranska et al., 2018 ). In addition, not all EVs will present a cup‐shaped morphology in negative stain TEM, making it difficult to distinguish EVs from lipoprotein (Malenica et al., 2021 ). Negative stain TEM gives a good characterisation of the sample; however, to properly be able to distinguish between EVs and lipoproteins we recommend cryo‐TEM, in which the double membranes of EVs and the single membranes of lipoproteins can be identified, or immunogold negative stain‐TEM in which EVs are labelled for EV enriched markers (Malenica et al., 2021 ).…”

“…In addition, not all EVs will present a cup‐shaped morphology in negative stain TEM, making it difficult to distinguish EVs from lipoprotein (Malenica et al., 2021 ). Negative stain TEM gives a good characterisation of the sample; however, to properly be able to distinguish between EVs and lipoproteins we recommend cryo‐TEM, in which the double membranes of EVs and the single membranes of lipoproteins can be identified, or immunogold negative stain‐TEM in which EVs are labelled for EV enriched markers (Malenica et al., 2021 ). Lastly, determining the size distribution of your particles using negative stain TEM and NTA both have their drawbacks, as negative stain TEM shrinks vesicles upon dehydration and NTA is not able to accurately detect particles in biological samples smaller than 50 nm (Dragovic et al., 2011 ).…”

Extracellular Vesicles and Their Relationship with the Heart–Kidney Axis, Uremia and Peritoneal Dialysis

“…This method consists of an electron beam which passes through an ultra-thin sample, and the scattered electrons are detected by the analyzers forming an image of the EVs. [153,154]. Compounds such as osmium tetroxide can be used to improve the contrast in TEM [155].…”