We also discuss some general aspects of sample preparation protocols, but also point to certain specificities depending on the applied microscopy mode. Next, we describe imaging options for different electron and atomic force microscopy modes and describe the main challenges of these methods when imaging EVs present in cerebrospinal fluid (CSF) or isolated by size-exclusion chromatography (SEC). Thus, we first provide description of general properties of EVs followed by an overview of EV isolation methods. Both the choice and performance of microscopy method on one side and EV form on the other side, including whether a biofluid or a purified sample is used as an EV source, can significantly impact visualisation of EVs. 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.Īdvanced and optimised microscopy methods are required to visualise and characterise morphology of extracellular vesicles (EVs), a heterogenous groups of nanoparticles (NPs) secreted by cells and regarded as highly promising source of diagnostic, prognostic, and therapeutic tools. We also demonstrate the effects of some of these factors by using cerebrospinal fluid as an example of human biofluid with a simpler composition. 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. 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. 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). Because EV composition reflects the state of its parental tissue, EVs possess an enormous diagnostic/prognostic potential to reveal pathophysiological conditions. Extracellular vesicles (EVs) are nanometric membranous structures secreted from almost every cell and present in biofluids.
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