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.

Hot spots are defined as nanostructures of noble metal able to locally enhance the electromagnetic field of several orders of magnitude and to confine this effect to a region for several orders of magnitude smaller than the light wavelength. Hot spots are particularly important for the surface enhanced Raman spectroscopy applications, in which the field enhancement is used to amplify the usually weak Raman scattering signal. The hot spots are mostly generated between two or more plasmonic nanostructures separated by nanometric gaps. Several strategies are used to design and realize the hot spots, both in solution, using the noble metal nanoparticles, and on surfaces, using nanolithography and evaporation. In this paper, we demonstrated the fabrication of a nanomechanical plasmonic device for Raman spectroscopy, in which the hot spots are switched on when biased at the resonant frequency and switched off when the actuation signal is removed.