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.

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 10¹⁰ particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 10⁹ particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

Bacterial contamination of the membranes used during guided bone regeneration directly influences the outcome of this procedure. In this study, we analyzed the early stages of bacterial adhesion on two commercial dense polytetrafluoroethylene (d-PTFE) membranes in order to identify microstructural features that led to different adhesion strengths. The microstructure was investigated by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR). The surface properties were analyzed by atomic force microscopy (AFM), scanning electron microscopy (SEM), and surface free energy (SFE) measurements. Bacterial properties were determined using the microbial adhesion to solvents (MATS) assay, and bacterial surface free energy (SFE) was measured spectrophotometrically. The adhesion of four species of oral bacteria (Streptococcus mutans, Streptococcus oralis, Aggregatibacter actinomycetemcomitas, and Veilonella parvula) was studied on surfaces with or without the artificial saliva coating. The results indicated that the degree of crystallinity (78.6% vs. 34.2%, with average crystallite size 50.54 nm vs. 32.86 nm) is the principal feature promoting the adhesion strength, through lower nanoscale roughness and possibly higher surface stiffness. The spherical crystallites (“warts”), observed on the surface of the highly crystalline sample, were also identified as a contributor. All bacterial species adhered better to a highly crystalline membrane (around 1 log₁₀CFU/mL difference), both with and without artificial saliva coating. Our results show that the changes in polymer microstructure result in different antimicrobial properties even for chemically identical PTFE membranes.

Klebsiella pneumoniae is an emerging multidrug-resistant pathogen that can contaminate hospital surfaces in the form of a biofilm which is hard to remove with standard disinfectants. Because of biofilm resistance to conservative disinfectants, the application of new disinfection technologies is becoming more frequent. Ozone gas has antimicrobial activity but there is lack of data on its action against K. pneumoniae biofilm. The aim of this study was to investigate the effects and mechanisms of action of gaseous ozone on the OXA-48-procuding K. pneumoniae biofilm. A 24 h biofilm of K. pneumoniae formed on ceramic tiles was subsequently exposed to different concentrations of ozone during one and two hours to determine the optimal ozone concentration. Afterwards, the total bacteria count, total biomass and oxidative stress levels were monitored. A total of 25 ppm of gaseous ozone was determined to be optimal ozone concentration and caused reduction in total bacteria number in all strains of K. pneumoniae for 2.0 log₁₀ CFU/cm², followed by reduction in total biomass up to 88.15%. Reactive oxygen species levels significantly increased after the ozone treatment at 182% for the representative K. pneumoniae NCTC 13442 strain. Ozone gas in the concentration of 25 ppm caused significant biofilm reduction but did not completely eradicate the K. pneumoniae biofilm formed on ceramics. In conclusion, ozone gas has great potential to be used as an additional hygiene measure in joint combat against biofilm in hospital environments.