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.

We report the fabrication and application of multi-functional hybrid bio-aerogels based on cellulose nanofibers (CNFs) and chitin nanocrystals (CNCs) developed through an environmentally friendly freeze-drying process. Herein, an organic non-solvent approach such as acid hydrolysis was used to extract CNFs and CNCs from corn husks and shrimp shells respectively followed by characterisation using IR spectroscopy and electron microscopy. The usual wire-like and a rare semi-square morphology was detected for the CNFs and CNCs respectively during electron microscopic analysis. Aerogels with two different quantities of CNCs on the CNFs were prepared (AR1 and AR2) and compared with reference to the neat CNF aerogel (neat AR). Interestingly, for AR2, under an electron microscope, a maple seed like morphology was observed compared to the neat AR and AR1. Their ability in water treatment was determined by conducting adsorption experiments using methylene blue and Rhodamine 6G, and it was found that AR2 had an enhanced removal percentage and adsorption capacity. To address their economic viability, we conducted several rounds of studies and evaluated the reusability of AR2. The antibacterial and antioxidant activities of the aerogels were studied using a disc diffusion method against four bacterial species, including S. aureus, E. coli, S. typhimurium and B. cereus, and the best conditions for the determination of minimal inhibitory concentration were carefully discussed. The prepared material having a higher CNC content (AR2) shows multi-functional behaviour and from further studies it is anticipated that they may be produced on an industrial scale for commercial applications in the future.

Chitosan and pectin films were enriched with blackcurrant pomace powder (10 and 20% (w/w)), as bio-based material, to minimize food production losses and to increase the functional properties of produced films aimed at food coatings and wrappers. Water vapor permeability of active films increased up to 25%, moisture content for 27% in pectin-based ones, but water solubility was not significantly modified. Mechanical properties (tensile strength, elongation at break and Young’s modulus) were mainly decreased due to the residual insoluble particles present in blackcurrant waste. FTIR analysis showed no significant changes between the film samples. The degradation temperatures, determined by DSC, were reduced by 18 °C for chitosan-based samples and of 32 °C lower for the pectin-based samples with blackcurrant powder, indicating a disturbance in polymer stability. The antioxidant activity of active films was increased up to 30-fold. Lightness and redness of dry films significantly changed depending on the polymer type. Significant color changes, especially in chitosan film formulations, were observed after exposure to different pH buffers. This effect is further explored in formulations that were used as color change indicators for intelligent biopackaging.