Dr. Anna Kashkanova

Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accurate sizing and quantification of EVs remain problematic, given their nanometre size range and small scattering cross-sections. This is compounded by the fact that common EV isolation methods result in co-isolation of particles with comparable features. Especially in blood plasma, similarly-sized lipoproteins outnumber EVs to a great extent. Recently, interferometric nanoparticle tracking analysis (iNTA) was introduced as a particle analysis method that enables determining the size and refractive index of nanoparticles with high sensitivity and precision. In this work, we apply iNTA to differentiate between EVs and lipoproteins, and compare its performance to conventional nanoparticle tracking analysis (NTA). We show that iNTA can accurately quantify EVs in artificial EV-lipoprotein mixtures and in plasma-derived EV samples of varying complexity. Conventional NTA could not report on EV numbers, as it was not able to distinguish EVs from lipoproteins. iNTA has the potential to become a new standard for label-free EV characterization in suspension.

Bright-field light microscopy and related techniques continue to play a key role in life sciences because they provide a facile and label-free insight into biological specimen. However, lack of three-dimensional imaging and low sensitivity to nanoscopic features hamper their application in high-end quantitative studies. Here, we remedy these shortcomings by employing confocal interferometric scattering (iSCAT) microscopy. We demonstrate the performance of this label-free technique in a selection of case studies in live cells and benchmark our findings against simultaneously acquired fluorescence images. We reveal the nanometric topography of the nuclear envelope, quantify the dynamics of the endoplasmic reticulum, detect single microtubules, and map nanoscopic diffusion of clathrin-coated pits undergoing endocytosis. Furthermore, we introduce the combination of confocal and wide-field iSCAT modalities for simultaneous imaging of cellular structures and high-speed tracking of nanoscopic entities such as single SARS-CoV2 virions. Confocal iSCAT can be readily implemented as an additional contrast mechanism in existing laser scanning microscopes.