Dr. Anna Kashkanova

Label-free discrimination of extracellular vesicles from large lipoproteins

Anna D. Kashkanova, Martin Blessing, Marie Reischke, Jan-Ole Baur, Andreas S. Baur, Vahid Sandoghdar, Jan Van Deun

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.

Confocal Interferometric Scattering Microscopy Reveals 3D Nanoscopic Structure and Dynamics in Live Cells

Michelle Küppers, David Albrecht, Anna D. Kashkanova, Jennifer Lühr, Vahid Sandoghdar

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.

Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions

Anna D. Kashkanova, Martin Blessing, André Gemeinhardt, Didier Soulat, Vahid Sandoghdar

Characterization of the size and material properties of particles in liquid suspensions is in very high demand, for example, in the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, existing methods are limited in their ability to decipher the constituents of realistic samples. Here we introduce iNTA as a new method that combines interferometric detection of scattering with nanoparticle tracking analysis to reach unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multicomponent and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

Precision single-particle localization using radial variance transform

Anna D. Kashkanova, Alexey Shkarin, Reza Gholami Mahmoodabadi, Martin Blessing, Yazgan Tuna, André Gemeinhardt, Vahid Sandoghdar

We introduce an image transform designed to highlight features with high degree of radial symmetry for identification and subpixel localization of particles in microscopy images. The transform is based on analyzing pixel value variations in radial and angular directions. We compare the subpixel localization performance of this algorithm to other common methods based on radial or mirror symmetry (such as fast radial symmetry transform, orientation alignment transform, XCorr, and quadrant interpolation), using both synthetic and experimentally obtained data. We find that in all cases it achieves the same or lower localization error, frequently reaching the theoretical limit.