André Gemeinhardt M.Sc.

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.

Optimized analysis for sensitive detection and analysis of single proteins via interferometric scattering microscopy

Houman Mirzaalian Dastjerdi, Mahyar Dahmardeh, André Gemeinhardt, Reza Gholami Mahmoodabadi, Harald Köstler, Vahid Sandoghdar

It has been shown that interferometric detection of Rayleigh scattering (iSCAT) can reach an exquisite sensitivity for label-free detection of nano-matter, down to single proteins. The sensitivity of iSCAT detection is intrinsically limited by shot noise, which can be indefinitely improved by employing higher illumination power or longer integration times. In practice, however, a large speckle-like background and technical issues in the experimental setup limit the attainable signal-to-noise ratio. Strategies and algorithms in data analysis are, thus, crucial for extracting quantitative results from weak signals, e.g. regarding the mass (size) of the detected nano-objects or their positions. In this article, we elaborate on some algorithms for processing iSCAT data and identify some key technical as well as conceptual issues that have to be considered when recording and interpreting the data. The discussed methods and analyses are made available in the extensive python-based platform, PiSCAT.

Precision single-particle localization using radial variance transform

Anna 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.

Label-Free Imaging of Single Proteins Secreted from Living Cells via ISCAT Microscopy

André Gemeinhardt, Matthew Paul McDonald, Katharina König, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

Visualizing single-cell secretion dynamics with single protein sensitivity

Matthew Paul McDonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Völkl, Andreas Mackensen, Vahid Sandoghdar

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.