Reza Gholami M.Sc.

PiSCAT: A Python Package for Interferometric Scattering Microscopy

Houman Mirzaalian Dastjerdi, Reza Gholami Mahmoodabadi, Matthias Bär, Vahid Sandoghdar, Harald Köstler

Interferometric scattering (iSCAT) microscopy allows one to image and track nano-objects with a nanometer spatial and microsecond temporal resolution over arbitrarily long measurement times (Lindfors et al., 2004; Taylor & Sandoghdar, 2019b, 2019a). A key advantage of this technique over the well-established fluorescence methods is the indefinite photostability of the scattering phenomenon in contrast to the photobleaching of fluorophores. This means that one can perform very long measurements. Moreover, scattering processes are linear and thus do not saturate. This leads to larger signals than is possible from a single fluorophore. As a result, one can image at a much faster rate than in fluorescence microscopy. Furthermore, the higher signal makes it possible to localize a nano-object with much better spatial precision.<br>The remarkable sensitivity of iSCAT, however, also brings about the drawback that one obtains a rich speckle-like background from other nano-objects in the field of view.

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 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.

Differential diffusional properties in loose and tight docking prior to membrane fusion

Agata Witkowska, Susann Spindler, Reza Gholami Mahmoodabadi, Vahid Sandoghdar, Reinhard Jahn

Fusion of biological membranes, although mediated by divergent proteins, is believed to follow a common pathway. It proceeds through distinct steps including docking, merger of proximal leaflets (stalk formation), and formation of a fusion pore. However, the structure of these intermediates is difficult to study due to their short lifetime. Previously, we observed a loosely and tightly docked state preceding leaflet merger using arresting point mutations in SNARE proteins, but the nature of these states remained elusive. Here we used interferometric scattering (iSCAT) microscopy to monitor diffusion of single vesicles across the surface of giant unilamellar vesicles (GUVs). We observed that the diffusion coefficients of arrested vesicles decreased during progression through the intermediate states. Modeling allowed for predicting the number of tethering SNARE complexes upon loose docking and the size of the interacting membrane patches upon tight docking. These results shed new light on the nature of membrane-membrane interactions immediately before fusion.

High-precision protein-tracking with interferometric scattering microscopy

Richard W. Taylor, Cornelia Holler, Reza Gholami Mahmoodabadi, Michelle Küppers, Houman Mirzaalian Dastjerdi, Vasily Zaburdaev, Alexandra Schambony, Vahid Sandoghdar

The mobility of proteins and lipids within the cell, sculpted oftentimes by the organisation of the membrane, reveals a great wealth of information on the function and interaction of these molecules as well as the membrane itself. Single particle tracking has proven to be a vital tool to study the mobility of individual molecules and unravel details of their behaviour. Interferometric scattering (iSCAT) microscopy is an emerging technique well suited for visualising the diffusion of gold nanoparticle-labelled membrane proteins to a spatial and temporal resolution beyond the means of traditional fluorescent labels. We discuss the applicability of interferometric single particle tracking (iSPT) microscopy to investigate the minutia in the motion of a protein through measurements visualising the mobility of the epidermal growth factor receptor in various biological scenarios on the live cell.

Point spread function in interferometric scattering microscopy (iSCAT). Part I: aberrations in defocusing and axial localization

Reza Gholami Mahmoodabadi, Richard W. Taylor, Martin Kaller, Susann Spindler, Mahdi Mazaheri, Kiarash Kasaian, Vahid Sandoghdar

Interferometric scattering (iSCAT) microscopy is an emerging label-free technique optimized for the sensitive detection of nano-matter. Previous iSCAT studies have approximated the point spread function in iSCAT by a Gaussian intensity distribution. However, recent efforts to track the mobility of nanoparticles in challenging speckle environments and over extended axial ranges has necessitated a quantitative description of the interferometric point spread function (iPSF). We present a robust vectorial diffraction model for the iPSF in tandem with experimental measurements and rigorous FDTD simulations. We examine the iPSF under various imaging scenarios to understand how aberrations due to the experimental configuration encode information about the nanoparticle. We show that the lateral shape of the iPSF can be used to achieve nanometric three-dimensional localization over an extended axial range on the order of 10 µm either by means of a fit to an analytical model or calibration-free unsupervised machine learning. Our results have immediate implications for three-dimensional single particle tracking in complex scattering media.

Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane

Richard W. Taylor, Reza Gholami Mahmoodabadi, Verena Rauschenberger, Andreas Giessl, Alexandra Schambony, Vahid Sandoghdar

Much of the biological functions of a cell are dictated by the intricate motion of proteins within its membrane over a spatial range of nanometers to tens of micrometers and time intervals of microseconds to minutes. While this rich parameter space is not accessible to fluorescence microscopy, it can be within reach of interferometric scattering (iSCAT) particle tracking. Being sensitive even to single unlabeled proteins, however, iSCAT is easily accompanied by a large speckle-like background, which poses a substantial challenge for its application to cellular imaging. Here, we show that these difficulties can be overcome and demonstrate tracking of transmembrane epidermal growth factor receptors (EGFR) with nanometer precision in all three dimensions at up to microsecond speeds and tens of minutes duration. We provide unprecedented examples of nanoscale motion and confinement in ubiquitous processes such as diffusion in the plasma membrane, transport on filopodia, and endocytosis.

High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes

Susann Spindler, Jeremias Sibold, Reza Gholami Mahmoodabadi, Claudia Steinem, Vahid Sandoghdar

Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoylsn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (l,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-l-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 mu m is found to be free. In the case of functionalization with gold and 6-mercapto-l-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.