Biology subjects

Gholami Mahmoodabadi, R.

Publications and source records attributed to Gholami Mahmoodabadi, R..

4 recordsLinked to original sources

bioRxiv2021

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

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[§].

biophysics↗

bioRxiv2020

High-precision protein-tracking with interferometric scattering microscopy

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.

biophysics↗

bioRxiv2020

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

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.

biophysics↗

bioRxiv2018

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

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.

biophysics↗