Confocal interferometric scattering microscopy reveals 3D nanoscopic structure and dynamics in live cells.

Bright-field light microscopy and related phase-sensitive techniques play an important role in life sciences because they provide facile and label-free insights into biological specimens. However, lack of three-dimensional imaging and low sensitivity to nanoscopic features hamper their application in many high-end quantitative studies. Here, we demonstrate that interferometric scattering (iSCAT) microscopy operated in the confocal mode provides unique label-free solutions for live-cell studies. 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-CoV-2 virions. We benchmark our findings against simultaneously acquired fluorescence images. Confocal iSCAT can be readily implemented as an additional contrast mechanism in existing laser scanning microscopes. The method is ideally suited for live studies on primary cells that face labeling challenges and for very long measurements beyond photobleaching times.

High-Precision Protein-Tracking With Interferometric Scattering Microscopy.

The mobility of proteins and lipids within the cell, sculpted oftentimes by the organization 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 behavior. Interferometric scattering (iSCAT) microscopy is an emerging technique well-suited for visualizing the diffusion of gold nanoparticle-labeled 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 visualizing the mobility of the epidermal growth factor receptor in various biological scenarios on the live cell.