Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

Abstract

Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins

“…their mass, allowing the characterization of mass distributions of macromolecules in solution (Young et al, 2018;Hundt, 2021). Furthermore, the quantification of complex assembly formation (Häußermann et al, 2019;Soltermann et al, 2020;Wu and Piszczek, 2020) and the characterization of membrane proteins using different solubilization approaches has successfully been applied (Olerinyova et al, 2020;Heermann et al, 2021;Steiert et al, 2022).…”

“…The technique provides a batch average of spherical modelled particles in suspension, and only those populations for which hydrodynamic radii differ by a factor of 3 are efficiently resolved. Finally, MP linearly correlates the interference of the scattered light of single particles landing on the measuring surface with their mass, allowing the characterization of mass distributions of macromolecules in solution ( Young et al, 2018 ; Hundt, 2021 ). Furthermore, the quantification of complex assembly formation ( Häußermann et al, 2019 ; Soltermann et al, 2020 ; Wu and Piszczek, 2020 ) and the characterization of membrane proteins using different solubilization approaches has successfully been applied ( Olerinyova et al, 2020 ; Heermann et al, 2021 ; Steiert et al, 2022 ).…”

Biochemistry: one molecule at a time

“…single-ion channel recordings using patch-clamp [1], the single-molecule toolkit has expanded considerably and continues to evolve. Single-molecule detection and manipulation techniques can broadly be divided into the following categories: (i) light microscopy approaches, including fluorescence imaging and spectroscopy [2][3][4][5][6][7], and interferometric scattering [8], (ii) electrical conductance measurements, including patch-clamp and nanopore-based detection [9,10], and (iii) force-based approaches, including optical [11] and magnetic tweezers [12], and atomic force microscopy [13]. Different single-molecule methods have different capabilities, applications and throughput, and offer different time and spatial resolutions.…”