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

Abstract

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.

Label-Free Imaging of DNA Interactions with 2D Materials

“…33−36 In this work we harness interferometric scattering (iSCAT) microscopy 37,38 molecular weights of 10 kDa, 40,41 and the products of other biomolecular reactions 42 as well as the dynamics of more complex cellular processes. 43 To study the effects of surface properties on DNA binding, we study its interactions with pristine hBN as well as engineered defects and nanostructures. 44 ■…”

“…Liberated from the limited photon budget of fluorescent dyes, the technique allows millisecond temporal resolution and theoretically indefinite imaging times. So far, iSCAT has been used to study the mass distribution of short single-stranded and double-stranded DNA, protein oligomers down to molecular weights of 10 kDa, , and the products of other biomolecular reactions as well as the dynamics of more complex cellular processes . To study the effects of surface properties on DNA binding, we study its interactions with pristine hBN as well as engineered defects and nanostructures …”

Scanning ion conductance microscopy revealed cisplatin-induced morphological changes related to apoptosis in single adenocarcinoma cells

“…10 Most of the light microscopy methods cannot provide three-dimensional (3D) topographical details, and studying small portions of the cell membrane at high resolution is not feasible. 11,12 White light interferometry is a label-free optical technique with high axial resolution that uses coherent light waves to generate a 3D prole of a surface. 13 However, imaging the complexity of cell surfaces such as microvilli is beyond the capability of this technique, and its potential applications in microbiology measurements remain unexplored.…”

Dual-Angle Interferometric Scattering Microscopy for Optical Multiparametric Particle Characterization

“…Moreover, the DAISY radius shows a negligible dependence on the refractive index of the surrounding media and is complementary to the hydrodynamic radius, allowing the combination of the DAISY and hydrodynamic radii to provide particle morphology estimates. Thus, DAISY opens up for multiparametric analysis for both suspended particles and of nanoparticles in biological environments, extending what is possible using holography and iSCAT as separate techniques. , Note also that current precision in size estimation is limited by statistical uncertainty from having an average track length of around 50–100 frames, where a higher frame rate will significantly improve the precision of the particle sizing. Moreover, kHz imaging would also enable analysis of nonrotational asymmetric particles based on the fluctuations in the measured scattering signal, which could be used to further the shape analysis of DAISY.…”