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

Abstract

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-Throughput, High-Resolution Interferometric Light Microscopy of Biological Nanoparticles

“…The speckle pattern produces contrast variations in the background signal comparable to the interferometric signal. 15 The speckle noise can be reduced by using computational algorithms and optical instruments which are typically expensive and complex. 20 Therefore, SPIR microscopy employs LED as the light-source which is a simple and low-cost solution for the interferometric detection of nanoparticles.…”

“…Recent advances in optical microscopy enable highly-sensitive direct imaging of BNPs by various contrast enhancement methods. [15][16][17][18][19][20][21] Wide-field interferometric imaging techniques enhance the weak scattering signal by interfering the signal with a strong reference field and have demonstrated the label-free detection of very small BNPs such as exosomes, 19 viruses, 22,23 and synthetic nanoparticles. 21,[24][25][26] Furthermore, Ünlü's group previously demonstrated the detection of viruses in complex media including cell media solution 27 and serum.…”

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

“…The size of the LUVs was estimated from the observed iSCAT contrast to be about 90 nm (see Figure 1c). Thus, an LUV appears as the iSCAT point spread function (PSF) of microscope, which consists of few lobes encoding the 3D position of the scatterer (24). This PSF could be fitted with a physical model to extract the third dimension (25), however here we fitted the main lobe for the lateral localization and used the geometry of the GUVs to extract the axial position of the vesicles (Figure 1b).…”

Toward High‐Speed Nanoscopic Particle Tracking via Time‐Resolved Detection of Directional Scattering

“…In the past, multiple approaches for the localization of single nanoparticles or multi-particle structures have emerged from the growing interest in ultra-precise positioning techniques for nanometrological applications. [1] These include scanning probe microscopy, which always involves a probe tip placed close to the sample, [2] but also all-optical solutions that aim to go beyond the diffraction limit, such as fluorescence microscopy approaches, [3][4][5][6][7] camera-based interferometric detection of light scattered by a particle, utilizing the diffraction-limited optical image of a point-like emitter, [8][9][10][11] photodetector-based localization of a particle in an optical trap [12,13] or via detection of the total intensity of scattered light with two separate detectors. [14] DOI: 10.1002/lpor.202000110…”