Revealing compartmentalised membrane diffusion in living cells with interferometric scattering microscopy

Abstract

Abstract:Single-particle tracking is a powerful tool for studying single molecule behaviour involving plasma membrane-associated events in cells. Here, we show that interferometric scattering microscopy (iSCAT) combined with gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons. The unique combination of microsecond temporal resolution and nanometer spatial precision reveals signatures of a compartmentalised plasma membrane in neurons.. CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/091736 doi: bioRxiv preprint first posted online Dec. 5, 2016;Single-particle tracking (SPT) generally involves tagging a single molecule with a probe, and detecting its position in a series of images, using computational image processing to achieve sub-pixel localisation precision.1 These trajectories contain information on the diffusion coefficient of the molecule, its momentum and possible underlying forces controlling its motion. [2][3][4][5] Furthermore, the trajectory can be correlated to features of the cell or the motion of interacting partners, providing a means to analyse dynamic changes in the behaviour of single molecules. While single particle tracking was first established using gold beads 6 , current implementations also make use of quantum dots (QDs) or single fluorescent dyes as probes, which have lead to the development of high-density methods. 7-9The enormous advantage of fluorescence emission as a contrast mechanism in terms of maximal background suppression is somewhat offset by restrictions in terms of the achievable simultaneous imaging speed and precision. Scattering labels, such as gold nanoparticles, are not subject to the photophysical and photochemical limitations of fluorescent dyes and can thus in principle achieve much higher spatiotemporal precision. Gold particles have been used extensively in the past for tracking fast nanoscale dynamics, 10 including SPT in living cells with up to 20 µs temporal resolution and about 15 nm spatial precision revealing signatures of hop diffusion. 11More recently, interferometric scattering microscopy (iSCAT) 12 has demonstrated even higher spatiotemporal capabilities down to few nm precision 13 and a few µs temporal resolution in vitro. 14 Here, we use gold nanoparticles (AuNPs) coupled to GFP-tagged or YFP-tagged membrane proteins to demonstrate iSCAT-based SPT of membrane proteins with nanometer precision at kHz speeds in live epithelial cells and cultured hippocampal neurons. We use this approach to study if the increase in spatiotemporal precision allows for the detection of anomalous diffusion at the nanoscale. Such effects could arise from interactions between the . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (whic...