Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging

Abstract

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Anomalous Subdiffusion in Living Cells: Bridging the Gap Between Experiments and Realistic Models Through Collaborative Challenges

“…This has enabled to directly identify deviations from pure Brownian motion in the plasma membrane of cells [41] or anomalous diffusion occurring, either during first order lipid phase transition [42] or in non-homogeneous fluids, gels and crowded solutions [43,44]. It has been recently extended to the line-scanning STED-FCS [45] and to Im-FCS [46].…”

Editorial: ImmunoPhysics and ImmunoEngineering

“…For this purpose, in this special issue, we aimed to provide a broad and interdisciplinary forum for researchers to present their personal views on the field, point to future challenges, and show their latest empirical or theoretical observations or method developments, with biomedical implications in vivo, in vitro, and in silico. Initially, we eagerly sought contributions employing highly advanced static or dynamic quantitative techniques spanning from conventional to super-or ultra-resolved microscopy in space and time to unravel protein clustering [1][2][3][4][5][6][7][8] or to disentangle the involvement of actin cortex dynamics and its mechanics in T-cell activation and synapse formation [9][10][11][12][13][14][15]; we also welcomed contributions applying steady and non-equilibrium quantitative fluorescence spectroscopic approaches to resolve receptor protein oligomerization and lipid-protein interactions and dynamics [16,17] that can be applied to resolve immunerelevant events [18,19]. Investigations employing systems immunology approaches, including quantitative and highthroughput assessment of the immune status and mathematical modeling of immune interactions [20][21][22][23][24][25] were also sought after; finally, we were also keen to include breakthroughs in the design or utilization of engineered tools and methods with applications to T-cell biology or immunotherapy [26][27][28][29][30][31].…”

The ciliary membrane of polarized epithelial cells stems from a midbody remnant-associated membrane patch with condensed nanodomains

“…Thus, by combining STED with RICS (STED-RICS microscopy), we can assess the spatial molecular localization and the lipid lateral mobility at the nanoscale resolution (Hedde, et al, 2013). Based on a recently reported technique in which alternating line scanning was used instead of RICS (Schneider, et al, 2018), we applied the same principle to RICS and simultaneously acquired confocal and STED images on a line-by-line basis to examine lipid nanodomain dynamics at RAMPs (Fig. 2C).…”

“…2C). By resolving the diffusion of the molecules simultaneously in diffraction-limited (D) and super-resolution (D STED ) modes, different mobility modes can be discerned (Schneider et al, 2018). For instance, the ratio between the quantified diffusion values (D STED /D), hereafter referred to as Drat, allows discrimination among free diffusion, hindered or trapped diffusion, and active diffusion.…”