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    Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells

    Year: 2021

    Journal: Small Methods, Volume 5, SEP

    Authors: Lorizate, Maier; Terrones, Oihana; Nieto-Garai, Jon Ander; Rojo-Bartolome, Iratxe; Ciceri, Dalila; Morana, Ornella; Olazar-Intxausti, June; Arboleya, Aroa; Martin, Alexia; Szynkiewicz, Marta; Calleja-Felipe, Maria; Bernardino de la Serna, Jorge; Contreras, F. -Xabier

    Organizations: Spanish Ministry of Science Innovation and Universities [BFU-2015-68981-P]; Basque Government [IT1264-19]; Bill and Melinda Gates Foundation [INV-016631]; BBSRC [BB/V019791/1]; Fundacion Biofisica Bizkaia (FBB); Basque Excellence Research Centre (BERC) program of the Basque Government; FI predoctoral fellowship from the Basque Government; FBB

    Keywords: bioorthogonal reactions; cholesterol; lipid raft; membranes; nanoprobes; nanoscale lipid heterogeneity; super-resolution microscopy

    Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N-3) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N-3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N-3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.
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