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

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‐N3) 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‐N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live‐cell super‐resolution microscopy, chol‐N3 demonstrates the existence of cholesterol‐rich nanodomains of