Biophysical Imaging

The eukaryotic cell plasma membrane is highly complex, and its organization is critical to multiple biological processes, such as virus infection. Still, the role of membrane heterogeneity remains poorly understood due to the lack of techniques showing both high spatial and temporal resolution. Here, we assess the capacity of MINFLUX (minimal photon fluxes) microscopy to investigate lipid membrane organization quantitatively. We show that MINFLUX imaging combined with exchangeable solvatochromic dyes allows quantifying lipid packing in live cell plasma membranes with nanometre resolution. Further, we use MINFLUX in combination with fluorescent lipid analogues to follow their path over space and time. We explore the time resolution of such single lipid tracking experiments as well as apply it to investigate Gag-induced lipid sorting on HIV virus assembly sites at the plasma membrane. To separate lipids diffusing in the plasma membrane from those located in a virus budding site, we develop an open- source MINFLUX track segmentation and analysis pipeline based on a confocal fluorescent image reference. In conclusion, we highlight the applicability of MINFLUX imaging and tracking to investigate lipid organization and dynamics in live cell membranes.