Biophysical Imaging

Observation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. Using lipid analogues tagged with a hybrid fluorescent tag-gold nanoparticle construct, in this work we directly compare results from STED-FCS and iSCAT measurements of phospholipid diffusion in a supported lipid bilayer. Compared to dye-tagged lipid analogues, gold nanoparticle tagging introduced a 50-fold reduction in the diffusion coefficient. Nevertheless, the mode of diffusion remained free, at least on the spatial scales probed (>40nm). These FCS measurements of hybrid fluorescent tag-gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. A direct comparison of STED-FCS and iSCAT revealed a 2-3-fold difference in diffusion coefficients between both approaches, but the relative differences in mobility between different lipid analogues were identical for both. In conclusion, our experiments reveal that large scattering tags introduce a significant slow-down in diffusion but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.