RT Journal Article
SR Electronic
T1 Complementary studies of lipid membrane dynamics using iSCAT and super-resolved Fluorescence Correlation Spectroscopy
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 235564
DO 10.1101/235564
A1 Francesco Reina
A1 Silvia Galiani
A1 Dilip Shrestha
A1 Erdinc Sezgin
A1 Gabrielle de Wit
A1 Daniel Cole
A1 B.Christoffer Lagerholm
A1 Philip Kukura
A1 Christian Eggeling
YR 2018
UL http://biorxiv.org/content/early/2018/03/08/235564.abstract
AB 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 compared results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). Compared to dye-tagged lipid analogues, tagging with 20 to 40-nm large gold nanoparticles introduced a 20- to 60-fold reduction in the diffusion coefficient, depending on nanoparticle size. 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. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.