Abstract
Observation techniques with high spatial and temporal resolution, such as single-particle tracking 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. In this work, we have used lipid analogues tagged with a hybrid fluorescent tag–gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous supported lipid bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 ⩽ t ⩽ 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye tagged lipid analogues. 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 and potentially cross-linking 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.
Highlights
The study of cellular membrane dynamics is a subject of intense research in biophysics, due to its relevance for many cellular processes, such as cellular signaling
The aim of this work was to compare the performance of two experimental techniques, STED-fluorescence correlation spectroscopy (FCS) and interferometric scattering (iSCAT)-based single-particle tracking (SPT), and explore the viability of protein-coated gold nanoparticles as tags for SPT by detecting the diffusion characteristics of gold nanoparticle tagged biotinylated lipids on simple, homogeneous model membranes
We conclude that a large influence on the general mobility results from the cross-linking of several biotinylated lipids by the gold nanoparticle tags
Summary
The study of cellular membrane dynamics is a subject of intense research in biophysics, due to its relevance for many cellular processes, such as cellular signaling Important questions in this field revolve around the functionality of membrane-associated protein-protein and protein-lipid interactions, for example, the formation and the function of lipid nanodomains or ‘rafts’ [1]. It is possible to overcome this limitation either by extrapolating the results from diffraction-limited FCS recordings to the nanoscale [10], or by combining superresolution STED microscopy with FCS (STED-FCS) [11, 12] The latter provides a direct method for tuning the observation spot size of FCS [12, 13], and allows direct monitoring of molecular dynamics at sub-diffraction length scales (30–200 nm). While SPT experiments have highlighted important features of molecular motions, they are ultimatively limited by issues such as low signal-to-noise ratios, low temporal resolution and photobleaching, which in turn leads to limited recording times and short trajectories
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