Complementary studies of lipid membrane dynamics using iSCAT and super-resolved fluorescence correlation spectroscopy

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.

Interferometric Scattering Microscopy: Seeing Single Nanoparticles and Molecules via Rayleigh Scattering

“…40 Here, we emphasize that it is challenging to carry out quantitative comparative studies of different measurement methods because each labeling strategy might introduce a systematic bias, leading to variations in the diffusion coefficients obtained. 41…”

Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

“…In previous work, higher particle densities have indeed been employed to aggregate membrane proteins in a controlled manner, e.g., to elicit receptor signaling 21 , 34 . A further common issue of using nanoparticles for SPT is potential crosslinking of the targeted membrane molecules through multiple binding sites on the nanoparticle 35 , 36 , which could lead to unwanted effects depending on the target molecule. For the live-cell experiments, we addressed this by using a monovalent nanobody at a 1:1 stoichiometry per particle.…”

High-Precision Protein-Tracking With Interferometric Scattering Microscopy

“…A key advantage of iSCAT microscopy is that the high sensitivity of detection permits imaging with excellent signal-tonoise levels even at extremely high speeds, with framerates in the range of 100-1,000,000 frames per second (fps) reported in the investigation of membrane diffusion (Lin et al, 2014;Spindler et al, 2016;Wu et al, 2016;Huang et al, 2017a;de Wit et al, 2018;Reina et al, 2018;. For imaging protein mobility on live cells, high framerates are particularly important in order to observe transient nanoscale molecular interactions which occur at swift milli-to microsecond time scales that may otherwise be not observable at lower framerates (Ritchie et al, 2005).…”

“…In addition, because the localization precision now can approach the few-nm length scale, the influence of the GNP-to-ligand linker becomes of interest, in particular, its length and flexibilty, and how these might affect the accuracy in localization of the associated protein. Many of these questions are not fully answered, but recent studies have investigated these issues of multi-valent labeling and efforts to control it (Reina et al, 2018;Liao et al, 2019), as well as the influence of colloidal probe size (Ritchie et al, 2005;Clausen and Lagerholm, 2011;Etoc et al, 2018). In the case of the latter, the size of the probe may not have significant effects on diffusion within the cell as one would have initially assumed.…”