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Complementary studies of lipid membrane dynamics using iSCAT and super-resolved fluorescence correlation spectroscopy
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.
Magnetooptical response of permalloy multilayer structures on different substrate in the IR-VIS-UV spectral range
The magnetooptical (MO) response of Ru/Py/Ta thin film stacks with 4, 8, and 17 nm thick Ni81Fe19 permalloy (Py) films on a SiO2/Si and a ZnO substrate was measured by vector magnetooptical generalized ellipsometry. The MO response from VMOGE was modelled using a 4 × 4 Mueller matrix algorithm. The wavelength-dependent, substrate-independent and thickness-independent complex MO coupling constant (Q) of Py in the Ru/Py/Ta thin film stacks was extracted by fitting Mueller matrix difference spectra in the spectral range from 300 nm to 1000 nm. Although the composition-dependent saturation magnetization of NixFe1−x alloys (x = 0.0...1.0), e.g. of Ni81Fe19, is predictable from the two saturation magnetization end points, the MO coupling constant of NixFe1−x is not predictable from the two Q end points. However, in a small alloy range (0.0 < x < 0.2 and 0.8 < x < 1.0) the composition-dependent Q of NixFe1−x can be interpolated from a sufficiently high number of analyzed NixFe1−x alloys. The available complex MO coupling constants of six different NixFe1−x (x = 1.0 to 0.0) alloys were used to interpolate MO response of binary NixFe1−x alloys in the range from x = 0.0 to x = 1.0.