Circle scanning STED fluorescence correlation spectroscopy to quantify membrane dynamics and compartmentalization

Methods ◽  
2018 ◽  
Vol 140-141 ◽  
pp. 188-197 ◽  
Author(s):  
Riccardo Maraspini ◽  
Oliver Beutel ◽  
Alf Honigmann
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

scholarly journals Spatial-Temporal Studies of Membrane Dynamics: Scanning Fluorescence Correlation Spectroscopy (SFCS)

2004 ◽  
Vol 87 (2) ◽  
pp. 1260-1267 ◽  
Author(s):  
Qiaoqiao Ruan ◽  
Melanie A. Cheng ◽  
Moshe Levi ◽  
Enrico Gratton ◽  
William W. Mantulin
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

scholarly journals Complementary studies of lipid membrane dynamics using iSCAT and super-resolved Fluorescence Correlation Spectroscopy

2017 ◽  
Author(s):  
Francesco Reina ◽  
Silvia Galiani ◽  
Dilip Shrestha ◽  
Erdinc Sezgin ◽  
Gabrielle de Wit ◽  
...  
Keyword(s):  
Gold Nanoparticle ◽  
Fluorescence Correlation Spectroscopy ◽  
Membrane Dynamics ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Molecular Organization ◽  
Complementary Information ◽  
Fluorescence Correlation ◽  
Apparent Diffusion Coefficients ◽  
Fluorescent Tag

AbstractObservation 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. 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 crosslinking 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.

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

2018 ◽  
Vol 51 (23) ◽  
pp. 235401 ◽  
Author(s):  
Francesco Reina ◽  
Silvia Galiani ◽  
Dilip Shrestha ◽  
Erdinc Sezgin ◽  
Gabrielle de Wit ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Lipid Membrane ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation
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