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

Area of Science:

• Membrane biophysics
• Advanced microscopy techniques
• Nanoparticle applications

Background:

• High-resolution microscopy techniques like interferometric scattering (iSCAT) and super-resolution STED fluorescence correlation spectroscopy (STED-FCS) offer insights into membrane molecular organization.
• These methods have distinct requirements: STED-FCS uses fluorescent probes, while iSCAT requires larger gold nanoparticle tags.
• Direct comparison of these techniques is crucial for understanding their limitations and potential.

Purpose of the Study:

• To directly compare STED-FCS and iSCAT measurements of phospholipid diffusion on supported lipid bilayers (SLBs).
• To investigate the impact of gold nanoparticle size on diffusion measurements.
• To develop a hybrid labeling strategy for complementary use of STED-FCS and iSCAT.

Main Methods:

• Utilized lipid analogues with hybrid fluorescent tag-gold nanoparticle constructs.
• Performed comparative diffusion measurements on homogeneous SLBs using both STED-FCS and iSCAT.
• Analyzed diffusion coefficients and relative mobility differences between techniques.

Main Results:

• Phospholipid diffusion mode remained free, but diffusion coefficients were reduced 20-60 fold with 20-40 nm gold nanoparticles compared to dye-tagged analogues.
• STED-FCS revealed free streptavidin in commercially supplied streptavidin-coated gold nanoparticles.
• Apparent diffusion coefficients differed by 2-3 fold between STED-FCS and iSCAT, yet relative mobility differences were consistent.

Conclusions:

• Large scattering tags significantly slow diffusion on SLBs without introducing bias.
• The hybrid labeling approach allows for the exploitation of complementary information from STED-FCS and iSCAT.
• This work provides a method for more comprehensive membrane studies using combined advanced microscopy techniques.