Interrogating Unsupported Lipid Bilayers With Scanning Confocal Single-Molecule Fluorescence Microscopy
D L. Burden, John J. Kasianowicz
We describe the use of scanning confocal fluorescence microscopy to probe the structure and temporal dynamics of unsupported planar lipid bilayer membranes. At high fluorescent label concentration, the shape and stability of the membranes are evaluated with submicron spatial resolution. At low label concentration, we observe individual molecules moving into and out of the confocal laser beam in real time. Lipid diffusion coefficients are measured via fluctuation correlation spectroscopy (FCS) and indicate nominal values of (1.0 0.2) x 10-7 cm2/s homogeneously distributed over the central portion of the bilayer. We also compare the real-time single-molecule fluorescence signatures to simulated photon emission patterns generated by a two-dimensional random-walk model. Interestingly, analysis of the interval between fluorescence events suggests the presence of a diffusion bias that can be explained by a weak optical trapping mechanism. The trap causes labeled lipids to return to the confocal detection region more frequently than pure Einsteinian diffusion predicts.
SPIE proceedings series: Scanning and Force Microscopies for Biomedical Applications II
and Kasianowicz, J.
Interrogating Unsupported Lipid Bilayers With Scanning Confocal Single-Molecule Fluorescence Microscopy, SPIE proceedings series: Scanning and Force Microscopies for Biomedical Applications II
(Accessed February 22, 2024)