Interrogating unsupported lipid bilayers with scanning confocal single-molecule fluorescence microscopy

Daniel L. Burden; John J. Kasianowicz

doi:10.1117/12.383357

21 April 2000 Interrogating unsupported lipid bilayers with scanning confocal single-molecule fluorescence microscopy

Daniel L. Burden, John J. Kasianowicz

Proceedings Volume 3922, Scanning and Force Microscopies for Biomedical Applications II; (2000) https://doi.org/10.1117/12.383357
Event: BiOS 2000 The International Symposium on Biomedical Optics, 2000, San Jose, CA, United States

Abstract

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 (0.1 +/- 0.2) X 10^-7 cm²/sec 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 2D 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.

Citation Download Citation

Daniel L. Burden and John J. Kasianowicz "Interrogating unsupported lipid bilayers with scanning confocal single-molecule fluorescence microscopy", Proc. SPIE 3922, Scanning and Force Microscopies for Biomedical Applications II, (21 April 2000); https://doi.org/10.1117/12.383357

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KEYWORDS

Diffusion

Confocal microscopy

Molecules

Luminescence

Computer simulations

Microscopy

3D modeling

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