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Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study
Authors:Lin Guo  Jia Yi Har  Jagadish Sankaran  Yimian Hong  Balakrishnan Kannan Dr  Thorsten Wohland Dr
Institution:1. Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore), Fax: (+65)?65161248;2. These two authors contributed equally to this work.;3. Singapore–MIT Alliance, E4‐04‐10, 4 Engineering Drive 3, Singapore 117576 (Singapore)
Abstract:Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single‐particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 μm?2) and very low surface concentrations (single‐molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well‐suited for the intermediate concentration range of about 0.1–100 μm?2. However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z‐scan FCS, is calibration‐free for membrane measurements, but requires several experiments at different well‐controlled focusing positions. A recently established FCS method, electron‐multiplying charge‐coupled‐device‐based total internal reflection FCS (TIR‐FCS), referred to here as imaging TIR‐FCS (ITIR–FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR–FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.
Keywords:bilayers  diffusion coefficients  fluorescence spectroscopy  lipids  membranes  vesicles
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