Premium
Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study
Author(s) -
Guo Lin,
Har Jia Yi,
Sankaran Jagadish,
Hong Yimian,
Kannan Balakrishnan,
Wohland Thorsten
Publication year - 2008
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.200700611
Subject(s) - fluorescence correlation spectroscopy , total internal reflection fluorescence microscope , diffusion , chemistry , fluorescence recovery after photobleaching , lipid bilayer , calibration , analytical chemistry (journal) , vesicle , membrane , molecular diffusion , photobleaching , biological membrane , fluorescence , molecule , chromatography , optics , physics , thermodynamics , biochemistry , quantum mechanics , metric (unit) , operations management , organic chemistry , economics
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.