title: On the Equivalence of FCS and FRAP: Simultaneous Lipid Membrane Measurements.
abstract: Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are widely used methods to determine diffusion coefficients. However, they often do not yield the same results. With the advent of camera-based imaging FCS, which measures the diffusion coefficient in each pixel of an image, and proper bleaching corrections, it is now possible to measure the diffusion coefficient by FRAP and FCS in the exact same images. We thus performed simultaneous FCS and FRAP measurements on supported lipid bilayers and live cell membranes to test how far the two methods differ in their results and whether the methodological differences, in particular the high bleach intensity in FRAP, the bleach corrections, and the fitting procedures in the two methods explain observed differences. Overall, we find that the FRAP bleach intensity does not measurably influence the diffusion in the samples, but that bleach correction and fitting introduce large uncertainties in FRAP. We confirm our results by simulations.
authors.affiliation: Department of Biological Sciences and Centre for BioImaging Sciences, National University of Singapore, Singapore.
authors.forename: Radek
authors.initials: R
authors.lastname: Macháň
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore.
authors.forename: Yong Hwee
authors.initials: YH
authors.lastname: Foo
authors.affiliation: Department of Biological Sciences and Centre for BioImaging Sciences, National University of Singapore, Singapore; Department of Chemistry, National University of Singapore, Singapore. Electronic address: twohland@nus.edu.sg.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wohland
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D008051:Lipid Bilayers / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D008051:Lipid Bilayers