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Tackling Sample‐Related Artifacts in Membrane FCS Using Parallel SAF and UAF Detection
Author(s) -
Winterflood Christian M.,
Ruckstuhl Thomas,
Reynolds Nicholas P.,
Seeger Stefan
Publication year - 2012
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.201200395
Subject(s) - membrane , fluorescence correlation spectroscopy , flatness (cosmology) , fluorescence , chemistry , analytical chemistry (journal) , diffusion , supercritical fluid , materials science , penetration (warfare) , biophysics , biological system , optics , chromatography , physics , biology , thermodynamics , mathematics , biochemistry , organic chemistry , cosmology , quantum mechanics , operations research
The complex shape and plasticity of cells is an intricate issue for the measurement of molecular diffusion in plasma membranes by fluorescence correlation spectroscopy (FCS). An important precondition for accurate diffusion measurements is a sufficient flatness of the membrane over the considered region and the absence of non‐membrane‐bound fluorescence diffusion. A method is presented to identify axial motion components caused by a non‐ideal geometry of the membrane based on simultaneous measurement of the fluorescence emitted above and below the critical angle of the specimen/glass interface. Thereby, two detection volumes are generated that are laterally coincident, but differ in their axial penetration of the specimen. The similarity between the intensity tracks of the supercritical angle fluorescence (SAF) and the undercritical angle fluorescence (UAF) strongly depends on the membrane flatness and intracellular fluorescence, and can help to avoid sample‐related artifacts in the diffusion measurement.