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Tackling Sample-Related Artifacts in Membrane FCS Using Parallel SAF and UAF Detection


Winterflood, Christian M; Ruckstuhl, Thomas; Reynolds, Nicholas P; Seeger, Stefan (2012). Tackling Sample-Related Artifacts in Membrane FCS Using Parallel SAF and UAF Detection. ChemPhysChem, 13(16):3655-3660.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:2012
Deposited On:13 Feb 2013 13:41
Last Modified:17 Feb 2018 00:54
Publisher:Wiley-Blackwell
ISSN:1439-4235
OA Status:Closed
Publisher DOI:https://doi.org/10.1002/cphc.201200395

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