Quantifying lipid diffusion by fluorescence correlation spectroscopy: A critical treatise

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

Abstract

Fluorescence correlation spectroscopy (FCS) measurements are widely used for determination of diffusion coefficients of lipids and proteins in biological membranes. In recent years, several variants of FCS have been introduced. However, a comprehensive comparison of these methods on identical systems has so far been lacking. In addition, there exist no consistent values of already determined diffusion coefficients for well-known or widely used membrane systems. This study aims to contribute to a better comparability of FCS experiments on membranes by determining the absolute diffusion coefficient of the fluorescent lipid analog 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) made of dioleoylphosphatidylcholine (DOPC), which can in future studies be used as a reference value. For this purpose, five FCS variants, employing different calibration methods, were compared. Potential error sources for each particular FCS method and strategies to avoid them are discussed. The obtained absolute diffusion coefficients for DiD in DOPC were in good agreement for all investigated FCS variants. An average diffusion coefficient of D = 10.0 ± 0.4 μm2 s-1 at 23.5 ± 1.5 °C was obtained. The independent confirmation with different methods indicates that this value can be safely used for calibration purposes. Moreover, the comparability of the methods also in the case of slow diffusion was verified by measuring diffusion coefficients of DiD in GUVs consisting of DOPC and cholesterol.

Details

OriginalspracheEnglisch
Seiten (von - bis)13395-13404
Seitenumfang10
FachzeitschriftLangmuir
Jahrgang28
Ausgabenummer37
PublikationsstatusVeröffentlicht - 18 Sept. 2012
Peer-Review-StatusJa

Externe IDs

PubMed 22891610