Quantifying Lipid Diffusion by Fluorescence Correlation Spectroscopy: A Critical Treatise Fabian Heinemann, †,‡,§ Viktoria Betaneli, †,‡ Franziska A. Thomas, ‡ and Petra Schwille* ,‡,§ ‡ Biophysics Institute, Biotec/Technische Universitä t Dresden, Tatzberg 47-51, 01307 Dresden, Germany § Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany * S Supporting Information 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 diffu- sion coefficient of the fluorescent lipid analog 1,1′-dioctadecyl- 3,3,3′,3′-tetramethylindodicarbocyanine (DiD) in giant uni- lamellar 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 μm 2 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. ■ INTRODUCTION The fluidity of the plasma membrane and thus lateral diffusion of lipids and membrane proteins is an essential property of living cells. Diffusion enables the distribution of membrane constituents and is a prerequisite for diffusion-limited chemical reactions inside the membrane plane. 1 According to the in- fluential 40 year old Singer and Nicholson fluid-mosaic model 2 and its biophysical description according to Saffman and Delbrü ck, 3 the plasma membrane can be described as a two- dimensional viscous fluid with a free lateral diffusion of lipids and embedded proteins. Nowadays, it is known that the situa- tion is more complex. 4 Lateral diffusion of membrane proteins and lipids is modulated by various factors, such as crowding due to the high protein content in the membrane (for example, 23% of the area in the erythrocyte membrane is occupied by proteins). 5 Furthermore, the proposed sphingolipid- and cholesterol-enriched lipid nanodomains 6 represent dynamic obstacles or traps for diffusing membrane species. 7-9 Also, membrane-associated parts of the cytoskeleton are supposed to interfere with membrane di ffusion 8,10 by dividing the membrane into compartments of typically 40-200 nm in diameter. 11,10 The question of how precisely lateral membrane diffusion is modulated is currently under intensive investigation. Minimal systems as well as native cell membranes are used for these studies. A widely used technique to measure lateral diffusion co- efficients D in biological membranes is fluorescence correlation spectroscopy (FCS), as recently discussed. 12-14 Besides the classical method of “point” FCS with a steady confocal volume, 15-18 several modifications like dual-focus FCS, 19-21 z-scan FCS, 22 one or two focus scanning FCS (1f SFCS, 2f SFCS), 23 circular scanning FCS, 24 and line-scan FCS (LSFCS) 25 have been developed. These new variants of FCS address some of the notorious problems related to FCS on membranes: membrane movements, fluorophore bleaching, and the requirement of a calibration of the detection volume. Despite this diversity of methods, there is a lack of consensus values of diffusion coefficients. This is reflected in the discrepancy of published diffusion coefficients for equivalent experimental conditions. As an example, the reported diffusion coefficients D of the frequently used fluorescent lipid analog 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) in giant unilamellar vesicles (GUVs) composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) range from D = 5.8 ± 0.3 18 to 8.7 ± 0.7 μm 2 s -1 26 (in both cases using point FCS). Another result for DOPC GUVs was obtained by nuclear Received: June 27, 2012 Revised: August 13, 2012 Published: August 14, 2012 Article pubs.acs.org/Langmuir © 2012 American Chemical Society 13395 dx.doi.org/10.1021/la302596h | Langmuir 2012, 28, 13395-13404