Abstract
Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.
Highlights
To dissect the complexity of cell membranes and to isolate the behavior of lipid bilayers in simple systems under controlled conditions, giant unilamellar vesicles (GUVs) are widely utilized model systems [1]
In many studies, where a liposome serves as model system [3] it is preferable that the giant vesicle (GV) should consist of a single lipid bilayer, i.e., be unilamellar, such as an actual cellular membrane
We have developed a believed-novel method for quantitative determination of GV lamellarity based on label-free differential interference contrast (DIC) microscopy
Summary
To dissect the complexity of cell membranes and to isolate the behavior of lipid bilayers in simple systems under controlled conditions, giant unilamellar vesicles (GUVs) are widely utilized model systems [1]. In many studies, where a liposome serves as model system [3] it is preferable that the giant vesicle (GV) should consist of a single lipid bilayer, i.e., be unilamellar, such as an actual cellular membrane. To determine the lamellarity from qDIC contrast images Ic, the fit procedure described in Section S3.2 in the Supporting. The parameters were initialized, and sequentially added to the fitted parameters using the following procedure: The GV center Rc and radius R0 were initialized using moments of Ic as described in Section S3.1 in the Supporting Material. The values ws and wp were set to the image resolution ws 1⁄4 l/(2NA) z 350 nm and wp 1⁄4 l/(NA) z 700 nm
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