Structural Characterization of Individual Vesicles using Fluorescence Microscopy

Abstract

Extrusion of hydrated lipid suspensions is frequently employed to produce vesicles of uniform size, and the resulting vesicles are often reported to be unilamellar. We describe a method for the quantitative fluorescence image analysis of individual vesicles to obtain information on the size, lamellarity, and structure of vesicles produced by extrusion. In contrast to methods for bulk analysis, fluorescence microscopy provides information about individual vesicles, rather than averaged results, and heterogeneities in vesicle populations can be characterized. Phosphatidylcholine vesicles containing small fractions of biotin-modified phospholipid and fluorescently labeled 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) phospholipid were immobilized through biotin-avidin-biotin binding to the surface of a biotin-modified glass coverslip. Biotin was attached to the surface in a mixed cyano-terminated silane monolayer. Initial fluorescence intensities for each immobilized vesicle were recorded, and a solution of membrane impermeable quencher was passed through the flow cell to quench the fluorescence of the outer layer. Fluorescence from individual vesicles was measured by fitting the spots to 2-dimensional Gaussian functions. The integrated signals under the peaks yielded a pre- and postquench intensity. From the fractional loss of intensity, the number and structure of the bilayers in individual vesicles could be quantified; the results showed that extruded vesicles exhibit a distribution of size, lamellarity, and structure.

Allosteric inhibition of individual enzyme molecules trapped in lipid vesicles

“…Thus, if the concentration is fixed, the volume variability implies that the number of molecules will be distributed. In principle, one should be able to correct for this effect by measuring the volume on a vesicle-by-vesicle basis (51,52). This was not done in the current experiment; nevertheless, our analysis suggests that vesicle volume variability cannot account for the whole width of the plateau level distribution.…”

Layer-by-layer cell membrane assembly

“…If each leaflet contains approximately the same surface area, fluorescence should increase linearly. Furthermore, selective external leaflet quenching of these multiply-labeled intermediates and final double bilayer product should yield a fraction of the construct total fluorescence that is inversely proportional to the number of depositions 28 . Our observations agree with linear and inverse proportionality for the layer-by-layer construction and selective external leaflet quenching experiments, respectively (Fig.…”

A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy

“…To overcome these problems with ensemble assays, it is necessary to obtain information on a large and representative number of individual vesicles, for example, by using single vesicle microscopy. Previous work along these lines showed that the size distribution of the vesicles could be determined by correlating the fluorescence intensity of a lipid marker in single vesicles with DLS data (Hatzakis et al, 2009; Malle et al, 2021; Lohr et al, 2009; Olsson et al, 2015), and that tightness and lamellarity could be assessed by bleaching of the marker (Heider et al, 2011). Other studies used step-wise photobleaching assays to determine the copy number of fluorescent proteins per vesicle (Hummert et al, 2021).…”