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.

Herein, we report on the experimental measurements for estimated relative mobility shifts caused by changes in mass distribution from isotopic substitutions in isotopologues and isotopomers with high-resolution cyclic ion mobility separations. By utilizing unlabeled and fully labeled isotopologues with the same isotopic substitutions (i.e., 2H or 13C), we created a highly precise mobility scale for each set analyzed to determine the magnitude of such mass distribution shifts and thus calculate estimated deviations from expected, theoretical reduced mass contributions. We observed relative mobility shifts in various isotopologues (e.g., hexadecyltrimethylammonium, sucrose, and palmitic acid species) that deviated from reduced mass theory, according to the Mason–Schamp relationship, ranging in estimated magnitude from ∼0.007% up to ∼0.1% in relative mobility. More interestingly, it was found that two deuterated palmitic acid isotopomers also differed by ∼0.03% from one another in their respective relative mobility shifts. Our results are the first report of isotopologue and isotopomer separations on a commercially available cyclic ion mobility spectrometry-mass spectrometry platform. We envision that our presented mobility scale methodology will have broad applicability in studying the effect of mass distribution changes from isotopic substitutions in other biomolecules and help pave the way for the improvement of ion mobility theory and collision cross section calculators.

Service-learning (SL) is an active learning approach that connects the knowledge a student acquires in the classroom to an application that benefits the community. Increasingly popular in the chemistry curriculum, service-learning is reported to provide student benefits including improved cognitive goals; increased academic, interpersonal, and leadership skills; increased ability to apply course concepts to real-world situations; and increased community engagement. For the work reported here, an analytical chemistry laboratory was modified to include a service-learning component with the goal of allowing students to apply their newly acquired analytical skills to relevant, real-world samples; to learn new analytical techniques; and to develop professional communication skills. Students implemented a study of the wastewater effluent at the Orlando Easterly Wetlands, an engineered water polishing facility that removes nutrients from treated wastewater. Students designed a sampling strategy, collected samples in the field, and performed standard analysis on the water, including pH, chloride, total dissolved solids, and phosphorus. Students also tested the water for the artificial sweetener, sucralose, and characterized the concentration throughout the flow path of the wetlands. Sucralose has been proposed as an indicator of contamination of natural waters by anthropogenic waste. This type of analysis has not been performed for this public utility until now, and the students shared the results in a public seminar. Student learning outcomes were compared to those in a conventional section, with SL students showing comparable subject mastery and improved self-efficacy.

Encapsulation of molecules in phospholipid vesicles provides unique opportunities to study chemical reactions in small volumes as well as the behavior of individual proteins, enzymes, and ribozymes in a confined region without requiring a tether to immobilize the molecule to a surface. These experiments generally depend on generating a predictable loading of vesicles with small numbers of target molecules and thus raise a significant measurement challenge, namely, to quantify molecular occupancy of vesicles at the single-molecule level. In this work, we describe an imaging experiment to measure the time-dependent fluorescence from individual dye molecules encapsulated in ~130 nm vesicles that are adhered to a glass surface. For determining a fit of the molecular occupancy data to a Poisson model, it is critical to count empty vesicles in the population since these dominate the sample when the mean occupancy is small, λ ≤ ~1. Counting empty vesicles was accomplished by subsequently labeling all the vesicles with a lipophilic dye and reimaging the sample. By counting both the empty vesicles and those containing fluors, and quantifying the number of fluors present, we demonstrate a self-consistent Poisson distribution of molecular occupancy for well-solvated molecules, as well as anomalies due to aggregation of dye, which can arise even at very low solution concentrations. By observation of many vesicles in parallel in an image, this approach provides quantitative information about the distribution of molecular occupancy in a population of vesicles.

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.