Raman Spectroscopy Study of Curvature-Mediated Lipid Packing and Sorting in Single Lipid Vesicles

Abstract

Cellular plasma membrane deformability and stability is important in a range of biological processes. Changes in local curvature of the membrane affect the lateral movement of lipids, affecting the biophysical properties of the membrane. An integrated holographic optical tweezers and Raman microscope was used to investigate the effect of curvature gradients induced by optically stretching individual giant unilamellar vesicles (GUVs) on lipid packing and lateral segregation of cholesterol in the bilayer. The spatially resolved Raman analysis enabled detection of induced phase separation and changes in lipid ordering in individual GUVs. Using deuterated cholesterol, the changes in lipid ordering and phase separation were linked to lateral sorting of cholesterol in the stretched GUVs. Stretching the GUVs in the range of elongation factors 1–1.3 led to an overall decrease in cholesterol concentration at the edges compared to the center of stretched GUVs. The Raman spectroscopy results were consistent with a model of the bilayer accounting for cholesterol sorting in both bilayer leaflets, with a compositional asymmetry of 0.63 ± 0.04 in favor of the outer leaflet. The results demonstrate the potential of the integrated holographic optical tweezers-Raman technique to induce deformations to individual lipid vesicles and to simultaneously provide quantitative and spatially resolved molecular information. Future studies can extend to include more realistic models of cell membranes and potentially live cells.

Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles

“…Therefore, RTM, with a typical processing capacity of 0.2 particles per min, is not considered a high-throughput methodology 71 . RTM can, however, be used to obtain interesting, unique information not only for EVs 57,60,61,[70][71][72][73][74][75][76][77] but also for many other bioparticles such as liposomes, lipid layers on synthetic nanoparticles and others [78][79][80][81][82][83] .…”

“…Direct visualization of EVs using microscopy lets researchers assess the shape and size of vesicles in their native state 156 and in various biological processes 157 , while other techniques, such as RTM, can be employed to examine further the morphology of bioparticles without direct visualization. For example, the effect of membrane lipid composition on the shape and size of giant unilamellar vesicles was first described using Raman tweezers, demonstrating that a decrease in cholesterol concentration increases the local membrane curvature and stretches the vesicle 82 .…”

Raman Spectroscopic Study of Phase Coexistence in Binary Phospholipid Bilayers

“…23 Also, it was reported that the apparent ratio I aCH /I sCH can slightly decrease with temperature increase, when the lipids are in a liquid-like state, and it can be changed with the vesicle deformation. 24,25 This complicates the analysis if only information about I aCH is used. On the other hand, a comparison of the expectation for I aCH , calculated in the spirit of Fig.…”

Automated Raman based cell sorting with 3D microfluidics

“…4,9,10 RACS in flow can be both non-invasive and have higher throughput than methods that rely on measuring spectra of cells that are (momentarily or permanently) stationary, followed by cell picking. Since spontaneous Raman spectra are often inherently weak, to date, most of the methods that have used cells in microfluidic systems have used a "trapand-release" approach, employing methods such as optical tweezing 4,[11][12][13][14][15] or dielectrophoresis. 16,17 Although "cell trapping" can meet the need for long signal acquisition, ultimately the trapping mechanism limits throughput, and the efficiency of the trap is often dependent on cell size, medium conductivity, refractive index and/or flow rate.…”