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.
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