Confocal Raman microscopy of single, optically-trapped unilamellar phospholipid vesicles

A new technique for the analysis of single, unilamellar phospholipid vesicles, or liposomes is reported that is based on a combination of Raman microscopy and optical-trapping. With the combination of optical trapping and confocal optics, detecting the phospholipid bilayer of a liposome in the optical trap is feasible because the contents of the vesicle can occupy the majority of the confocal volume where Raman scattering is collected with high efficiency. This optical efficiency leads to an excellent signal-to-noise ratio and allows for analysis of molecular species within the bilayer and differentiation of contents inside a trapped vesicle from the surrounding solution. This latter capability is used to measure the permeability of a membrane bilayer to small molecular ions that are initially inside the vesicle, and which can diffuse through the membrane when there are defects at boundaries between phase domains in the bilayer. The force and energy gradients that are responsible for immobilizing a vesicle in an optical trap are estimated from the dielectric contrast between the phospholipid molecules and the aqueous solution. The impact of these forces on the shapes of vesicles are measured, both for optically-trapped and surface-immobilized liposomes. Finally, the technique is applied for a real-time measurement of enzyme-catalyzed hydrolysis of the phospholipid bilayer of individual vesicles. Phospholipase A2 from cobra venom (Naja naja naja) is introduced to a liposome solution to catalyze the hydrolysis the phosphatidylcholine molecules at the sn-2 ester linkage. Changes in Raman scattering bands in the carbon-carbon stretching region of the spectrum are monitored over the course of the reaction and correlate with the rate of phospholipid hydrolysis based on mass spectrometry evidence. The change in the Raman spectra over time is analyzed to determine kinetic parameters, such as lag time and turnover rate. These parameters are extracted from the data acquired from the hydrolysis of molecules in the bilayer of a single liposome. The data are then analyzed by self-modeling curve resolution and changes in the Raman spectra can be observed to occur due to a reorganization of molecules in the optical trap.