Optical Trapping of Submicron DPPC Vesicles for Raman Spectroscopy Measurements

Extracellular vesicles are promising biomarkers for non-invasive diagnostics, but their heterogeneity in size and composition challenges conventional analysis. Raman spectroscopy with optical tweezers can characterize individual vesicles, yet the full capabilities of this approach remain underexplored. Here, we investigate Raman spectroscopy integrated with optical tweezers for real-time characterization of submicron particles, focusing on vesicle trapping kinetics. Using 100-nm dipalmitoylphosphatidylcholine (DPPC) vesicles, we examine the effects of objective type (water immersion, oil immersion, dry), laser power, concentration, and polarization on trapping dynamics and Raman signals. Vesicle trapping kinetics were satisfactorily described using a kinetic balance model with a stretched exponential that separates the contributions of particle influx, thermal escape, and finite trap capacity. Our measurements reveal distinct regimes with different objectives: with water immersion the signal rapidly saturates because the deep, stiff trap suppresses escape; with oil immersion spherical aberrations reduce trap stiffness and depth, leading to a continuous increase in equilibrium signal with power. The characteristic trapping time universally depends on total laser power, independent of objective. At elevated concentrations, the stretching exponent parameter β drops from 1 to ∼0.6 and large-amplitude steps appear, indicating vesicle aggregation as a source of kinetic dispersion. Numerical simulations qualitatively support the interpretation that trap stiffness and depth govern the observed kinetic regimes. These findings provide practical guidelines for single-vesicle Raman experiments and show that kinetic analysis offers a sensitive label-free indicator of sample polydispersity and aggregation that can be applied to real biological samples.