Flow-Through Optical Chromatography in Combination with Confocal Raman Microspectroscopy: A Novel Label-Free Approach...

Flow-through optical chromatography (FT-OC), an advanced mode of optical chromatography, achieved baseline separation of a mixture of silica microparticles (SiO₂, 1.00 and 2.50 μm) and a mixture of polystyrene microparticles (PS, 1.00, 2.00, and 3.00 μm) based on particle size. Comparisons made between experimentally determined velocities for the microparticles and theoretically derived velocities from Mie theory and Stokes' law validated the data collection setup and the data analysis for FT-OC. A population shift in live macrophages (cell line IC-21, ATCC TIB-186) responding to environmental stimuli was sensitively detected by FT-OC. The average velocity of macrophages stressed by nutritional deprivation was decreased considerably together with a small but statistically significant increase in cell size. Mie scattering calculations demonstrated that the small increase in cell size of macrophages stressed by nutritional deprivation was not entirely responsible for this decrease. Confocal fluorescence microscopy and atomic force microscopy (AFM) studies revealed morphological changes of macrophages induced by nutritional deprivation, and these changes were more likely responsible for the decrease in average velocity detected by FT-OC. Confocal Raman microspectroscopy was used to shed light upon biochemical transformations of macrophages suffering from nutritional deprivation.