Toward a Comprehensive Raman Analysis of Functional Cells With Structured Optical Traps

Optically trapping RBCs with conventional Gaussian laser beams in physiological media and studying their properties have long been of interest, as it can be beneficial for understanding RBC-related disorders and their manifestations. An ever-ignored fact of such studies is that the point excitation spot of a tightly focused Gaussian beam inevitably probes only a small part of the cell's interior region, mostly the cytoplasm. More importantly, it omits key components of the cell membrane, thereby lagging behind information from one of the most crucial organs of RBCs. Recent donut- and line focus trapping using a focused vortex beam and an optical sheet, respectively, have proven capable of extracting information from cell membranes. However, the donut and line focus trapping are distinctly different from each other in probing RBCs, as the former is most exclusive, and the latter is most inclusive in excitation of the cell membrane. This study aims to establish a clear and distinctive capability of point, donut, and line focus trapping in deriving Raman spectroscopy signatures from RBCs. Principal component analysis highlighted these differences by classifying spectra based on dimensionally reduced characteristic spectral features. ANOVA identified the significance of the membrane contribution in shaping the Raman spectra obtained using donut and line focus trapping. Line focus trapping also revealed that the spectra are rich in information, particularly in the 1220-1290 cm^-1 range, corresponding to the amide III band.