Optical sectioning in multifoci Raman hyperspectral imaging

In this study, we compared the depth discrimination and speed performance of multifoci Raman hyperspectral imaging with the reference standard of a single laser point confocal Raman mapping. A liquid crystal spatial light modulator was employed for the generation of multifoci laser beams, and a digital micromirror device was used as a software-configurable reflective pinhole array. The patterns of the laser foci and pinhole array can be rapidly changed without requiring any hardware alterations. Confocal patterns with different distance-to-size ratios were tested and compared. After optimization of the laser-foci pattern, we demonstrated the feasibility of multifoci Raman hyperspectral microscopy for recording depth-resolved molecular maps of biological cells (Acanthamoeba castellanii trophozoites). Micrometric depth discrimination and short acquisition times (20min for single plane confocal image) were achieved.