High-throughput spontaneous Raman imaging of biological specimens with water background reduction

In multiline-illumination Raman microscopy, background signals from the out-of-focus planes limit the throughput of cell imaging. Here, we improved the throughput of cell imaging by reducing background signals originating from the sample buffer solution and immersion medium of the objective lens. The background from water was suppressed by restricting the thickness of the sample buffer layer. In addition, the background signal in the CH stretching region was suppressed by replacing H₂O with D₂O as the immersion medium. These approaches successfully decreased background signals by 75%, enabling the same signal-to-noise ratio with a 2.2-fold shorter exposure time in cell imaging. Finally, we demonstrated high-throughput Raman imaging by visualizing bead uptake in living macrophages, successfully capturing 80 cells per frame within 3 min.