The acoustofluidic-focusing microfluidic chip was used for flowing and focusing cells in 2D (29). To flow cells, we used a syringe pump (Pump 11 Elite 70-4500, Harvard Apparatus) to push a syringe that contains cells dispersed in water. The syringe was connected to the inlet of the chip with a tube. The pump kept the injection amount constant in time, typically at 400 μl/min to set the flow speed of the cells at 20 cm/s. To focus the cells in 2D, we applied a radio frequency signal of 40 to 80 Vpp at 3.66 MHz to the PZT. The signal was generated by a function generator (WF1974, NF Corporation) and amplified by an amplifier (BA4850, NF Corporation). The resonant vibration of the PZT generated 2D acoustic standing waves in the square-shaped cross-sectional plane orthogonal to the flow direction in the microchannel. Cells were focused at the node of the standing waves that appear at the center of the microchannel. In FT-CARS measurements with the acoustic focusing, an artificial signal oscillating at 3.66 MHz, which gives a fake peak in the obtained Raman spectra, was detected. In the present setup however, the frequency of 3.66 MHz corresponded to a Raman shift of 1750 to 2000 cm−1, where no Raman signature is expected for the cells studied here.

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