The stability of the FT-CARS flow cytometer was evaluated by measuring bead-to-bead fluctuations of the peak position and intensity of the 1003 cm−1 mode of 16-μm PS beads as a reference. FT-CARS spectra of 4873 PS beads were acquired within 1 s. The spectrum of a single PS bead was obtained as an average of three spectra measured when the bead passed through the optical interrogation point (fig. S6A). The acquired peak positions and intensities were evaluated by fitting a Gaussian function to them (fig. S6B). The distribution of the peak positions showed fluctuations with an SD of 3.2 cm−1, which is larger than that of the static sample presented in fig. S6B, presumably because the time-domain interferogram was distorted by unwanted nonlinear effects due to the spherical shape of the beads. However, this level of fluctuations is small enough to probe the spectral shift originating from the conformational change of astaxanthin (Fig. 4D). The intensity fluctuations were also evaluated by fitting a Gaussian function to them (blue dots in fig. S6C). The SD of the intensity fluctuations was estimated to be ±53%, which is larger than that in fig. SC due to the original variations of the bead size (coefficient of variation, <10%) and the fluctuations of the position of the beads at the interrogation point (caused by cell focusing accuracy), although it is a problem not unique to our method but common in flow cytometry in general1. The background noise level was estimated by plotting the intensity at 900 cm−1, which is shown in green in fig. S6C. Even with the relatively large intensity fluctuations, we found that the Raman signals from single PS beads are distinguishable from the noise (fig. S6C).

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