1：Experimental Design and Method Selection:

The study employs a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) spectrometer integrated with a microfluidic chip for high-throughput, label-free single-cell analysis. The design leverages FT-CARS for fast, broadband spectral acquisition and acoustofluidic focusing for precise cell positioning.

2：Sample Selection and Data Sources:

Samples include polymer beads (PMMA and PS), Euglena gracilis microalgae, and Haematococcus lacustris microalgae under various culture conditions (e.g., nitrogen deficiency, isotope labeling). Cells were obtained from microbial culture collections and prepared in specific media.

3：List of Experimental Equipment and Materials:

Key equipment includes a Ti:Sapphire femtosecond laser, Michelson interferometer with resonant scanner, avalanche photodiode, high-speed digitizer, microfluidic chip with piezoelectric transducer, syringe pump, function generator, amplifier, and various optical components (e.g., polarizing beamsplitters, filters). Materials include polymer beads, cell culture media, and isotopes.

4：Experimental Procedures and Operational Workflow:

Cells are flowed through the microfluidic channel at high speed (e.g., 20 cm/s) using a syringe pump. Acoustic focusing ensures cells are centered for optical interrogation. Laser pulses excite molecular vibrations, and the resulting anti-Stokes signals are detected, digitized, and Fourier-transformed to obtain Raman spectra. Forward scattering and bright-field imaging are used for validation.

5：Data Analysis Methods:

Data analysis involves Fourier transformation of interferograms, singular value decomposition for spectral contribution extraction, Gaussian fitting for peak analysis, and statistical methods to evaluate fluctuations and classification accuracy.