Research ArticleCELL BIOLOGY

High-throughput label-free molecular fingerprinting flow cytometry

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Science Advances  16 Jan 2019:
Vol. 5, no. 1, eaau0241
DOI: 10.1126/sciadv.aau0241
  • Fig. 1 Schematic and principles of FT-CARS flow cytometry.

    (A) Schematic of the FT-CARS flow cytometer. (B) Principles of FT-CARS spectroscopy. A coherent molecular vibration in the target cell excited by the pump pulse induces a frequency shift to the probe pulse. Fourier transforming the measured time-domain interferogram gives the Raman spectrum of the cell. PBS, polarizing beamsplitter.

  • Fig. 2 Demonstration of FT-CARS flow cytometry.

    (A) Raman spectra and high-speed camera images of fast-flowing polymer beads of multiple species (PS and PMMA), demonstrating a throughput of 2700 events/s. See movie S1 for details. (B) Raman spectra and high-speed camera images of fast-flowing E. gracilis cells, demonstrating a high throughput of 1555 events/s. See movie S2 for details. (C) Scatterplot of the polymer beads in 1003 and 815 cm−1 intensities of their Raman spectra (n = 2514 PMMA beads and 4873 PS beads) with a high classification accuracy of >99.9%. (D) Scatterplot of the E. gracilis cells in 921 and 750 cm−1 intensities of their Raman spectra, enabling the quantification of intracellular chlorophyll content. a.u., arbitrary units.

  • Fig. 3 High-throughput label-free single-cell analysis of the astaxanthin productivity of H. lacustris.

    (A) Raman spectra of three single H. lacustris cells under nitrogen deficiency on day 0 through day 5. (B) Averaged Raman spectra of H. lacustris cells (n = 8000 for days 0, 1, 4, and 5; n = 6000 for days 2 and 3) under nitrogen deficiency, showing their gradual production of astaxanthin over time. (C) Scatterplot of H. lacustris cells (n = 8000 for each condition) under nitrogen-sufficient (day 0) and nitrogen-deficient (day 5) conditions in 1155- and 750-cm−1 Raman intensities, showing their separation by the different cultural conditions and heterogeneity in the productivity of astaxanthin. (D) Contour plot of H. lacustris cells under nitrogen deficiency for 0 to 5 days, showing their 5-day evolution. (E) Evolution of H. lacustris cells (n = 20,020) in the production of astaxanthin.

  • Fig. 4 High-throughput label-free single-cell analysis of the photosynthetic dynamics of H. lacustris.

    (A) Procedure of preparing H. lacustris cells with different degrees of isotope substitution of carbon dioxide. (B) Averaged Raman spectra of H. lacustris cells (n = 3000 for each condition) 0 to 10 days after the isotope substitution. (C) Scatterplot of H. lacustris cells (n = 2592) in Raman intensities of the two peaks, showing their 10-day evolution. (D) Scatterplot of H. lacustris cells (n = 2592) in Raman shifts (positions) of the two peaks, showing their 10-day evolution.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau0241/DC1

    Fig. S1. Figure of merit that compares our work and previous work by others.

    Fig. S2. Complete schematic of the FT-CARS flow cytometer.

    Fig. S3. Digital signal processing.

    Fig. S4. Structure of the acoustofluidic-focusing microfluidic chip.

    Fig. S5. Steps for fabricating the acoustofluidic-focusing microfluidic chip.

    Fig. S6. Stability of the FT-CARS flow cytometer.

    Fig. S7. Stability of the FT-CARS spectrometer.

    Fig. S8. Image of an E. gracilis cell under a conventional optical microscope.

    Fig. S9. Images of H. lacustris cells under the nitrogen deficiency stress obtained by a conventional optical microscope.

    Fig. S10. Raman spectra obtained by FT-CARS and conventional spontaneous Raman spectroscopy.

    Movie S1. High-speed imaging and FT-CARS flow cytometry of fast-flowing polymer beads of multiple species.

    Movie S2. High-speed imaging and FT-CARS flow cytometry of fast-flowing E. gracilis cells.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Figure of merit that compares our work and previous work by others.
    • Fig. S2. Complete schematic of the FT-CARS flow cytometer.
    • Fig. S3. Digital signal processing.
    • Fig. S4. Structure of the acoustofluidic-focusing microfluidic chip.
    • Fig. S5. Steps for fabricating the acoustofluidic-focusing microfluidic chip.
    • Fig. S6. Stability of the FT-CARS flow cytometer.
    • Fig. S7. Stability of the FT-CARS spectrometer.
    • Fig. S8. Image of an E. gracilis cell under a conventional optical microscope.
    • Fig. S9. Images of H. lacustris cells under the nitrogen deficiency stress obtained by a conventional optical microscope.
    • Fig. S10. Raman spectra obtained by FT-CARS and conventional spontaneous Raman spectroscopy.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). High-speed imaging and FT-CARS flow cytometry of fast-flowing polymer beads of multiple species.
    • Movie S2 (.mp4 format). High-speed imaging and FT-CARS flow cytometry of fast-flowing E. gracilis cells.

    Files in this Data Supplement:

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