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High-throughput imaging flow cytometry by optofluidic time-stretch microscopy

Abstract

The ability to rapidly assay morphological and intracellular molecular variations within large heterogeneous populations of cells is essential for understanding and exploiting cellular heterogeneity. Optofluidic time-stretch microscopy is a powerful method for meeting this goal, as it enables high-throughput imaging flow cytometry for large-scale single-cell analysis of various cell types ranging from human blood to algae, enabling a unique class of biological, medical, pharmaceutical, and green energy applications. Here, we describe how to perform high-throughput imaging flow cytometry by optofluidic time-stretch microscopy. Specifically, this protocol provides step-by-step instructions on how to build an optical time-stretch microscope and a cell-focusing microfluidic device for optofluidic time-stretch microscopy, use it for high-throughput single-cell image acquisition with sub-micrometer resolution at >10,000 cells per s, conduct image construction and enhancement, perform image analysis for large-scale single-cell analysis, and use computational tools such as compressive sensing and machine learning for handling the cellular ‘big data’. Assuming all components are readily available, a research team of three to four members with an intermediate level of experience with optics, electronics, microfluidics, digital signal processing, and sample preparation can complete this protocol in a time frame of 1 month.

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Fig. 1: Overview of the protocol for optofluidic time-stretch microscopy.
Fig. 2: Optical time-stretch microscope.
Fig. 3: Performance of optical time-stretch microscopy.
Fig. 4: Schematic illustration and working principles of cell focusing in microfluidic devices.
Fig. 5: Fabrication procedures.
Fig. 6: Procedure for fabricating a microfluidic device for hydrodynamic focusing or inertial focusing.
Fig. 7: Digital image processing.
Fig. 8: Graphical illustration of the MMD.
Fig. 9: Imaging flow cytometry and analysis of E. gracilis cells.
Fig. 10: Imaging flow cytometry and analysis of blood cells.
Fig. 11: Imaging flow cytometry and analysis of MCF-7 cells.

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Acknowledgements

This work was supported by the ImPACT Program of the Council for Science, Technology and Innovation (Cabinet Office, Government of Japan). The fabrication of the microfluidic devices was conducted at the University of Tokyo’s Center for Nano Lithography.

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Affiliations

Authors

Contributions

C.L. and K.G. designed the protocol. C.L., H.K., and Y.W. performed the experiments. M.L. and A.I. helped fabricate the microfluidic devices. A.Y. and T.I. helped prepare the blood and microalga samples. H.M., N.N., and T.S. helped prepare the figures and tables. M.Y., Y.Y., D.D.C., Y.O., and K.G. supervised the project. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Cheng Lei or Keisuke Goda.

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The authors declare no competing interests.

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Related links

1. Goda, K., Tsia, K. K. & Jalali, B. Serial time-encoded amplified imaging for real-time observation of fast dynamic phenomena. Nature 458, 1145–1149 (2009). https://doi.org/10.1038/nature07980

2. Goda, K. et al. High-throughput single-microparticle imaging flow analyzer. Proc. Natl. Acad. Sci. USA 109, 11630–11635 (2012). https://doi.org/10.1073/pnas.1204718109

3. Lei, C., Guo, B., Cheng, Z. & Goda, K. Optical time-stretch imaging: principles and applications. Appl. Phys. Rev. 3, 011102 (2016). https://doi.org/10.1063/1.4941050

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Design of microfluidic devices.

Design of (a) hydrodynamic focusing and (b) inertial focusing microfluidic devices. Scale bar = 100 µm.

Supplementary Figure 2 Photographs of microfluidic devices.

Photographs of (a) hydrodynamic-focusing and (b) inertial-focusing microfluidic devices used in the experiments for cell focusing.

Supplementary Figure 3 Procedure for preparing an E. gracilis sample.

(I) Stock culture of E. gracilis NIES-48 in the plant growth chamber. (II) Inoculate the stock cells of E. gracilis to fresh AF-6. (III) To prepare fresh E. gracilis cells, inoculate 3 × 104 cells to fresh AF-6. (IV) To prepare lipid-accumulated E. gracilis cells, collect cells using a centrifuge, and then replace the culture medium with AF-6‒N (nitrogen nutrient omitted from AF-6) for rinsing. After rinsing, collect and resuspend cells in fresh AF-6‒N. All the cultures are incubated in the same conditions except for the culture media and cell density.

Supplementary Figure 4 Procedure for preparing a human blood sample.

(I) Prepare a 21-guage butterfly needle and a 4.5-mL vacuum plasma separator tube with 0.5-mL of 3.2% sodium citrate, Venoject II. (II) Mix the blood sample and the anti-coagulant thoroughly and slowly by gentle inversion. (III) Transfer 100 µL of the blood sample to a 1.5-mL tube immediately after mixing it. (IV) Add 500 µL of OptiLyse C Lysing Solution to the blood sample and mix it gently by tapping. (V) Incubate it for 10 min at room temperature (18–25 °C). (VI) Add 500 µL of DPBS to the blood sample and mix it gently by tapping. (VII) Leave the blood sample for 10 min at room temperature. This study was approved by the Institutional Ethics Committee of the Faculty of Medicine, the University of Tokyo (#11049-[5]). Written informed consents were obtained from the blood donors.

Supplementary Figure 5 Procedure for preparing a breast cancer cell sample.

(I) Incubate MCF-7 cells seeded in a 12-well plate for 24 h with multiple concentrations of paclitaxel. (II) Aspirate the cell medium in the 12-well plate, wash the cells with 1 mL of DPBS, and aspirate it again. (III) Add 100 µL of trypsin to the sample and incubate it for ~5 min at 37 °C. (IV) Add 1 mL of complete medium to the sample to dilute the cell suspension and mix it by pipetting up and down a few times with a micropipette to break up any clumps of cells. (V) Place a 40-µm cell strainer on top of a 2-mL conical tube. Pass cells through the cell strainer to remove clumps and debris. (VI) Put the single-cell suspension into a 1-mL syringe and load the syringe on a syringe pump.

Supplementary information

Combined Supplementary Information

Supplementary Figures 1–5 and Supplementary Methods

Reporting Summary

Supplementary Data 1

MATLAB scripts for image construction

Supplementary Data 2

AutoCAD design files for hydrodynamic and inertial focusing microfluidic devices

Supplementary Data 3

MATLAB scripts for image segmentation

Supplementary Video 1

Procedures for microfluidic device fabrication

Supplementary Video 2

Procedures for high-throughput imaging flow cytometry by optofluidic time-stretch microscopy

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Lei, C., Kobayashi, H., Wu, Y. et al. High-throughput imaging flow cytometry by optofluidic time-stretch microscopy. Nat Protoc 13, 1603–1631 (2018). https://doi.org/10.1038/s41596-018-0008-7

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