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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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.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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
Design of (a) hydrodynamic focusing and (b) inertial focusing microfluidic devices. Scale bar = 100 µm.
Photographs of (a) hydrodynamic-focusing and (b) inertial-focusing microfluidic devices used in the experiments for cell focusing.
(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.
(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-). Written informed consents were obtained from the blood donors.
(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 Figures 1–5 and Supplementary Methods
MATLAB scripts for image construction
AutoCAD design files for hydrodynamic and inertial focusing microfluidic devices
MATLAB scripts for image segmentation
Procedures for microfluidic device fabrication
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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