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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.
Details
; Kobayashi, Hirofumi 1 ; Wu, Yi 2 ; Li, Ming 3 ; Isozaki, Akihiro 1 ; Yasumoto, Atsushi 4 ; Mikami, Hideharu 1 ; Ito, Takuro 5 ; Nitta, Nao 5
; Sugimura, Takeaki 5 ; Yamada, Makoto 6 ; Yatomi, Yutaka 4 ; Dino Di Carlo 7 ; Ozeki, Yasuyuki 8 ; Goda, Keisuke 9 1 Department of Chemistry, The University of Tokyo, Tokyo, Japan
2 Department of Chemistry, The University of Tokyo, Tokyo, Japan; Department of Computational Biology, Carnegie Mellon University, Pittsburgh, PA, USA
3 Department of Electrical Engineering, University of California, Los Angeles, Los Angeles, CA, USA; Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, USA
4 Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
5 Japan Science and Technology Agency, Kawaguchi, Japan
6 Centre for Advanced Intelligence Project, RIKEN, Tokyo, Japan
7 Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA; Jonsson Comprehensive Cancer Centre, University of California, Los Angeles, Los Angeles, CA, USA
8 Department of Electrical Engineering and Information Systems, The University of Tokyo, Tokyo, Japan
9 Department of Chemistry, The University of Tokyo, Tokyo, Japan; Department of Electrical Engineering, University of California, Los Angeles, Los Angeles, CA, USA; Japan Science and Technology Agency, Kawaguchi, Japan