Color-scalable flow cytometry with Raman tags

Author:

Nishiyama Ryo1ORCID,Hiramatsu Kotaro123ORCID,Kawamura Shintaro45ORCID,Dodo Kosuke45ORCID,Furuya Kei1,de Pablo Julia Gala1,Takizawa Shigekazu1,Min Wei6ORCID,Sodeoka Mikiko45ORCID,Goda Keisuke178ORCID

Affiliation:

1. Department of Chemistry, The University of Tokyo , Tokyo 113-0033 , Japan

2. Research Centre for Spectrochemistry, The University of Tokyo , Tokyo 113-0033 , Japan

3. PRESTO, Japan Science and Technology Agency , Saitama 332-0012 , Japan

4. Synthetic Organic Chemistry Laboratory, RIKEN Cluster for Pioneering Research , Saitama 351-0198 , Japan

5. RIKEN Center for Sustainable Resource Science , Saitama 351-0198 , Japan

6. Department of Chemistry, Columbia University , New York , NY 10027 , USA

7. Department of Bioengineering, University of California , CA 90095 , USA

8. Institute of Technological Sciences, Wuhan University , Wuhan 430072 , China

Abstract

AbstractFlow cytometry is an indispensable tool in biology and medicine for counting and analyzing cells in large heterogeneous populations. It identifies multiple characteristics of every single cell, typically via fluorescent probes that specifically bind to target molecules on the cell surface or within the cell. However, flow cytometry has a critical limitation: the color barrier. The number of chemical traits that can be simultaneously resolved is typically limited to several due to the spectral overlap between fluorescence signals from different fluorescent probes. Here, we present color-scalable flow cytometry based on coherent Raman flow cytometry with Raman tags to break the color barrier. This is made possible by combining a broadband Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) flow cytometer, resonance-enhanced cyanine-based Raman tags, and Raman-active dots (Rdots). Specifically, we synthesized 20 cyanine-based Raman tags whose Raman spectra are linearly independent in the fingerprint region (400 to 1,600 cm−1). For highly sensitive detection, we produced Rdots composed of 12 different Raman tags in polymer nanoparticles whose detection limit was as low as 12 nM for a short FT-CARS signal integration time of 420 µs. We performed multiplex flow cytometry of MCF-7 breast cancer cells stained by 12 different Rdots with a high classification accuracy of 98%. Moreover, we demonstrated a large-scale time-course analysis of endocytosis via the multiplex Raman flow cytometer. Our method can theoretically achieve flow cytometry of live cells with >140 colors based on a single excitation laser and a single detector without increasing instrument size, cost, or complexity.

Funder

Japan Science and Technology Corporation

Japan Society for the Promotion of Science

Publisher

Oxford University Press (OUP)

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