Virtual-freezing fluorescence imaging flow cytometry

Hideharu Mikami,Hiroshi Watarai,Kazuya Nagasawa ,Hirotoshi Matsumura ,Suguru Ueno ,Chia‐Liang Sun ,Shunsuke Uemura ,Takeshi Sugimura ,Makoto Kawaguchi,Kangrui Huang,Shinsuke Ohnuki,Takanori Maeno,Hiromi Kurokawa,Satoshi Matsusaka,Taichi Miura,Yoshikazu Ohya,Yasuyuki Ozeki,Cheng Lei,Chun-Jung Huang,Takuro Ito,Mai Yamagishi,Keisuke Goda

doi:10.1038/s41467-020-14929-2

Abstract

By virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. However, the performance and utility of IFC are severely limited by the fundamental trade-off between throughput, sensitivity, and spatial resolution. Here we present an optomechanical imaging method that overcomes the trade-off by virtually freezing the motion of flowing cells on the image sensor to effectively achieve 1000 times longer exposure time for microscopy-grade fluorescence image acquisition. Consequently, it enables high-throughput IFC of single cells at >10,000 cells s−1 without sacrificing sensitivity and spatial resolution. The availability of numerous information-rich fluorescence cell images allows high-dimensional statistical analysis and accurate classification with deep learning, as evidenced by our demonstration of unique applications in hematology and microbiology.

Highlights

By virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology
The imaging speed and field of view (FOV) in the y direction can be improved by replacing the scientific complementary metal–oxide–semiconductor (sCMOS) camera with a high-speed camera at the expense of imaging sensitivity
With the flexibility and scalability of virtual-freezing fluorescence imaging (VIFFI) flow cytometry, its capabilities can further be enhanced in various directions, thereby extending the range of applications and discoveries which are accessible by them

Summary

Introduction

By virtue of the combined merits of flow cytometry and fluorescence microscopy, imaging flow cytometry (IFC) has become an established tool for cell analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. We present an optomechanical imaging method that overcomes the trade-off by virtually freezing the motion of flowing cells on the image sensor to effectively achieve 1000 times longer exposure time for microscopygrade fluorescence image acquisition. It enables high-throughput IFC of single cells at >10,000 cells s−1 without sacrificing sensitivity and spatial resolution. The CCD suffers from large readout noise (typically tens of photoelectrons), limiting its detection sensitivity Another approach to overcoming the trade-off is single-pixel imaging that achieves both high-throughput and high-spatial resolution[20,21,22], but comes at the expense of sensitivity. A common trait of these techniques is the compromise of one of the key parameters in favor of the others, limiting the utility of IFC to niche applications

Methods

Results

Conclusion