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4 March 2019 Fluorescence imaging flow cytometry with ultrahigh sensitivity enabled by virtual motion freezing (Conference Presentation)
Hideharu Mikami, Makoto Kawaguchi, Chun-Jung Huang, Cheng Lei, Shunnosuke Ueno, Taichi Miura, Takuro Ito, Kazumichi Nagasawa, Takanori Maeno, Hiroshi Watarai, Chia-Wei Sun, Yasuyuki Ozeki, Keisuke Goda
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Proceedings Volume 10889, High-Speed Biomedical Imaging and Spectroscopy IV; 108890T (2019) https://doi.org/10.1117/12.2507577
Event: SPIE BiOS, 2019, San Francisco, California, United States
Abstract
While fluorescence imaging flow cytometry is a promising method for high-throughput single-cell analysis, it has not been suitable for analysis of large populations of cells (e.g., blood samples) due to its low imaging sensitivity at a high cell throughput. Here we present fluorescence imaging flow cytometry with an ultrahigh imaging sensitivity, which is enabled by virtual motion freezing. In this method, we prepare a wide-field imaging system with a CMOS camera and scan images of flowing cells by a scanning device, such as a polygon scanner, equipped in the imaging system so that the motion of the cells is canceled in the imaging plane, thus significantly extending the exposure time of the camera without suffering from motion blur. Additionally, we scan a loosely focused excitation beam during the exposure time of the camera in the direction opposite to the cell flow using a beam scanner such as an acousto-optic deflector, which significantly reduces motion cancellation errors caused by the image distortion of the imaging system and hence allows further extension of the exposure time. Consequently, our method improves imaging sensitivity by a factor of ~1,000 compared with a conventional wide-field excitation method, enabling acquisition of microscopy-grade images of fast flowing cells. As a proof-of-concept, we obtained fluorescence images of nuclei of murine white blood cells stained by SYTO16 at a flow speed of 1 m/s (corresponding to a cell throughput of 10,000 cells/s assuming the 100-μm cell spacing) and determined the population of nuclear lobulation from the high-signal-to-noise-ratio images obtained.
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Hideharu Mikami, Makoto Kawaguchi, Chun-Jung Huang, Cheng Lei, Shunnosuke Ueno, Taichi Miura, Takuro Ito, Kazumichi Nagasawa, Takanori Maeno, Hiroshi Watarai, Chia-Wei Sun, Yasuyuki Ozeki, and Keisuke Goda "Fluorescence imaging flow cytometry with ultrahigh sensitivity enabled by virtual motion freezing (Conference Presentation)", Proc. SPIE 10889, High-Speed Biomedical Imaging and Spectroscopy IV, 108890T (4 March 2019); https://doi.org/10.1117/12.2507577
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KEYWORDS
Luminescence
Flow cytometry
Imaging systems
Blood
Cameras
Statistical analysis
Acousto-optics
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