Fluorescence imaging flow cytometry with ultrahigh sensitivity enabled by virtual motion freezing (Conference Presentation)

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.