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Review
, 16 (24), 4639-4647

Review: Imaging Technologies for Flow Cytometry

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Review

Review: Imaging Technologies for Flow Cytometry

Yuanyuan Han et al. Lab Chip.

Abstract

High-throughput single cell imaging is a critical enabling and driving technology in molecular and cellular biology, biotechnology, medicine and related areas. Imaging flow cytometry combines the single-cell imaging capabilities of microscopy with the high-throughput capabilities of conventional flow cytometry. Recent advances in imaging flow cytometry are remarkably revolutionizing single-cell analysis. This article describes recent imaging flow cytometry technologies and their challenges.

Figures

Figure 1
Figure 1
Optics of ImageStream. Reproduced from Ref. with permission from the EMD Millipore Corporation.
Figure 2
Figure 2
Multiple Field of View Imaging Flow Cytometer. (a) Diffractive lens wide field imaging system. L1 collimates the LED, L2 is a condenser that images Iris 1 onto the object plane. DL is the diffractive lens providing multiple field of view. L3 is a relay lens. (b) 16 object planes. (c) 16 imaging planes. Sample: 3.5um latex beads. Reproduced from Ref. with permission from the Royal Society of Chemistry.
Figure 3
Figure 3
Coded excitation fluorescence microscope. (a) Chopper wheel that modulates the excitation beam with a pseudo-random code. (b) a microfluidic device imaged by 40X fluorescent imaging microscope. (c) Raw blur encoded images captured by the camera and decoded images after computational approach. Reproduced from Ref. with permission from the Optical Society.
Figure 4
Figure 4
Schematic of the STEAM flow analyzer. Imaging and illumination optics takes blur-free images by encoding object location information into spectral domain. ADFT converts spectral information into time domain through time-stretch method and processed by real-time imaging processor. Reproduced from Ref. with permission from the PNAS.
Figure 5
Figure 5
FIRE microscopy. a, Schematic diagram of the FIRE microscope. BS, beamsplitter; AOD, acousto-optic deflector; AOFS, acousto-optic frequency shifter; DM, dichroic mirror; EF, fluorescence emission filter; OL, objective lens; PMT, photomultiplier tube; DIG, 250 MS digital recording oscilloscope; RS, resonant scanning mirror. Upper inset: the AOD produces a single diffracted first-order beam for each radiofrequency comb frequency. Lower inset: beat frequency generation at the MZI output. b, Gabor lattice diagram of FIRE’s frequency-domain multiplexing approach. Points in same horizontal line are excited in parallel at distinct radiofrequencies. The horizontal line is scanned by a galvo-mirror to acquire a 2D image. Reproduced from Ref. with permission from the Nature Publishing Group.
Figure 6
Figure 6
Implementation of spatial-temporal transformation-based IFC. (a) Schematic diagram of the imaging flow cytometer system. DM, dichroic mirror; SF, spatial filter; EF, emission filter; PMT, photomultiplier tube. (b) Spatial filter design that has ten 100 μm by 1 mm slits positioned apart in the way of one is immediately after another in both x-direction and y-direction. (c) Experimental result: time-domain PMT output signal of fluorescent light from a A549 cell stained with CellTrace CFSE, corresponding original fluorescence image restored by algorithm, and corresponding resized fluorescence image to show the real size of the cell. The numbered regions segmented by dashed lines demonstrate the correspondence between the time-domain signal and the resulting image. Size is labelled in figure. Reproduced from Ref. with permission from the Nature Publishing Group.
Figure 7
Figure 7
Demonstration of bright-field, two-color fluorescence and backscattering cell images. Scale bars are 5 μm. (a) Bright-field images of MDA-MB-231 human breast cancer cells flowing at 0.2 m/s. (b) Representative two-color fluorescent images of MDAMB-231 human breast cancer cells stained with CellTrace CFSE, cell membrane bond with 1 μm fluorescent beads, flowing in the microfluidic channel at 0.25 m/s. Ref. (b) Representative fluorescence plus backscattering cell images from spatial filter based imaging flow cytometry. All images are of A549 human lung adenocarcinoma epithelial cells, stained with CellTrace CFSE, flowing at a velocity of 0.2 m/s. Reproduced from Ref. with permission from the Nature Publishing Group.

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