Ultra High-Throughput Multiparametric Imaging Flow Cytometry: Towards Diffraction-Limited Sub-Cellular Detection

ABSTRACTFlow cytometry is widely recognized as the gold-standard technique for the analysis and enumeration of heterogeneous cellular populations and has become an indispensable tool in diagnostics,1 rare-cell detection2 and single-cell proteomics.3 Although contemporary flow cytometers are able to analyse many thousands of cells per second, with classification based on scattering or fluorescence criteria, the vast majority require unacceptably large sample volumes, and do not allow the acquisition of spatial information. Herein, we report a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence and brightfield detection at analytical throughputs in excess of 60,000 cells/s and 400,000 cells per second respectively. Our imaging platform is capable of multi-parametric fluorescence quantification and subcellular (co-)localization analysis of cellular structures down to 500 nm with microscopy image quality. We demonstrate the efficacy of our approach by performing challenging high-throughput localization analysis of cytoplasmic RNA granules in yeast and human cells. Results suggest significant utility of the imaging flow cytometer in the screening of rare events at the subcellular level for diagnostic applications.

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Webcam-based flow cytometer using wide-field imaging for low cell number detection at high throughput

The Analyst ◽

10.1039/c4an00669k ◽

2014 ◽

Vol 139 (17) ◽

pp. 4322-4329 ◽

Cited By ~ 10

Author(s):

Joshua Balsam ◽

Hugh Alan Bruck ◽

Avraham Rasooly

Keyword(s):

Flow Cytometry ◽

High Throughput ◽

Large Volume ◽

Current Flow ◽

Low Cost ◽

Cell Number ◽

Flow Cytometer ◽

Wide Field ◽

Field Imaging ◽

Wide Field Imaging

Here we describe a novel low-cost high throughput flow cytometer based on a webcam capable of low cell number detection in a large volume which may overcome the limitations of current flow cytometry.

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High-throughput label-free molecular fingerprinting flow cytometry

Science Advances ◽

10.1126/sciadv.aau0241 ◽

2019 ◽

Vol 5 (1) ◽

pp. eaau0241 ◽

Cited By ~ 37

Author(s):

Kotaro Hiramatsu ◽

Takuro Ideguchi ◽

Yusuke Yonamine ◽

SangWook Lee ◽

Yizhi Luo ◽

...

Keyword(s):

Flow Cytometry ◽

Single Cell ◽

High Throughput ◽

Single Cell Analysis ◽

Single Cells ◽

Fluorescent Labeling ◽

Live Cells ◽

Molecular Fingerprinting ◽

Label Free ◽

Indispensable Tool

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm−1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics ofHaematococcus lacustris.

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HTPS Flow Cytometry: A Novel Platform for Automated High Throughput Drug Discovery and Characterization

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710100600204 ◽

2001 ◽

Vol 6 (2) ◽

pp. 83-90 ◽

Cited By ~ 23

Author(s):

Bruce S. Edwards ◽

Frederick W. Kuckuck ◽

Erick R. Prossnitz ◽

John T. Ransom ◽

Larry A. Sklar

Keyword(s):

Flow Cytometry ◽

Drug Discovery ◽

Intracellular Calcium ◽

High Throughput ◽

Combinatorial Libraries ◽

Flow Cytometer ◽

Automated System ◽

Primary Screening ◽

Formyl Peptide ◽

Multifactorial Approach

The flow cytometer is unique among biomedical analysis instruments because it makes simultaneous and multiple optical measurements on individual cells or particles at high rates. High throughput flow cytometry represents a potentially important multifactorial approach for screening large combinatorial libraries of compounds. Limiting this approach has been the availability of instrumentation and methods in flow cytometry for automated sample handling on the scale required for drug discovery applications. Here, we describe an automated system in which a novel patented fluidics-based pharmacology platform, the HTPS (High Throughput Pharmacological System), is coupled to a flow cytometer using a recently described plug flow-coupling valve technology. Individual samples are aspirated sequentially from microplate wells and delivered to a flow cytometer for rapid multiparametric analysis. For primary screening to detect and quantify cell fluorescence in endpoint assays, a high-speed no-wash protocol enabled processing of 9-10 cell samples/min from 96-well microplates. In an alternate primary screening format, soluble receptor ligands were sampled from microplate wells at rates of 3-4 samples/minute and successfully assessed for the ability to elicit intracellular calcium responses. Experiments with fluorescent beads validated the accurate automated production by the HTPS of exponential and linear gradients of soluble compounds. This feature enabled rapid (2- to 3-min) characterization of the intracellular calcium dose response of myeloid cells to formyl peptide as well as the quantitative relationship between formyl peptide receptor occupancy and cell response. HTPS flow cytometry thus represents a powerful high throughput multifactorial approach to increase the efficiency with which novel bioresponse-modifying drugs may be identified and characterized.

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Impact of the New Beckman Coulter Cytomics FC 500 5-Color Flow Cytometer on a Regional Flow Cytometry Clinical Laboratory Service

Laboratory Hematology ◽

10.1532/lh96.04121 ◽

2004 ◽

Vol 10 (2) ◽

pp. 102-108 ◽

Cited By ~ 12

Author(s):

J. Luider ◽

M. Cyfra ◽

P. Johnson ◽

I. Auer

Keyword(s):

Flow Cytometry ◽

Clinical Laboratory ◽

Flow Cytometer ◽

Color Flow ◽

Laboratory Service ◽

Regional Flow

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High Throughput Microfluidic Electrical Impedance Flow Cytometry for Assay of Micro Particles

Micro and Nanosystems ◽

10.2174/1876402911002040298 ◽

2010 ◽

Vol 2 (4) ◽

pp. 298-308 ◽

Cited By ~ 2

Author(s):

Ashish V. Jagtiani ◽

Jiang Zhe

Keyword(s):

Flow Cytometry ◽

High Throughput ◽

Electrical Impedance ◽

Micro Particles

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A deep learning-based concept for high throughput image flow cytometry

Applied Physics Letters ◽

10.1063/5.0037336 ◽

2021 ◽

Vol 118 (12) ◽

pp. 123701

Author(s):

Julie Martin-Wortham ◽

Steffen M. Recktenwald ◽

Marcelle G. M. Lopes ◽

Lars Kaestner ◽

Christian Wagner ◽

...

Keyword(s):

Flow Cytometry ◽

Deep Learning ◽

High Throughput ◽

Image Flow

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High-throughput multiparametric imaging flow cytometry: toward diffraction-limited sub-cellular detection and monitoring of sub-cellular processes

Cell Reports ◽

10.1016/j.celrep.2021.108824 ◽

2021 ◽

Vol 34 (10) ◽

pp. 108824

Author(s):

Gregor Holzner ◽

Bogdan Mateescu ◽

Daniel van Leeuwen ◽

Gea Cereghetti ◽

Reinhard Dechant ◽

...

Keyword(s):

Flow Cytometry ◽

High Throughput ◽

Multiparametric Imaging ◽

Cellular Processes ◽

Imaging Flow Cytometry

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Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽

10.3390/diagnostics11081320 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1320

Author(s):

Kristýna Pekárková ◽

Jakub Soukup ◽

Marie Kostelanská ◽

Jan Širc ◽

Zbyněk Straňák ◽

...

Keyword(s):

Flow Cytometry ◽

Cord Blood ◽

Extracellular Vesicles ◽

Correlation Coefficients ◽

Flow Cytometer ◽

Liquid Biopsies ◽

Physiological Conditions ◽

Flow Cytometric ◽

Term Births ◽

And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

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