scholarly journals High-throughput multicolor 3D localization in live cells by depth-encoding imaging flow cytometry

2019 ◽  
Author(s):  
Lucien E. Weiss ◽  
Yael Shalev Ezra ◽  
Sarah E. Goldberg ◽  
Boris Ferdman ◽  
Yoav Shechtman
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Imaging System ◽  
Live Cells ◽  
Flow Profile ◽  
Imaging Flow Cytometry ◽  
3D Localization ◽  
Large Populations ◽  
Point Spread Function Engineering

ABSTRACTImaging flow cytometry replaces the canonical point-source detector of flow cytometry with a camera, unveiling subsample details in 2D images while maintaining high-throughput. Here we show that the technique is inherently compatible with 3D localization microscopy by point-spread-function engineering, namely the encoding of emitter depth in the emission pattern captured by a camera. By exploiting the laminar-flow profile in microfluidics, 3D positions can be extracted from cells or other objects of interest by calibrating the depth-dependent response of the imaging system using fluorescent microspheres mixed with the sample buffer. We demonstrate this approach for measuring fluorescently-labeled DNA in vitro and the chromosomal compaction state in large populations of live cells, collecting thousands of samples each minute. Furthermore, our approach is fully compatible with existing commercial apparatus, and can extend the imaging volume of the device, enabling faster flowrates thereby increasing throughput.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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

Cell Reports ◽  
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

scholarly journals High‐Throughput Particle Uptake Analysis by Imaging Flow Cytometry

2017 ◽  
Vol 80 (1) ◽  
Author(s):  
Asya Smirnov ◽  
Michael D. Solga ◽  
Joanne Lannigan ◽  
Alison K. Criss
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Particle Uptake ◽  
Imaging Flow Cytometry

scholarly journals Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro

Mutagenesis ◽  
2018 ◽  
Vol 33 (4) ◽  
pp. 283-289 ◽  
Author(s):  
Jatin R Verma ◽  
Danielle S G Harte ◽  
Ume-Kulsoom Shah ◽  
Huw Summers ◽  
Catherine A Thornton ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lymphoblastoid Cells ◽  
Imaging Flow Cytometry ◽  
Chemically Induced

scholarly journals High-Throughput Screen Identifies Cyclic Nucleotide Analogs That Inhibit Prostatic Acid Phosphatase

2012 ◽  
Vol 18 (4) ◽  
pp. 481-489 ◽  
Author(s):  
Eric S. McCoy ◽  
Wendy A. Lea ◽  
Bryan T. Mott ◽  
David J. Maloney ◽  
Ajit Jadhav ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
High Throughput ◽  
Cyclic Nucleotide ◽  
Prostatic Acid Phosphatase ◽  
Live Cells ◽  
Nucleotide Analogs ◽  
Small Molecule Modulators ◽  
Pharmacologically Active ◽  
Kinetic Mode

The secretory and transmembrane isoforms of prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5′-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small-molecule modulators of PAP, we used a 1536-well–based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC1280) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.

Optofluidic time-stretch imaging – an emerging tool for high-throughput imaging flow cytometry

Lab on a Chip ◽  
2016 ◽  
Vol 16 (10) ◽  
pp. 1743-1756 ◽  
Author(s):  
Andy K. S. Lau ◽  
Ho Cheung Shum ◽  
Kenneth K. Y. Wong ◽  
Kevin K. Tsia
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Cell Imaging ◽  
Imaging Flow Cytometry ◽  
Single Cell Imaging ◽  
Optical Time ◽  
High Throughput Imaging

Optical time-stretch imaging is now proven for ultrahigh-throughput optofluidic single-cell imaging, at least 10–100 times faster.

scholarly journals Label Free Identification of Peripheral Blood Eosinophils Using High-Throughput Imaging Flow Cytometry

2017 ◽  
Vol 139 (2) ◽  
pp. AB163
Author(s):  
Justyna Piasecka ◽  
Holger Hennig ◽  
Fabian J. Theis ◽  
Paul Rees ◽  
Huw D. Summers ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Peripheral Blood ◽  
Label Free ◽  
Imaging Flow Cytometry ◽  
Blood Eosinophils ◽  
High Throughput Imaging
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