Cited by 116Cell Image Analysis TechniquesAdvanced Fluorescence Microscopy TechniquesCCD and CMOS Imaging Sensors

Sensitivity measurement and compensation in spectral imaging

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Ortyn, William E., Hall, Brian E., George, Thaddeus C., Frost, Keith, Basiji, David A., Perry, David J., Zimmerman, Cathleen A., Coder, David +1 more·2006·Cytometry Part A

Abstract

Background: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. Methods: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. Results: Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT‐based flow cytometer. A methodology for addressing spectral overlap, individual pixel anomalies, and multiple imaging modalities was demonstrated for spectral compensation of K562 cells. Imagery is shown pre‐ and post‐compensation. Conclusions: Unlike intensity measurements made with conventional flow cytometers, object size impacts both dynamic range and fluorescence sensitivity in systems that utilize pixilated detection. Simultaneous imaging of alternate modalities can be employed to increase fluorescent sensitivity. Effective compensation of complex multimode imagery spanning six spectral bands is accomplished in a semi‐automated manner. © 2006 International Society for Analytical Cytology

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Crossref · 10.1002/cyto.a.20306

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  • Single color controls for each fluorochrome were acquired to generate the compensation matrix that was applied to each sample prior to analysis.

    “Single color controls for each fluorochrome were acquired to generate the compensation matrix that was applied to each sample prior to analysis (63).”

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Authors
Ortyn, W. E., Hall, B. E., George, T. C., Frost, K., Basiji, D. A., Perry, D. J., Zimmerman, C. A., Coder, D., & Morrissey, P. J.
Corresponding author
Ortyn, William E.
Venue
Cytometry Part A
Publisher
Wiley
Published
2006-08
Volume
69A
Issue
8
Pages
852-862
ISSN
  • 1552-4922 (ISSN-L)
  • 1552-4930
OpenAlex
W2149072868
Cited by
115
References
30
Citations by year
2026: 3 · 2025: 2 · 2024: 3 · 2023: 3 · 2022: 4 · 2021: 6 · 2020: 2 · 2019: 2 · 2018: 5 · 2017: 1 · 2016: 3 · 2015: 15 · 2014: 5 · 2013: 7 · 2012: 9
FWCI
5.94
Type
journal article
Language
en
Open access
Locations
  • Cytometry Part A (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Cancer Institute (9 R44 CA01798-02, R44CA001798)
  • National Institute of General Medical Sciences (R43GM058956, Q R43 GM58956-01)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Physical Sciences › Engineering › Electrical and Electronic Engineering › CCD and CMOS Imaging Sensors
Keywords
spectral imaging, flow cytometry, fluorescence sensitivity, dynamic range, spectral compensation, spectral overlap, multispectral imaging, K562 cells, fluorescent microspheres, CCD detection, multimodal imaging
MeSH terms
Diagnostic Imaging, Flow Cytometry, Fluorescence, Fluorescent Antibody Technique, Humans, Mathematics, Microspheres, Sensitivity and Specificity, Spectrometry, Fluorescence, Spectrum Analysis, Image Cytometry, Cell Line, Tumor

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