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Multiphoton Flow Cytometry to Assess Intrinsic and Extrinsic Fluorescence in Cellular Aggregates: Applications to Stem Cells

Published online by Cambridge University Press:  05 August 2010

David G. Buschke ,
Jayne M. Squirrell ,
Hidayath Ansari ,
Michael A. Smith ,
Curtis T. Rueden ,
Justin C. Williams ,
Gary E. Lyons ,
Timothy J. Kamp ,
Kevin W. Eliceiri  and
Brenda M. Ogle
David G. Buschke
Affiliation:
Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Jayne M. Squirrell
Affiliation:
Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Hidayath Ansari
Affiliation:
Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Michael A. Smith
Affiliation:
Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Curtis T. Rueden
Affiliation:
Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Justin C. Williams
Affiliation:
Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Material Sciences Program, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Gary E. Lyons
Affiliation:
Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Department of Anatomy, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Timothy J. Kamp
Affiliation:
Departments of Medicine, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Kevin W. Eliceiri
Affiliation:
Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
Brenda M. Ogle*
Affiliation:
Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA Material Sciences Program, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA
*
Corresponding author. E-mail: ogle@wisc.edu
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Abstract

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

Keywords

stem cellsendogenous fluorophoresfluorescencemultiphoton microscopyflow cytometrydifferentiation
Type
Research Article
Information
Microscopy and Microanalysis , Volume 17 , Issue 4 , August 2011 , pp. 540 - 554
Copyright
Copyright © Microscopy Society of America 2010

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References

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