Multiphoton flow cytometry to assess intrinsic and extrinsic fluorescence in cellular aggregates: Applications to stem cells

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, Brenda M. Ogle

Research output: Contribution to journalArticle

18 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)540-554
Number of pages15
JournalMicroscopy and Microanalysis
Volume17
Issue number4
DOIs
StatePublished - Aug 1 2011

Fingerprint

cytometry
Flow cytometry
stem cells
Stem cells
Fluorescence
fluorescence
cells
Throughput
excitation
disrupting
Fluorophores
biomarkers
Biomarkers
Microscopic examination
Screening
screening
Cells
Tissue
microscopy
Scanning

Keywords

  • differentiation
  • endogenous fluorophores
  • flow cytometry
  • fluorescence
  • multiphoton microscopy
  • stem cells

Cite this

Multiphoton flow cytometry to assess intrinsic and extrinsic fluorescence in cellular aggregates : Applications to stem cells. / Buschke, David G.; Squirrell, Jayne M.; Ansari, Hidayath; Smith, Michael A.; Rueden, Curtis T.; Williams, Justin C.; Lyons, Gary E.; Kamp, Timothy J.; Eliceiri, Kevin W.; Ogle, Brenda M.

In: Microscopy and Microanalysis, Vol. 17, No. 4, 01.08.2011, p. 540-554.

Research output: Contribution to journalArticle

Buschke, DG, Squirrell, JM, Ansari, H, Smith, MA, Rueden, CT, Williams, JC, Lyons, GE, Kamp, TJ, Eliceiri, KW & Ogle, BM 2011, 'Multiphoton flow cytometry to assess intrinsic and extrinsic fluorescence in cellular aggregates: Applications to stem cells', Microscopy and Microanalysis, vol. 17, no. 4, pp. 540-554. https://doi.org/10.1017/S1431927610000280
Buschke, David G. ; Squirrell, Jayne M. ; Ansari, Hidayath ; Smith, Michael A. ; Rueden, Curtis T. ; Williams, Justin C. ; Lyons, Gary E. ; Kamp, Timothy J. ; Eliceiri, Kevin W. ; Ogle, Brenda M. / Multiphoton flow cytometry to assess intrinsic and extrinsic fluorescence in cellular aggregates : Applications to stem cells. In: Microscopy and Microanalysis. 2011 ; Vol. 17, No. 4. pp. 540-554.
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