Microscopy and Microanalysis

Stem Cell Special Section

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

David G. Buschkea1a4, Jayne M. Squirrella4, Hidayath Ansaria4, Michael A. Smitha4, Curtis T. Ruedena4, Justin C. Williamsa1a4a5, Gary E. Lyonsa1a2a4, Timothy J. Kampa3a4, Kevin W. Eliceiria1a4 and Brenda M. Oglea1a4a5 c1

a1 Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA

a2 Department of Anatomy, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA

a3 Departments of Medicine, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA

a4 Laboratory for Optical and Computational Instrumentation, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA

a5 Material Sciences Program, University of Wisconsin at Madison, Madison, Wisconsin 53706, USA

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.

(Received August 06 2009)

(Accepted March 08 2010)

(Online publication August 05 2010)

Correspondence:

c1 Corresponding author. E-mail: ogle@wisc.edu