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

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.

Endogenous Optical Signals Reveal Changes of Elastin and Collagen Organization During Differentiation of Mouse Embryonic Stem Cells

“…[31][32][33] Leukemia inhibitory factor (Millipore) and bone morphogenetic protein 4 (R&D Systems) were added to media at 2000 U/ mL and 10 ng/mL, respectively, to maintain pluripotency. 34 Mouse EBs were prepared using the hanging drop method.…”

“…Mouse EBs were imaged using multiphoton laser scanning microscopy (MPLSM) 32 with the following excitation and emission settings: for endogenous Eln-780 nm excitation, 520/35 nm emission; for ColI SHG (backward)-890 nm excitation, 445/20 nm emission; for Eln and ColI Ab-890 nm excitation, 520/35 nm emission. Images were collected with a Plan Apo VC, 20 · air, 0.75 NA, 1.0-mm WD objective (Nikon), at a resolution of 512 · 512.…”

‘Go with the flow ’: A review of methods and advancements in blood flow imaging

“…Yet, despite these limiting factors, confocal and two-photon imaging have been used in a wide variety of applications: rodent cerebral microcirculation investigations [275][276][277][278]; used in conjunction with particle image velocimetry (see § 4.1) to investigate micro-channel blood flow [279]; as a quantitative method in the study of embryonic vasculature dynamics [280,281]; dynamic studies of Alzheimer's disease and epilepsy [274]; and in cytometric studies of the relationships between stem cells [282], tumour cells [283] and their tissue environments [284,285].…”

A hardware accelerated approach for imaging flow cytometry

“…In particular, in imaging cytometry technology to analyze cells and micro particles [2,3]. Buschke et al present a cell analysis system for image cytometry using a DSP and FPGA [3].…”