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Journal of Biomedical Optics

Quantitative two-photon flow cytometry—in vitro and in vivo
Author(s): Cheng Zhong; Eric R. Tkaczyk; Thommey P. Thomas; Jing Yong Ye; Andrzej Myc; Anna Bielinska; Zhengyi Cao; Istvan J. Majoros; Balazs Keszler; James R. Baker; Theodore B. Norris
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Paper Abstract

Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.

Paper Details

Date Published: 1 May 2008
PDF: 19 pages
J. Biomed. Opt. 13(3) 034008 doi: 10.1117/1.2931077
Published in: Journal of Biomedical Optics Volume 13, Issue 3
Show Author Affiliations
Cheng Zhong, Univ. of Michigan (United States)
Eric R. Tkaczyk, Univ. of Michigan (United States)
Thommey P. Thomas, Univ. of Michigan (United States)
Jing Yong Ye, Univ. of Michigan (United States)
Andrzej Myc, Univ. of Michigan (United States)
Anna Bielinska, Univ. of Michigan (United States)
Zhengyi Cao, Univ. of Michigan (United States)
Istvan J. Majoros, Univ. of Michigan (United States)
Balazs Keszler, Univ. of Michigan (United States)
James R. Baker, Univ. of Michigan (United States)
Theodore B. Norris, Univ. of Michigan (United States)


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