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Two-Photon Flow CytometryFlow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.
Document ID
20040111394
Acquisition Source
Ames Research Center
Document Type
Other
Authors
Zhog, Cheng Frank
(Michigan Univ. Ann Arbor, MI, United States)
Ye, Jing Yong
(Michigan Univ. Ann Arbor, MI, United States)
Norris, Theodore B.
(Michigan Univ. Ann Arbor, MI, United States)
Myc, Andrzej
(Michigan Univ. Ann Arbor, MI, United States)
Cao, Zhengyl
(Michigan Univ. Ann Arbor, MI, United States)
Bielinska, Anna
(Michigan Univ. Ann Arbor, MI, United States)
Thomas, Thommey
(Michigan Univ. Ann Arbor, MI, United States)
Baker, James R., Jr.
(Michigan Univ. Ann Arbor, MI, United States)
Date Acquired
August 21, 2013
Publication Date
January 1, 2004
Subject Category
Fluid Mechanics And Thermodynamics
Funding Number(s)
CONTRACT_GRANT: NAS2-02069
Distribution Limits
Public
Copyright
Work of the US Gov. Public Use Permitted.

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