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Proceedings Paper

Two-photon flow cytometry
Author(s): Cheng Frank Zhong; Jing Yong Ye; Andrzej Myc; Thommey P. Thomas; Anna Bielinska; James R. Baker; Theodore B. Norris
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Paper Abstract

Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantization 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 (T3FC) 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.

Paper Details

Date Published: 30 March 2005
PDF: 12 pages
Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, (30 March 2005); doi: 10.1117/12.589620
Show Author Affiliations
Cheng Frank Zhong, Univ. of Michigan (United States)
Jing Yong Ye, Univ. of Michigan (United States)
Andrzej Myc, Univ. of Michigan Medical School (United States)
Thommey P. Thomas, Univ. of Michigan Medical School (United States)
Anna Bielinska, Univ. of Michigan Medical School (United States)
James R. Baker, Univ. of Michigan Medical School (United States)
Theodore B. Norris, Univ. of Michigan (United States)


Published in SPIE Proceedings Vol. 5700:
Multiphoton Microscopy in the Biomedical Sciences V
Ammasi Periasamy; Peter T. C. So, Editor(s)

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