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30 March 2005 Two-photon flow cytometry
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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.
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Cheng Frank Zhong, Jing Yong Ye, Andrzej Myc, Thommey P. Thomas, Anna Bielinska, James R. Baker Jr., and Theodore B. Norris "Two-photon flow cytometry", Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, (30 March 2005); https://doi.org/10.1117/12.589620
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