Research Papers

Quantitative two-photon flow cytometry—in vitro and in vivo

[+] Author Affiliations
Cheng Frank Zhong

University of Michigan, Electrical Engineering and Computer Science Department, Center for Ultrafast Optical Science, 2200 Bonisteel Boulevard, Ann Arbor, Michigan 48109-2099

Eric R. Tkaczyk

University of Michigan, Electrical Engineering and Computer Science Department, Center for Ultrafast Optical Science, 2200 Bonisteel Boulevard, Ann Arbor, Michigan 48109-2099 and University of Michigan, Michigan Nanotechnology Institute for Medicine and Biological Sciences, Ann Arbor, Michigan 48109-0648

Thommey Thomas

University of Michigan, Michigan Nanotechnology Institute for Medicine and Biological Sciences, Ann Arbor, Michigan 48109-0648

Jing Yong Ye

University of Michigan, Electrical Engineering and Computer Science Department, Center for Ultrafast Optical Science, 2200 Bonisteel Boulevard, Ann Arbor, Michigan 48109-2099 and University of Michigan, Michigan Nanotechnology Institute for Medicine and Biological Sciences, Ann Arbor, Michigan 48109-0648

Andrzej Myc, Anna U. Bielinska, Zhengyi Cao, Istvan Majoros, Balazs Keszler, James R. Baker

University of Michigan, Michigan Nanotechnology Institute for Medicine and Biological Sciences, Ann Arbor, Michigan 48109-0648

Theodore B. Norris

University of Michigan, Electrical Engineering and Computer Science Department, Center for Ultrafast Optical Science, 2200 Bonisteel Boulevard, Ann Arbor, Michigan 48109-2099 and University of Michigan, Michigan Nanotechnology Institute for Medicine and Biological Sciences, Ann Arbor, Michigan 48109-0648

J. Biomed. Opt. 13(3), 034008 (June 20, 2008). doi:10.1117/1.2931077
History: Received October 04, 2007; Revised January 24, 2008; Accepted January 29, 2008; Published June 20, 2008; October 09, 2008
Text Size: A A A

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.

Figures in this Article
© 2008 Society of Photo-Optical Instrumentation Engineers

Citation

Cheng Frank Zhong ; Eric R. Tkaczyk ; Thommey Thomas ; Jing Yong Ye ; Andrzej Myc, et al.
"Quantitative two-photon flow cytometry—in vitro and in vivo", J. Biomed. Opt. 13(3), 034008 (June 20, 2008). ; http://dx.doi.org/10.1117/1.2931077


Access This Article
Sign in or Create a personal account to Buy this article ($20 for members, $25 for non-members).

Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging & repositioning the boxes below.

Related Book Chapters

Topic Collections

PubMed Articles
Advertisement
  • Don't have an account?
  • Subscribe to the SPIE Digital Library
  • Create a FREE account to sign up for Digital Library content alerts and gain access to institutional subscriptions remotely.
Access This Article
Sign in or Create a personal account to Buy this article ($20 for members, $25 for non-members).
Access This Proceeding
Sign in or Create a personal account to Buy this article ($15 for members, $18 for non-members).
Access This Chapter

Access to SPIE eBooks is limited to subscribing institutions and is not available as part of a personal subscription. Print or electronic versions of individual SPIE books may be purchased via SPIE.org.