Quantitative two-photon flow cytometry--in vitro and in vivo
Cheng Frank Zhong1, Eric R Tkaczyk, Thommey Thomas
1University of Michigan, Electrical Engineering and Computer Science Department, Center for Ultrafast Optical Science, 2200 Bonisteel Boulevard, Ann Arbor, Michigan 48109-2099, USA.
Journal of Biomedical Optics
|July 8, 2008
Summary
This study introduces quantitative flow cytometry using two-photon excitation and nanoparticle-based multidye labeling. This method achieves accurate cell analysis even in non-uniform flow conditions, enhancing fluorescence detection.
Area of Science:
- Biophysics
- Optical Engineering
- Cell Biology
Background:
- Flow cytometry traditionally requires uniform flow for accurate quantitative fluorescence measurements.
- Two-photon excitation offers advantages like reduced background fluorescence and simultaneous multi-dye excitation, improving signal-to-noise ratio (SNR).
Purpose of the Study:
- To develop a quantitative flow cytometry technique robust to non-uniform flow conditions.
- To enable precise cell detection, differentiation, and in vivo monitoring using two-photon excitation.
Main Methods:
- Utilized two-photon excitation for optical interrogation of cells.
- Employed targeted multidye labeling with nanoparticles for signal normalization.
- Matched excitation volume to cell size for single-cell detection.
- Implemented a two-beam scanning method for cell size measurement.
Main Results:
- Demonstrated quantitative flow cytometry in non-uniform flow (capillary and in vivo).
- Achieved accurate detection and differentiation of particles and cells.
- Enabled in vivo monitoring of fluorescent dye labeling dynamics.
- Successfully measured cell size in non-uniform flow.
Conclusions:
- Two-photon excitation with multidye nanoparticle labeling enables quantitative flow cytometry in challenging, non-uniform flow environments.
- The technique is suitable for in vitro and in vivo applications, including real-time monitoring and cell sizing.
