The research goal of the ROO phase of this Pathway to Independence Award is to develop a general purpose multiplexing fluorescence microscopic platform with single monomer sensitivity. The instrument will perform lifetime and radiometric measurements on excitation and emission simultaneously, which allows quantitative measurements on multi-color FOrster Resoiiant Energy Transfer (FRET). In the K99 phase of the award, we worked on developing a high-speed Simultaneous Fluorescence Lifetime Excitation Emission (SFLEE) spectrometer. In the ROO phase, we will improve the sensitivity of the SFLEE technique, and apply the technique to multi-color FRET investigations. Fast lifetime and intensity excitation-emission matrices obtained by the SLFEE instrument will allow real time measurements of FOrster Resonant Energy Transfer between more than three fluorescent labels. To demonstrate the instrument's capability, we perform quantitative four-color FRET on single DNA Holliday junction. Specifically, we will study structure dynamics of single DNA Holliday junctions undergoing global confirmation switching and branch migration. The long term objective of this research is to provide a high-throughput multiplexed imaging tool for both fundamental and biomedical research.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Transition Award (R00)
Project #
4R00EB008737-02
Application #
7890714
Study Section
Special Emphasis Panel (NSS)
Program Officer
Zhang, Yantian
Project Start
2008-08-01
Project End
2012-07-31
Budget Start
2009-08-01
Budget End
2010-07-31
Support Year
2
Fiscal Year
2009
Total Cost
$248,502
Indirect Cost
Name
University of Arizona
Department
Type
Other Domestic Higher Education
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Zhao, Ming; Wan, Xiaoyang; Li, Yu et al. (2015) Multiplexed 3D FRET imaging in deep tissue of live embryos. Sci Rep 5:13991
Zhao, Ming; Li, Yu; Peng, Leilei (2014) Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging. Opt Express 22:10221-32
Zhao, Ming; Li, Yu; Peng, Leilei (2014) FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging. Opt Express 22:23073-85
Kim, Sung-Eun; Huang, He; Zhao, Ming et al. (2013) Wnt stabilization of ?-catenin reveals principles for morphogen receptor-scaffold assemblies. Science 340:867-70
Zhao, Ming; Huang, Run; Peng, Leilei (2012) Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy. Opt Express 20:26806-27
Zhang, Han; Zhao, Ming; Peng, Leilei (2011) Nonlinear structured illumination microscopy by surface plasmon enhanced stimulated emission depletion. Opt Express 19:24783-94
Zhao, Ming; Peng, Leilei (2010) Multiplexed fluorescence lifetime measurements by frequency-sweeping Fourier spectroscopy. Opt Lett 35:2910-2