Fluorescence microscopy has poor spatial resolution (~200nm for the best confocal microscope) owing to the diffraction limit, but provides exquisite dynamical information on live cells. With the development of novel fluorescent sensors and molecules in the past decade, the usage of fluorescence microscopy (especially confocal microscopy) for live-cell studies has greatly increased. The goal of our project is to increase the resolution of confocal imaging techniques to a few tens of nanometers in spatial resolution, using a new microscopy technique that we termed """"""""Difference Deconvolution Microscopy (DDM)"""""""" Furthermore, we plan to increase the temporal resolution of DDM beyond that of traditional scanning confocal microscopy, so we can access even faster dynamical information than currently possible using a standard scanning confocal microscope. We believe DDM, when successfully developed, will increase dramatically the resolution and capability of fluorescence microscopy, particularly for applications in live- cell imaging where laser power needs to be sufficiently low to avoid cellular damage and the time resolution must be sufficiently fast to resolve the dynamics of interest. The goal of this project is to increase the resolution of confocal imaging techniques to the tens of nanometers in length scale and with sub-millisecond time resolution for live-cell imaging, using a new microscopy method that we termed """"""""Difference Deconvolution Microscopy (DDM)"""""""". DDM uses comparable experimental parameters (e.g. similar laser powers) as confocal microscopy, and thus is inherently suited to use with fragile samples, such as live mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085485-02
Application #
7664280
Study Section
Special Emphasis Panel (ZGM1-GDB-7 (EU))
Program Officer
Deatherage, James F
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
$297,322
Indirect Cost
Name
University of Washington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Ye, Fangmao; Smith, Polina B; Chiu, Daniel T (2015) Ultrasensitive Protein Detection on Dot Blots and Western Blots with Semiconducting Polymer Dots. Methods Mol Biol 1314:131-7
Rong, Yu; Yu, Jiangbo; Zhang, Xuanjun et al. (2014) Yellow Fluorescent Semiconducting Polymer Dots with High Brightness, Small Size, and Narrow Emission for Biological Applications. ACS Macro Lett 3:1051-1054
Zhang, Yong; Yu, Jiangbo; Gallina, Maria Elena et al. (2013) Highly luminescent, fluorinated semiconducting polymer dots for cellular imaging and analysis. Chem Commun (Camb) 49:8256-8
Sun, Wei; Ye, Fangmao; Gallina, Maria E et al. (2013) Lyophilization of semiconducting polymer dot bioconjugates. Anal Chem 85:4316-20
Neupane, Bhanu; Chen, Fang; Sun, Wei et al. (2013) Tuning donut profile for spatial resolution in stimulated emission depletion microscopy. Rev Sci Instrum 84:043701
Zhang, Xuanjun; Yu, Jiangbo; Rong, Yu et al. (2013) High-intensity near-IR fluorescence in semiconducting polymer dots achieved by cascade FRET strategy. Chem Sci 4:2143-2151
Wu, Changfeng; Chiu, Daniel T (2013) Highly fluorescent semiconducting polymer dots for biology and medicine. Angew Chem Int Ed Engl 52:3086-109
Sun, Wei; Yu, Jiangbo; Deng, Ruiping et al. (2013) Semiconducting polymer dots doped with europium complexes showing ultranarrow emission and long luminescence lifetime for time-gated cellular imaging. Angew Chem Int Ed Engl 52:11294-7
Zeigler, Maxwell B; Chiu, Daniel T (2013) Single-cell nanosurgery. Methods Mol Biol 991:139-48
Ye, Fangmao; Smith, Polina B; Wu, Changfeng et al. (2013) Ultrasensitive detection of proteins on Western blots with semiconducting polymer dots. Macromol Rapid Commun 34:785-90

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