? Over the past year and a half, we have developed a new interferometric technique in fluorescent imaging called spectral self-interference fluorescent microscopy (SFM). The technique utilizes the spectral oscillations emitted by a fluorophore located a distance of several wavelengths above a reflecting surface. These spectral oscillations are due to the self-interference from the direct and reflected emitted light, analysis of which yields the vertical position of the fluorophore to within a few nanometers. This proposal is to demonstrate SFM as a nanometer resolution fluorescent microscopy technique in both artificial and biological model systems, apply its unique resolution capability to novel biological questions in vivo, and finally, extend the capabilities to arbitrary sectioning and eventual 3D, real-time nanoscale fluorescent imaging. The overall, long-term goal is to develop, demonstrate and apply in vivo subcellular microscopy at an ultimate resolution of 10 nanometers. ? ? The precise three-dimensional localization of proteins within prokaryotic cells is key to many cellular functions, including cell cycle, DNA replication, development, motility, and adhesion. As yet, the basic mechanisms that mediate three-dimensional targeting of proteins in prokaryotes remain largely unknown. Dr. MB Goldberg's laboratory has shown that the targeting of the Shigella actin assembly protein IcsA to the bacterial old pole occurs in the bacterial cytoplasm and involves two specific regions of the polypeptide. We will apply the SFM developed in this proposal to analyze with sub-cellular resolution in live bacteria specific interactions of IcsA with proteins involved in its secretion. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB000756-02
Application #
6660391
Study Section
Special Emphasis Panel (ZRG1-SSS-X (30))
Program Officer
Mclaughlin, Alan Charles
Project Start
2002-09-30
Project End
2007-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
2
Fiscal Year
2003
Total Cost
$320,725
Indirect Cost
Name
Boston University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
049435266
City
Boston
State
MA
Country
United States
Zip Code
02215
Dogan, Mehmet; Aksun, M Irsadi; Swan, Anna K et al. (2009) Closed-form representations of field components of fluorescent emitters in layered media. J Opt Soc Am A Opt Image Sci Vis 26:1458-66
Davis, Brynmor J; Dogan, Mehmet; Goldberg, Bennett B et al. (2007) 4Pi spectral self-interference microscopy. J Opt Soc Am A Opt Image Sci Vis 24:3762-71
Davis, Brynmor J; Swan, Anna K; Unlu, M Selim et al. (2007) Spectral self-interference microscopy for low-signal nanoscale axial imaging. J Opt Soc Am A Opt Image Sci Vis 24:3587-99
Moiseev, Lev; Unlu, M Selim; Swan, Anna K et al. (2006) DNA conformation on surfaces measured by fluorescence self-interference. Proc Natl Acad Sci U S A 103:2623-8