Abstract

The proposed research builds on recent extensions to our hardware and software that allows, in a generalized resolution overlapping fashion, 3-dimensional reconstructions of subcellular structures at both the light and electron microscopic levels. We apply this methodology to the analysis of higher-order chromosome structure and function using the unique interphase and anaphase chromosomes of Drosophila melanogaster. New technology utilizing structured illumination optical microscopy (SIM) and electron microscopy tomography (EMT) allows novel new insights into 3-dimensional chromosome structure. A 3-dimensional Bar Code provides for a systematic approach to the study of 3 dimension diploid interphase chromosome topography in the nucleus on a cell-by-cell basis. Chromosome dynamics, in living nuclei, can now be studied using green fluorescent protein (GFP) to label specific chromosomal loci.
The specific aims of this proposal are: A) Determine the 3 dimensional diploid chromosome topography in Drosophila early embryo nuclei using the 3-dimensional Bar Code methodology. B) Study the 4-dimensional dynamics of diploid chromosome behavior using the newly available GFP chromosome locus labeling methodology. C) Continue the determination of the 3-dimensional structure of anaphase chromosomes using SIM and EMT and their extensions.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025101-27
Application #
6944806
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Lewis, Catherine D
Project Start
1978-04-01
Project End
2007-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
27
Fiscal Year
2005
Total Cost
$641,931
Indirect Cost

  Institution

Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143

  Publications

Shao, L; Winoto, L; Agard, D A et al. (2012) Interferometer-based structured-illumination microscopy utilizing complementary phase relationship through constructive and destructive image detection by two cameras. J Microsc 246:229-36
Kner, Peter; Winoto, Lukman; Agard, David A et al. (2010) Closed loop adaptive optics for microscopy without a wavefront sensor. Proc SPIE Int Soc Opt Eng 7570:
Kner, P; Sedat, J W; Agard, D A et al. (2010) High-resolution wide-field microscopy with adaptive optics for spherical aberration correction and motionless focusing. J Microsc 237:136-47
Matsuda, Atsushi; Shao, Lin; Boulanger, Jerome et al. (2010) Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones. PLoS One 5:e12768
Chang, Henry H; Falick, Arnold M; Carlton, Peter M et al. (2008) N-terminal processing of proteins exported by malaria parasites. Mol Biochem Parasitol 160:107-15
Schermelleh, Lothar; Carlton, Peter M; Haase, Sebastian et al. (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332-6
Gustafsson, Mats G L; Shao, Lin; Carlton, Peter M et al. (2008) Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys J 94:4957-70
Zheng, Shawn Q; Kollman, Justin M; Braunfeld, Michael B et al. (2007) Automated acquisition of electron microscopic random conical tilt sets. J Struct Biol 157:148-55
Xu, Jingsong; Van Keymeulen, Alexandra; Wakida, Nicole M et al. (2007) Polarity reveals intrinsic cell chirality. Proc Natl Acad Sci U S A 104:9296-300
Krug, Roland; Carballido-Gamio, Julio; Burghardt, Andrew J et al. (2007) Wavelet-based characterization of vertebral trabecular bone structure from magnetic resonance images at 3 T compared with micro-computed tomographic measurements. Magn Reson Imaging 25:392-8

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