Given adequate sensitivity, polarizing microscopes provide the means to measure submicroscopic molecular arrangements in living cells and other specimens. The polarizing microscope can thus provide information which is otherwise only available with more intrusive techniques such as electron microscopy. We have been developing and applying a new polarized light microscope (Pol-Scope) which dramatically enhances the unique capabilities of the traditional polarizing microscope. The Pol- Scope incorporates a universal compensator, made from computer driven liquid crystal devices, to measure the birefringent fine structure for all orientations of the birefringence axis within the plane of focus. We propose to develop a 2nd generation Pol-Scope by improving the design and function of the universal compensator and by adding the capability for measuring the magnitude and orientation of birefringences in all three dimensions, including those structures whose birefringence axis do not lie in the focal plane. To increase sensitivity, we will refine the design of the universal compensator by using sectored liquid crystal plates to rectify polarization errors introduced by high numerical aperture lenses. With the rectified Pol-Scope we expect to be able to measure extremely low levels of birefringence such as exhibited by single actin filaments. Also, the rectified system is needed for the 3-D capability of birefringence measurement. To realize the capability of measuring the complete 3-D birefringence indicatix for each resolvable area in the specimen, we will place a rotatable, asymmetric mask (or its equivalent sectored liquid crystal plate) into the back focal plane of the condenser lens. The asymmetric mask will effectively tilt the light path through the specimen. Thus, data for birefringence components parallel to the optical axis of the microscope will become available. Data will be recorded for several mask positions and image records will be combined using digital processing for a complete 3-D analysis of molecular orientation and birefringent fine structure. To test the accuracy and sensitivity of the 2nd generation Pol- Scope, we will use a variety of man-made and biological model systems, including small birefringent crystals and isolated asters. We will extend our collaborative applications projects on cytoskeletal dynamics in motile cells, DNA tertiary structure in chromosomes, and spindles and zona pellucida of mammalian oocytes and their relation to oocyte infertility.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM049210-07A2
Application #
6052377
Study Section
Special Emphasis Panel (ZRG1-SSS-I (02))
Project Start
1992-08-12
Project End
2002-08-31
Budget Start
1999-09-30
Budget End
2000-08-31
Support Year
7
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Marine Biological Laboratory
Department
Type
DUNS #
001933779
City
Woods Hole
State
MA
Country
United States
Zip Code
02543
Janicke, Marie A; Lasko, Loren; Oldenbourg, Rudolf et al. (2007) Chromosome malorientations after meiosis II arrest cause nondisjunction. Mol Biol Cell 18:1645-56
LaFountain Jr, James R; Oldenbourg, Rudolf (2004) Maloriented bivalents have metaphase positions at the spindle equator with more kinetochore microtubules to one pole than to the other. Mol Biol Cell 15:5346-55
Shribak, Michael; Oldenbourg, Rudolf (2003) Three-dimensional birefringence distribution in reconstituted asters of Spisula oocytes revealed by scanned aperture polarized light microscopy. Biol Bull 205:194-5
Shribak, Michael; Oldenbourg, Rudolf (2003) Techniques for fast and sensitive measurements of two-dimensional birefringence distributions. Appl Opt 42:3009-17
LaFountain Jr, J R; Oldenbourg, R; Cole, R W et al. (2001) Microtubule flux mediates poleward motion of acentric chromosome fragments during meiosis in insect spermatocytes. Mol Biol Cell 12:4054-65
Oldenbourg, R; Katoh, K; Danuser, G (2000) Mechanism of lateral movement of filopodia and radial actin bundles across neuronal growth cones. Biophys J 78:1176-82
Katoh, K; Hammar, K; Smith, P J et al. (1999) Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones. Mol Biol Cell 10:197-210
Katoh, K; Hammar, K; Smith, P J et al. (1999) Arrangement of radial actin bundles in the growth cone of Aplysia bag cell neurons shows the immediate past history of filopodial behavior. Proc Natl Acad Sci U S A 96:7928-31
Oldenbourg, R (1999) Polarized light microscopy of spindles. Methods Cell Biol 61:175-208
Silva, C P; Kommineni, K; Oldenbourg, R et al. (1999) The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes. Fertil Steril 71:719-21

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