We are developing a new and automated polarized light microscope (""""""""pol- scope"""""""") which dramatically enhances the unique capabilities of the traditional pol-scope. Current capabilities of the new system include: Measurement of specimen retardance, regardless of orientation and concurrently for the whole field of view, at the full spatial resolution of the light microscope, with a sensitivity of 0. l nm retardance magnitude and angular resolution of 1 degree. Complete image records at a single focus level are obtained in 0.43 seconds documenting fine structural and molecular organization within a thin optical section of the specimen. We propose to enhance the capabilities of the existing instrument in at least three significant areas: speed of data acquisition (elapsed time equals approximately 10 ms), sensitivity (0.02 nm retardance), and spatial resolution (0.2 micromoles in all 3 dimensions). Enhanced spatial resolution of retardance measurements will be achieved through the development of 3-D deconvolution algorithms for optical sections of polarized light images. All these improvements, from which other types of light microscopy will benefit as well, will substantially broaden the applications of the new pol-scope, especially for the study of 3- dimensional architectural dynamics in living cells. Specifically, we propose to measure with the improved system: (a) microtubule retardance (in vitro and in vivo) as a function of microtubule dynamics and association with other proteins (MAP's); (b)microtubule and other protein retardances as a function of wavelength to explore the potential of retardance dispersion as indicator for chemical composition; (c) the 3-D architecture of the mitotic spindle and its relationship to position and movement of spindle poles and chromosomes; (d) chromosome and DNA tertiary structure of (decondensing) sperm; and (e) spatial and temporal birefringence fluctuations in virus liquid crystals determining the dynamic and viscoelastic properties of well-characterized model systems of ordered arrays of molecular aggregates in aqueous suspension. We are thus proposing to continue and intensify collaborative efforts already underway in most of the named application areas.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049210-06
Application #
2459483
Study Section
Special Emphasis Panel (ZRG7-SSS-3 (10))
Project Start
1992-08-12
Project End
1999-07-31
Budget Start
1997-08-01
Budget End
1999-07-31
Support Year
6
Fiscal Year
1997
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
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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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