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.
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