DEVELOPMENT OF AUTOMATED POLARIZED HIGHT MICROSCOPE

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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.