DEVELOPMENT OF AUTOMATED POLARIZED HIGHT MICROSCOPE

如果有足够的灵敏度，偏光显微镜提供了测量活细胞和其他标本中亚微观分子排列的手段。因此，偏光显微镜可以提供只有通过电子显微镜等更具侵入性的技术才能获得的信息。我们一直在开发和应用一种新的偏振显微镜(POL-SCOPE)，它极大地增强了传统偏振显微镜的独特能力。Pol-Scope集成了一个由计算机驱动的液晶器件制成的通用补偿器，用于测量焦平面内双折射轴所有方向的双折射精细结构。我们建议通过改进万能补偿器的设计和功能，并增加在所有三个维度上测量双折射的大小和方向的能力，包括那些双折射轴不在焦平面内的结构，来开发第二代极镜。为了提高灵敏度，我们将改进通用补偿器的设计，使用扇形液晶板来校正由大数值孔径透镜引入的偏振误差。使用校正后的波尔显微镜，我们希望能够测量极低水平的双折射，例如单个肌动蛋白细丝所表现出的双折射。此外，为了实现双折射测量的三维能力，还需要校正后的系统。为了实现测量样品中每个可分辨区域的完整三维双折射指示值的能力，我们将在聚光透镜的后焦平面中放置一个可旋转的非对称掩模(或其等效的扇形液晶板)。非对称掩模将有效地倾斜穿过样品的光路。因此，平行于显微镜光轴的双折射分量的数据将变得可用。将记录几个掩模位置的数据，并使用数字处理将图像记录组合在一起，以完成分子取向和双折射精细结构的完整三维分析。为了测试第二代极镜的准确性和灵敏度，我们将使用各种人造和生物模型系统，包括小的双折射晶体和孤立的紫锥菊。我们将扩大我们在活动细胞的细胞骨架动力学、染色体的DNA三级结构、哺乳动物卵母细胞的纺锤体和透明带及其与卵母细胞不孕症的关系等方面的合作应用项目。

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.