Development of New Automated Polarized Light Microscope

描述(由申请人提供)： 我们正在建造和应用一种基于液晶技术的超灵敏和高分辨率偏振光显微镜(LC-PolScope)。我们使用独特的仪器来揭示和破译细胞骨架动态结构的功能作用，重点放在细胞分裂过程中的有丝分裂装置上。 该仪器延续了LC-PolScope的发展，极大地提高了偏振光在活体细胞成像中的应用。在上一次授权期间，我们在显微镜光学系统中增加了一个孔径扫描装置，从而消除了一个重要的限制。与包括LC PolScope在内的任何其他偏光显微镜不同，新的扫描Aperture PolScope可以揭示倾斜到焦平面的物体的双折射。因此，这项技术最终可以揭示具有任何方向的双折射对象，如有丝分裂纺锤体，并且它不限于偶然或通过特殊操作定向在焦平面上的对象。 在概念验证的基础上，我们现在建议通过修改孔径扫描器的设计并在显微镜的成像路径中增加第二个类似的设备来改进孔径扫描技术。这些变化和对光学装置的增加将使其能够(A)更准确和灵敏地测量双折射，以及(B)测量表现出偏振相关吸收的样品的二色性。此外，在成像路径上增加了高分辨率的干涉滤光片，该仪器将能够揭示荧光偏振。 双折射、二向色性和荧光偏振是局域分子有序各向异性的重要指标。结构各向异性经常被证明是建立生物功能的重要因素，例如在形成膜的过程中脂分子的排列或纺锤体微管的线性排列，以在细胞分裂过程中定向分离染色体。通过利用扫描孔径PolScope的新的和改进的能力，我们将研究昆虫精母细胞减数分裂过程中染色体分离的机制。SIEARA和鹤苍蝇的精母细胞有机会将正常染色体分离的结果与染色体的结果进行比较，这些结果表明“它们的分离行为要么是自然的(Sciara中的单极纺锤体，而鹤苍蝇中的性染色体晚期分离)，要么是诱导的(冷处理或药物暴露)分离行为异常。

DESCRIPTION (provided by applicant): We are building and applying an ultra-sensitive and high-resolution polarized light microscope on liquid based crystal technology (the LC-PolScope). We use the unique instrumentation to reveal and decipher the functional role of the dynamic architecture of the cytoskeleton, with a focus on the mitotic apparatus during cell division. The proposed instrumentation continues the development of the LC-PolScope that has greatly enhanced the utility of polarized light for live cell imaging. During the last grant period, we removed an important limitation by adding an aperture scanning device to the microscope optical train. Unlike any other polarizing microscopes, including the LCPolScope, the new Scanned Aperture PolScope can reveal the birefringence of objects that are inclined to the plane of focus. Therefore, this technique can finally reveal birefringent objects, such as mitotic spindles, that have any orientation, and it is not limited to objects that either by chance or by special manipulation are oriented in the plane of focus. Building on the proof of concept, we now propose to refine the aperture scanning technique by modifying the design of the aperture scanner and adding a second, similar device in the imaging path of the microscope. These changes and additions to the optical set-up will make it possible (a) to measure birefringence more accurately and sensitively, and (b) measure dichroism in specimens that exhibit polarization-dependent absorption. Furthermore, with the addition of highly discriminating interference filters to the imaging path, the instrument will be capable of revealing fluorescence polarization. Birefringence, dichroism, and fluorescence polarization are important indicators of anisotropy in local molecular order. Structural anisotropy often proves to be important in establishing biological function, such as the alignment of lipid molecules in forming membranes or the linear arrangement of spindle microtubules for the directed segregation of chromosomes during cell division. By taking advantage of the new and improved capabilities of the Scanned Aperture PolScope, we will investigate mechanisms of chromosome segregation during meiosis in insect spermatocytes. Spermatocytes of Sciara and crane flies provide the opportunity to compare the results from normal chromosome segregation with results obtained with chromosomes that show" either natural (monopolar spindle in Sciara and late segregation of sex chromosomes in crane fly) or induced (cold treatment or drug exposure) abnormalities in their segregation behavior.