Orientation independent DIC and polarization microscopy

项目描述（申请人提供）：本项目旨在开发和应用一种新型的、与取向无关的微分干涉对比显微镜（OI-DIC），该显微镜的对比度与标本的取向无关，适用于活体生物标本的研究。扩展了使用传统的DIG与样品旋转相结合的初步研究，通过该研究验证了所提出技术的理论有效性，提出了不需要样品或DIG棱镜旋转的OI-DIC系统。使用新系统（专利申请），可以在2秒或更短的时间内获得OI-DIC图像。因此，结合新系统的显微镜将允许对细胞器形态、运动性以及干质量分布进行精确分析。此外，还将研制出一种常规DIG和不依赖取向偏振（LC-Pol）显微镜的组合。这种新系统将产生样品薄光学切片的两个互补相位图像：由于细胞结构的结构或内部各向异性，光路梯度的分布和双折射的分布。将DIG和LC-Pol设备组合成一个单元，将允许在两种模式之间快速切换，而无需移动任何光学元件，因此可以几乎同时捕获两幅图像，并且不会出现错位，在大约0.3秒内生成组合图像。将所提出的技术与不移动任何光学元件的近同期荧光图像相结合的可能性将被探索。这种组合将提供标本的独特视图，其中结构信息可以与分子特异性标签连接。这些新设备将用于研究活体生物标本的结构和分子动力学，重点是与有丝分裂和减数分裂相关的事件；OI-DIC提供了增强的染色体图像，LC-Pol图像定量描述了纺锤体纤维和星光射线中微管的动态组装-拆卸和分布。新的显微镜通常可以对细胞器运动和亚细胞器各向异性进行高级分析，这些分析可以直接反映活细胞中亚微观和正在进行的分子事件。相关性：为了更好地了解健康和病理受损组织的动态行为和分子事件，我们将开发用于光学显微镜的新光学和处理软件，以便在不破坏或染色细胞的情况下快速捕获信息图像。新系统将使细胞器，包括它们的变化和运动更加可见，允许测量它们的干质量，同时揭示分子组装和排列的变化，所有这些都是非侵入性的，而不会扰乱细胞的状态。

DESCRIPTION (provided by applicant): This project aims to develop and apply a new, orientation-independent differential interference contrast (OI-DIC) microscope, whose contrast is independent of specimen orientation and is appropriate for investigation of live biological specimens. Extending the preliminary studies using conventional DIG coupled with specimen rotation, by which the theoretical validity of the proposed technique was validated, OI-DIC systems are proposed which require no rotation of either the specimen or the DIG prisms. With the new systems (patent applied), an OI-DIC image should be obtained in 2 seconds or less. Thus microscopes incorporating the new system will allow precise analyses of organellar morphology, motility, as well as dry mass distribution. In addition, a combination of regular DIG and orientation independent polarization (LC-Pol) microscope will be developed. This new system will yield two complementary phase images of thin optical sections of the specimen: distribution of optical path gradients and distribution of birefringence due to structural or internal anisotropy of the cell structure. The DIG and LC-Pol devices combined into one unit will allow rapid switching between the two modes without the need to move any optical components so that both images can be captured nearly simultaneously and without misalignment, yielding a combined image in ca. 0.3 seconds. The possibilities of combining the proposed technique with near-contemporaneous fluorescence images without moving any optical elements will be explored. This combination will give unique views of a specimen, where structural information can be joined with molecularly specific labels. The new devices will be used to study the architectural and molecular dynamics of live biological specimen, with emphasis on events associated with mitosis and meiosis; the OI-DIC providing enhanced chromosome images while the LC-Pol images quantitatively depicting the dynamic assembly-disassembly and distribution of microtubules in the spindle fibers and astral rays. The new microscope should generally allow advanced analyses of organellar movement and sub-organellar anisotropy that reflect the submicroscopic and ongoing molecular events, directly in living cells. RELEVANCE: In order to gain improved insight into the dynamic behavior and molecular events underlying healthy and pathologically impaired tissues, we will develop new optics and processing software for light microscopes that allow speedy capture of informative images without the need to destroy or stain the cells. The new systems will make cell organelles, including their changes and movement more visible, allow measurement of their dry mass, and concurrently reveal changes in molecular assembly and alignment, all non-invasively without perturbing the state of the cells.