Orientation Independent DIC and Polarization Microscopy

项目摘要 该项目旨在开发和应用新的定量差分干涉对比度和偏振光 显微镜技术，这两种技术的对比度都与样品方向无关。在上一次 在GRANT期间，我们建立了与取向无关的微分干涉衬度(OI-DIC)显微镜 快速改变剪切方向，无需任何机械移动部件。OI-DIC技术，它 我们已经开发，提供了最高质量的光程长度(干质量)图的薄光学截面 未染色和染色样品，横向分辨率~250-300 nm，轴向分辨深度~100 nm，以及 在波长546 nm处，光程灵敏度为0.5 nm。我们认为，具有如此高水平的图像 任何其他目前可用的干涉和相位显微镜技术都不能产生分辨率。 我们还建立了多色偏振光显微镜(偏振镜)，它采用了新的原理 产生干涉色并产生具有以下延迟性的双折射结构的彩色图像 几纳米，这在以前是不可能的。结构的色调指示其慢轴方向，而 结构的亮度与其延迟性成正比。我们将设计改进的高分辨率OI- 使用和开发两种主要新的OI-DIC方法的DIC。新的OI-DIC将与 与取向无关的偏振(LC-Polcope)和共聚焦荧光技术。改进后的OI- DIC和组合装置将用于研究活的生物标本的结构动力学， 强调与有丝分裂和减数分裂有关的事件。我们将建立一个即时的OI-DIC，它将 同时采集4幅剪切方向垂直的原始DIC图像。瞬时OI-DIC 这些技术将提供最佳的时间分辨率，并允许消除运动引起的伪影 细胞质和细胞器。我们将开发OI-DIC技术来恢复这两个干质量的三维分布 (与相位有关的信息)和折射率。有了这项技术，人们将能够确定干度 并观察其在分裂过程中的变化。新的高潮- 灵敏的多色偏振器将显示延迟小于1 nm的双折射结构。这个 将多色偏振镜与相位对比度和暗场技术相结合，使图像 亮度显示干质量分布，颜色描述分子取向。这些加在一起 将利用技术研究减数分裂I中期，并分析结构之间的对应关系 不同活体的特征，以及它们的遗传背景。我们将创建一个量化的 多色偏振器，它将提供样品的延迟和 慢轴方向。定量多色偏光显微镜将被用来研究胶原纤维 癌症组织。

Project Summary This project aims to develop and apply new quantitative differential interference contrast and polarized light microscope techniques, both of whose contrast is independent of specimen orientation. During the previous grant period we built the orientation-independent differential interference contrast (OI-DIC) microscope, which rapidly changes shear directions without any mechanically moved components. The OI-DIC technique, which we have developed, provides the highest quality of optical path length (dry mass) map of thin optical section of unstained and stained specimens with lateral resolution ~250-300nm, axial discrimination depth ~100nm, and optical path length sensitivity ~0.5nm at wavelength 546nm. We believe that images with such high level of resolution cannot be produced by any other currently available interference and phase microscopy techniques. We also built the polychromatic polarized light microscope (polscope), which employs new principle of generating interference color and produces a color image of the birefringent structures with retardances of several nm, which was not possible before. The hue of the structure indicates its slow axis orientation, and the brightness of the structure is proportional to its retardance. We will engineer the improved high-resolution OI- DIC with and exploit two principally new OI-DIC approaches. The new OI-DIC will be combined with the orientation-independent polarization (LC-polscope) and confocal fluorescence techniques. The improved OI- DIC and the combined setup will be used to study the architectural dynamics of live biological specimens, with emphasis on events associated with mitosis and meiosis. We will build an instantaneous OI-DIC, which will simultaneously capture 4 raw DIC images with the orthogonal shear directions. The instantaneous OI-DIC techniques will provide the best temporal resolution and allow the elimination of artifacts caused by movements of the cytosol and organelles. We will develop OI-DIC technique to restore the 3D distribution of both dry mass (phase-related information) and refractive index. With this technique, one will be able to determine the dry mass of chromosomes before division and observe its change during the division process. The new high- sensitive polychromatic polscope will visualize birefringent structures with retardances less than 1nm. The polychromatic polscope will be combined with phase contrast and dark field techniques, so that image brightness displays dry mass distribution and the color depicts molecular orientation. These combined techniques will be used to study metaphase of meiosis I, and analyze the correspondence between structural signatures in different live organisms, as well as their genetic background. We will create a quantitative polychromatic polscope, which will provide a two-dimensional distribution both the specimen’s retardance and slow axis orientation. The quantitative polychromatic polscope will be employed to study collagen fibers in cancer tissues.