Quantitative Optical Phase, Amplitude, and Polarization Microscopy

描述（由申请人提供）：拟议的研究将开发一种新的定量光学显微镜技术，提供透明，未染色的生物标本的高分辨率，高精度，相位，振幅和偏振图像。结合现有的定量荧光显微镜方法，并通过其直接和完整地获取目标的物理和化学/分子信息，它将导致生物标本的全数字/计算、全面、多模态光学显微镜，并大大提高当前光学方法的能力。新的显微镜将建立一个新的（演示）光学相位，振幅和偏振传感器，以现代商用显微镜底盘。传感器将使用标准技术（例如积分球）、初步工作中使用的特定技术的扩展和定制相显微镜目标进行校准。成像性能将在一组精心挑选的代表性透明标本上进行测试。所选择的成像目标将提供不同的光学显微镜挑战，这对于工程开发和获得解决重要生物学问题的新方法都是重要的：各种单层培养细胞，薄小鼠组织样本和不同发育阶段的秀丽隐杆线虫。通过所提出的方法，对当前光学相位显微镜的重大改进将包括：a)成像（不染色）低对比度细胞器，现有的相对比或DIC显微镜（例如高尔基体）看不见；B)通过一致地应用线性处理算法（数据在对象信息中是线性的）对定量多模态成像数据进行更强大的数字处理，以提供全新的信息/视图；c)利用衍射层析成像中的定量相位数据或z-stack重建算法获得透明、未染色标本的真实三维体积信息。这些改进将使，例如，用于高通量遗传筛选的更先进的光学显微镜，更快，更准确的（定量）自动化透明，未染色的组织样本组织学，以及包含全面，完全数字化，定量的特定表型的多模态光学显微镜数据的复杂遗传数据库成为可能。

DESCRIPTION (provided by applicant): Proposed research will develop a new quantitative optical microscopy technique providing high resolution, high accuracy, phase, amplitude and polarization images of transparent, unstained, biological specimens. In conjunction with existing methods of quantitative fluorescence microscopy and enabled by its direct and complete access to a target's physical and chemical/molecular information, it will lead to fully digital/computational, comprehensive, multi-modal optical microscopy of biological specimens and significantly advance the capabilities of current optical methods. The new microscope will be built by coupling a novel (demonstrated) optical phase, amplitude and polarization sensor to a modern commercially available microscope chassis. The sensor will be calibrated using a combination of standard techniques (e.g. integrating sphere), extensions of specific techniques used in preliminary work, and custom phase microscopy targets. Imaging performance will be tested on a carefully chosen representative set of transparent specimens. The chosen imaging targets will provide different optical microscopy challenges that are important both for the engineering development and for gaining insight into new ways of solving important biological problems: various monolayer cultured cells, thin mouse tissue samples, and C. elegans worms at different stages of development. Significant improvements to current optical phase microscopy, as enabled by the proposed method, will include: a) imaging (without staining) low contrast organelles, invisible by existing phase contrast or DIC microscopy (e.g. Golgi apparatus); b) vastly more powerful digital processing through consistent application of linear processing algorithms (the data is linear in object information) to quantitative multi-modal imaging data to provide entirely new information/views; and c) true 3D volume information of transparent, unstained, specimens by using the quantitative phase data in diffraction tomography or z-stack reconstruction algorithms. Such improvements will enable, for example, more advanced optical microscopy for high- throughput genetic screening, faster, more accurate (quantitative) automated histology of transparent, unstained, tissue samples, and complex genetic databases containing comprehensive, fully digital, quantitative multi-modal optical microscopy data on specific phenotypes. This research will develop a new quantitative optical microscopy technique capable of providing essential new information to biologists, in a manner amenable to wide use. By providing complete access to the physical/chemical properties of biological specimens, it will enable the application of advanced computer processing techniques to obtain and store (digitally) new information relevant for both basic and clinical research.