Quantitative Optical Phase, Amplitude, and Polarization Microscopy

描述(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、未染色的生物样品的高分辨率、高精度、相位、幅度和偏振图像。与现有的定量荧光显微镜方法相结合，并通过其直接和完全地获取目标的物理、化学/分子信息，它将导致生物标本的完全数字/计算、全面、多模式的光学显微镜，并显著提高当前光学方法的能力。这种新型显微镜将通过将一个新的(演示的)光学相位、幅度和偏振传感器连接到一个现代商业显微镜底盘上来制造。传感器将使用标准技术(例如积分球)、前期工作中使用的特定技术的扩展和定制的相位显微镜靶标的组合进行校准。成像性能将在精心挑选的一组具有代表性的透明样品上进行测试。所选择的成像目标将提供不同的光学显微镜挑战，这对于工程开发和洞察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、薄的小鼠组织样本和不同发育阶段的线虫。通过提出的方法，对当前光学相位显微镜的重大改进将包括：a)通过现有的相位对比或DIC显微镜(例如高尔基仪器)来成像(不染色)低对比度细胞器；b)通过将线性处理算法(对象信息中的数据是线性的)一致地应用于定量的多模式成像数据，以提供全新的信息/视图，从而大大增强数字处理能力；以及c)通过在衍射层析成像或z-Stack重建算法中使用定量相数据来获得透明的、未染色的样品的真实3D体积信息。例如，这些改进将使更先进的光学显微镜能够进行高通量基因筛查，更快、更准确(定量)的透明、未染色组织样本的自动化组织学，以及包含关于特定表型的全面、完全数字、定量的多模式光学显微镜数据的复杂遗传数据库。 这项研究将开发一种新的定量光学显微镜技术，能够以一种易于广泛使用的方式为生物学家提供必要的新信息。通过提供对生物样本物理/化学特性的全面访问，它将使先进的计算机处理技术的应用能够获得和(以数字方式)存储与基础和临床研究有关的新信息。

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.