Automation of quantitative DIC microscopy for 3D live-cell imaging using programm

描述（由申请人提供）：提出的主要目标是利用博尔德非线性系统（BNS）的空间光调制器（SLM）技术来自动化Cogswell集团最近开发的定量DIC显微镜（Q-DIC），并使其在市场上的生物学家。Q-DIC是一种全场（非扫描）相位成像技术，可提供指示厚度、密度或化学变化的光程长度测量。科罗拉多大学（CU）实验室Q-DIC系统的当前限制可以通过以这样一种方式适应用于显微镜的BNS空间光调制器来克服，即可以动态地改变三个必需的DIC光学设计参数（即相位偏置、剪切距离和剪切方向）。在此上下文中，SLM是电驱动的各向异性液晶装置，其具有动态改变入射光学波前的复相位的能力。使用SLM技术精确控制DIC参数可变性将实现Q-DIC图像的自动采集，用于非侵入性、实时定量相位（光程长度）成像。这种新的能力将允许Q-DIC的高分辨率生物成像的优势进行评估，也将加强DIC的比较荧光成像的效用。它在不引入染料（可能干扰细胞动力学）的情况下在活细胞图像中产生对比度的能力将引起细胞生物学界的特别兴趣。在首先证明了利用现有SLM技术自动化DIC图像采集的可行性之后，BNS提出设计和制造两种新的、截然不同的SLM配置，以比较和对比它们的性能，并确定哪一种（如果有的话）最适合在第二阶段进行进一步开发。一个适销对路的SLM设计必须满足现有商业显微镜改装的技术挑战，而不限制其多模功能或高分辨率光学质量。这些新的结构将通过我们的合作生物学家（T。Su）。这项第一阶段研究的成功将通过通过活细胞Q-DIC成像实验证明已知细胞发育过程的准确定量的能力来衡量。除了使CU实验室Q-DIC显微镜自动化之外，该提案还包括初步调查新的显微镜兼容SLM配置是否也可以更普遍地用于增强未来的光学显微镜设计和性能。将主动光学波前操纵的新方法直接耦合到用于数字图像处理的新方法具有激发优化光学和数字图像处理的开创性方法的潜力。例子包括扩展高分辨率物镜的景深、主动像差校正和超分辨率。这种创新可以帮助理解基本的生物学问题和生物医学研究。 公共卫生相关性：定量微分干涉衬度（Q-DIC）显微镜是一种全视场（非扫描）相位成像技术，其从非吸收（或部分吸收）对象的图像中的对比度提取指示厚度，密度或化学变化的光程长度测量，而不引入染料（可能干扰细胞动力学）。自动化Q-DIC显微镜将实现3D活细胞Q-DIC成像和细胞发育过程中动态特性的非侵入性研究，这可能会揭示疾病发生过程的新见解。

DESCRIPTION (provided by applicant): The primary objective proposed is to utilize Boulder Nonlinear System's (BNS) spatial light modulator (SLM) technology to automate the Cogswell group's recently-developed quantitative DIC microscope (Q-DIC) and make it accessible to biologists in the marketplace. Q-DIC is a full-field (non-scanning) phase imaging technique which provides optical path length measurements indicative of either thickness, density, or chemical variations. The present limitations of the University of Colorado (CU) lab Q-DIC system can be overcome by adapting BNS spatial light modulators for the microscope in such a way that it will be possible to dynamically vary the three requisite DIC optical design parameters (i.e. phase bias, shear distance and shear direction). A SLM in this context is an electrically driven, anisotropic liquid crystal device with the ability to dynamically change the complex phase of an incident optical wavefront. Precise control of DIC parameter variability using SLM technology will enable automated acquisition of Q-DIC images for non-invasive, real-time quantitative phase (optical path length) imaging. This new capability will allow evaluation of the advantages of Q-DIC for high-resolution biological imaging and will also strengthen the utility of DIC comparisons to fluorescence imaging. Its ability to produce contrast in live-cell images without introducing dyes (which potentially interfere with cellular dynamics) will be of special interest to the cellular biology community. After first proving the feasibility of automating DIC image acquisition with existing SLM technology, BNS proposes to design and fabricate two new and distinctly different SLM configurations in order to compare and contrast their performance and determine which (if either) is best-suited for further development in Phase II. A marketable SLM design must meet the technical challenges of retro-fitting to existing commercial microscopes without limiting their multimode functionality or their high-resolution optical quality. These new configurations will be tested and evaluated through in vitro imaging studies of developing Drosophila embryos, provided by our collaborating biologist (Dr. T. Su). The success of this Phase I research will be measured by the ability to demonstrate accurate quantification of a known cellular development process through a live-cell Q-DIC imaging experiment. In addition to automating the CU lab Q-DIC microscope, this proposal includes initial investigation into whether the new microscope-compatible SLM configurations can also be used more generally for enhancing optical microscope design and performance in the future. Directly coupling new methods of active optical wavefront manipulation to novel methods for digital image processing has the potential to inspire pioneering methods of optimized optical and digital imaging processing. Examples include extending the depth of field of high resolution objectives, active aberration correction, and super-resolution. Such innovations could aid understanding of basic biological problems and biomedical research. PUBLIC HEALTH RELEVANCE: Quantitative differential interference contrast (Q-DIC) microscopy is a full-field-of-view (non-scanning) phase imaging technique which extracts optical path length measurements indicative of either thickness, density, or chemical variations from contrast in images of non-absorbing (or partially absorbing) objects without introducing dyes (which potentially interfere with cellular dynamics). Automated Q-DIC microscopy will enable 3D live-cell Q-DIC imaging and the non-invasive study of dynamic properties during cell development which may reveal new insights into the processes through which the onset of disease takes place.