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

使用程序实现 3D 活细胞成像定量 DIC 显微镜自动化

• 批准号: 7803845
• 负责人: Carol Cogswell
• 金额: $ 34.94万
• 依托单位: BOULDER NONLINEAR SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7803845
• 关键词: Automation; Biological; Biomedical Research; Cells; Cellular biology; Chemicals; Colorado; Communities; Complex; Coupling; Development; Devices; Drosophila genus; Dyes; Embryo; Evaluation; Future; Image; Imaging Techniques; In Vitro; Investigation; Laboratories; Length; Life; Light; Marketing; Measurement; Measures; Mechanics; Methods; Microscope; Microscopy; Morphologic artifacts; Onset of illness; Optics; Performance; Phase; Positioning Attribute; Process; Property; Refractive Indices; Research; Resolution; Rotation; Scanning; Solutions; Speed; Staging; Staining method; Stains; System; Technology; Testing; Thick; Time; Universities; Variant; absorption; active method; cellular development; cellular imaging; cost; density; design; digital imaging; fluorescence imaging; image processing; innovation; insight; interest; liquid crystal; meetings; millisecond; novel; public health relevance; research study; success

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.

描述（由申请人提供）：提出的主要目标是利用 Boulder Nonlinear System (BNS) 空间光调制器 (SLM) 技术实现 Cogswell 小组最近开发的定量 DIC 显微镜 (Q-DIC) 的自动化，并使其可供市场上的生物学家使用。 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 系统目前的局限性可以通过为显微镜采用 BNS 空间光调制器来克服，这样就可以动态改变三个必要的 DIC 光学设计参数（即相位偏差、剪切距离和剪切方向）。在这种情况下，SLM 是一种电驱动的各向异性液晶器件，能够动态改变入射光波前的复杂相位。使用 SLM 技术精确控制 DIC 参数变异性将能够自动采集 Q-DIC 图像，以实现非侵入性、实时定量相位（光程长度）成像。这项新功能将允许评估 Q-DIC 在高分辨率生物成像方面的优势，并将加强 DIC 与荧光成像比较的实用性。它能够在活细胞图像中产生对比度而​​不引入染料（这可能会干扰细胞动力学），这将引起细胞生物学界的特别兴趣。在首次证明利用现有 SLM 技术自动采集 DIC 图像的可行性后，BNS 建议设计和制造两种截然不同的新 SLM 配置，以便比较和对比它们的性能，并确定哪种（如果有的话）最适合第二阶段的进一步开发。适销对路的 SLM 设计必须满足改造现有商用显微镜的技术挑战，同时不限制其多模功能或高分辨率光学质量。这些新的配置将通过我们的合作生物学家（T. Su 博士）提供的果蝇胚胎发育的体外成像研究进行测试和评估。这一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量化的能力来衡量。除了实现 CU 实验室 Q-DIC 显微镜的自动化之外，该提案还包括初步调查新的显微镜兼容 SLM 配置是否也可以更广泛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与数字图像处理的新方法直接耦合有可能激发优化光学和数字成像处理的开创性方法。示例包括扩展高分辨率物镜的景深、主动像差校正和超分辨率。这些创新可以帮助理解基本的生物学问题和生物医学研究。 公共健康相关性：定量微分干涉对比 (Q-DIC) 显微镜是一种全视场（非扫描）相位成像技术，可从非吸收（或部分吸收）物体的图像对比度中提取指示厚度、密度或化学变化的光路长度测量值，而不引入染料（可能会干扰细胞动力学）。自动化 Q-DIC 显微镜将实现 3D 活细胞 Q-DIC 成像以及细胞发育过程中动态特性的非侵入性研究，这可能揭示疾病发生过程的新见解。