Collaborative Research: Quantitative DIC Microscope for Measuring 3-Dimensional Cell Attributes

该奖项支持PI和科罗拉多大学研究人员之间的合作努力，以开发和评估增强型微分干涉对比（DIC）显微镜，该显微镜将允许活细胞动态过程的可视化和测量。活细胞分子成分浓度的细微变化可以提供大量潜在有用的信息。显微镜将检测并记录光相位的微小变化，这些变化是由于光通过样品的光程长度的变化所引起的。这些信息将以精确改变的图像强度可视化。该显微镜将能够将由于相位特性改变而引起的强度变化与由于吸收引起的强度变化分开，这是现有DIC仪器无法做到的。新仪器基于相移干涉测量原理，需要对DIC图像形成过程进行仔细建模，以便设计合适的数字图像重建程序。结果将是光学和数字处理技术的全自动组合，包括最近为荧光显微镜开发的新型扩展景深成像能力。生物学家将能够在一张图像中看到三维特征，否则这些特征会出现失焦。新型定量DIC显微镜在活细胞生物学领域具有广泛的应用前景。因此，仪器的初始测试将确定灵敏度，以检测非常微妙的细胞周期阶段（例如，S期的哪一阶段，或是否在早期开始有丝分裂），仅仅通过测量折射率。将用于开发和改进仪器的其他应用包括突触前和突触后神经元的比较以及由于蛋白质定位引起的变化率的测量。新系统将在标准商用DIC显微镜中实施，使用新颖的液晶装置设计，使仪器完全自动化，易于生物学家使用。该显微镜有望减少或消除对荧光探针的需求，因为荧光探针可以改变正常的细胞功能。调查人员与当地公司的关系有望促进新技术向商业发展的转移。该项目将为研究生和本科生提供培训机会，研究人员将把在项目中获得的知识纳入研究生和本科生的课程和一个公共网站。

This award supports a collaborative effort between the PI and investigators at the University of Colorado to develop and evaluate an enhanced differential interference contrast (DIC) microscope that will permit the visualization and measurement of dynamic processes in living cells. A wealth of potentially useful information can be provided by subtle variations in the concentrations of the molecular components of living cells. The microscope will detect and record minute changes in light phase resulting from the effect of the variation on the optical path length of light passing through the specimen. The information will be visualized as precisely altered image intensities. The microscope will be able to separate changes in intensity due to altered phase properties from those due to absorption, which existing DIC instruments are unable to do. The new instrument is based on phase-shifting interferometry principles that require careful modeling of the DIC image formation process in order to design appropriate digital image reconstruction routines. The result will be a fully automated combination of optical and digital processing technologies that include novel extended-depth-of-field imaging capabilities recently developed for fluorescence microscopy. Biologists will be able to view, within a single image, three-dimensional features that would otherwise appear out of focus. Potential applications of the new quantitative DIC microscope are plentiful in live-cell biology. Thus, initial tests of the instrument will determine sensitivity for detecting very subtle stages of the cell cycle (e.g. what stage of S phase, or whether in early onset of mitosis) simply by measuring refractive index. Other applications that will be used to develop and refine the instrument include comparison of pre- and post-synaptic neurons and measurement of the rate of change due to protein localization. The new system will be implemented within standard commercial DIC microscopes using a novel liquid crystal device design, making the instrument fully automated and easily accessible to biologists. The microscope is expected to minimize or eliminate the need for fluorescent probes, which can alter normal cell function. The investigators' relation with local companies is expected to facilitate transfer of new technology to commercial development. The project will provide training opportunities for graduate and undergraduate students, and the investigators will incorporate knowledge gained in the project into graduate and undergraduate courses and a public website.