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

该奖项支持一项合作努力，以开发和评估增强型微分干涉对比(DIC)显微镜，该显微镜将允许可视化和测量活细胞中的动态过程。通过活细胞分子成分浓度的细微变化，可以提供丰富的潜在有用信息。显微镜将检测并记录由于变化对通过样品的光的光程长度的影响而导致的光相的微小变化。这些信息将被可视化为精确改变的图像强度。该显微镜将能够将由于相性质改变而引起的强度变化与由于吸收而引起的强度变化区分开来，这是现有的DIC仪器无法做到的。新仪器基于相移干涉原理，需要对DIC图像形成过程进行仔细建模，以便设计适当的数字图像重建程序。其结果将是光学和数字处理技术的全自动组合，其中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原本看起来模糊的三维特征。这种新型的定量DIC显微镜在活细胞生物学中有着广泛的应用前景。因此，仪器的初始测试将仅通过测量折射率来确定检测细胞周期非常细微的阶段(例如，S的哪个阶段，或者是否处于有丝分裂早期)的灵敏度。其他将用于开发和改进该仪器的应用包括比较突触前和突触后神经元，以及测量由于蛋白质定位而导致的变化率。新系统将在标准的商用DIC显微镜上实施，使用一种新的液晶设备设计，使该仪器完全自动化，生物学家可以很容易地使用。该显微镜有望最大限度地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预计将有助于将新技术转移到商业开发中。该项目将为研究生和本科生提供培训机会，调查人员将把在该项目中学到的知识纳入研究生和本科生课程和一个公共网站。

This award supports a collaborative effort 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.