Real-time PolScope microscope for live-cell imaging

描述（由申请人提供）：I期 我们建议开发一种超灵敏的实时偏振光显微镜（“实时PolScope”），用于通过双折射成像（一种功能强大的定量新成像对比度技术）分析活细胞、整个生物体和无细胞模型系统中分子结构的动态。该仪器建立在PolScope平台上，该平台先前是由CRI公司和海洋生物实验室的Oldenbourg博士合作开发的。虽然原始PolScope系统的空间分辨率和分析能力仍然是最先进的，但成像系统的时间分辨率不足以解决和分析许多潜在的有趣细胞事件。 在第一阶段的应用中，我们将加快图像采集，从而大大提高双折射测量的保真度。我们计划的主要创新是开发一种四扇区液晶通用补偿器，该补偿器被添加到成像分束器中，该分束器将四个偏振图像并行投影到高分辨率数码相机上。同时记录四个图像进行偏振分析，预计将增加超过10倍的图像，我们可以获得每分钟的数量。此外，偏振分析图像的并行记录将消除由样品组分的内部运动引起的大部分伪影。理想地，计算的双折射图像的呈现将以与成像速度相称的速率发生。 总体而言，在第一阶段和第二阶段的拟议工作的目标是一个实时PolScope的时间分辨率至少比当前模型的时间分辨率好10倍，很少或没有牺牲当前仪器的灵敏度。在第二阶段，我们将添加软件和硬件组件，这将创建一个高度复杂的偏振和荧光分析工具，该仪器将提供急需的信息补充其他显微镜模式用于活细胞成像。为了测试生物材料的建议显微镜的效用，我们将继续我们的活细胞内的建筑动力学的研究，特别强调在有丝分裂和减数分裂过程中的微管含纺锤体装置，和生化信号转导途径的机械联系。 二期 第一阶段应该已经完成了近实时的双折射延迟成像系统的实施，利用现有的先进技术的以下组件：高速，敏感的数码相机;一个四路分束器，允许收集多达四个偏振图像同时;和一个超亮脉冲光源。这些组件将由一个新的四扇区通用补偿器补充，该补偿器在分束器内部使用，并由新的偏振算法补充，该算法可以在收集延迟图像时尽快计算和显示延迟图像。 在第二阶段，我们将扩展这些功能并添加工程和软件改进，以创建商业上可接受、经济实惠且有用的仪器平台。新的能力将包括近同步荧光成像;光学修改，以提供新的成像选项，并减少成像路径中的偏振像差;开发新的脉冲，多光谱光源，增加新的偏振分析模式的额外算法;和3D光学切片。其他任务将是在必要时更换第一阶段中使用的现成物品，以便改进功能或大幅度降低成本，同时沿着系统工程，以创建适合商业化的测试版。这包括用户界面和其他软件功能的进步。这些技术进步将在合作中部署。

DESCRIPTION (provided by applicant): Phase I We propose to develop an ultra-sensitive real-time polarized light microscope ("Real-Time PolScope") for analyzing the dynamics of the molecular architecture in living cells, in whole organisms and in cell-free model systems through birefringence imaging, a powerful and quantitative new imaging contrast technique. The instrumentation builds on the PolScope platform previously developed through a partnership between CRI, Inc and Dr. Oldenbourg at the Marine Biological Laboratory While the spatial resolution and analytical capabilities of the original PolScope system are still state-of-the-art, the temporal resolution of the imaging system is insufficient for resolving and analyzing many potentially interesting cellular events. In Phase I of this application we will speed up image acquisition and thereby greatly improve the fidelity of birefringence measurements. The major innovation we are planning is the development of a four-sector liquid crystal universal compensator that is added to an imaging beam splitter that projects in parallel four polarization images onto a high-resolution digital camera. The simultaneous recording of four images for polarization analysis is expected to increase by more then 10-fold the number of images that we can acquire per minute. Furthermore, the parallel recording of polarization-analyzed images will eliminate most of the artifacts that arise from the internal motion of sample components. Ideally, the presentation of calculated birefringence images will occur at a rate commensurate with the imaging speed. Overall, the goal of the proposed work in Phase I and II is a Real-Time PolScope with a temporal resolution of at least 10 times better than that of the current model with little or no sacrifice in the sensitivity of the current instrument. In Phase II we will add software and hardware components that will create a highly sophisticated polarization and fluorescence analysis tool This instrument will provide much-needed information complementary to other microscope modes used for live-cell imaging. To test the utility of the proposed microscope on biological material, we will continue our studies of the architectural dynamics within living cells, with specific emphasis on the microtubule-containing spindle apparatus during mitosis and meiosis, and mechanical links to biochemical signal transduction pathways. Phase II Phase I should have accomplished implementation of a near-real-time birefringence retardance imaging system by exploiting existing advanced technology of the following components: high-speed, sensitive digital camera; a four-way beam splitter to allow collection of up to four polarization images simultaneously; and an ultra-bright pulsed light source. These components will be complemented by a new four-sector universal compensator that is employed inside the beam splitter and by new polarimetric algorithms that can calculate and display retardances images as fast as they are collected. In Phase II we will extend these capabilities and add engineering and software refinements to create a commercially acceptable, affordable and useful instrument platform. New capabilities will include near simultaneous fluorescence imaging; optical modifications to provide new imaging options and to reduce polarization aberrations in the imaging path; development of a new pulsed, multi-spectral light source, additional algorithms that add new modes of polarization analysis; and 3-D optical sectioning. Other tasks will be directed towards replacing, where necessary, off-the-shelf items used in Phase I when either improved function or a dramatic decrease in cost can be achieved, along with systems engineering to create a beta version suitable for commercialization. This includes advances in the user interface and other software functionalities. These technical advances will be deployed in collaborations.