Real-time PolScope microscope for live-cell imaging

描述(申请人提供)：第一阶段 我们建议开发一种超灵敏实时偏振光显微镜(Real-Time PolScope)，通过双折射成像这一强大而定量的新成像对比技术来分析活细胞、整个生物体和无细胞模型系统中的分子结构动力学。该仪器建立在之前由CRI公司和海洋生物实验室的Oldenburg博士合作开发的PolScope平台上。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析和分析许多潜在的有趣的细胞事件。 在此应用程序的第一阶段，我们将加快图像采集速度，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平行投影到高分辨率数码相机上。同时记录四幅图像以进行偏振分析，预计将使我们每分钟可以获得的图像数量增加10倍以上。此外，偏振分析图像的并行记录将消除由于样品分量的内部运动而产生的大部分伪影。理想情况下，计算的双折射图像将以与成像速度相称的速率呈现。 总体而言，第一阶段和第二阶段拟议工作的目标是一种实时PolScope，其时间分辨率至少比当前模型好10倍，而对当前仪器的灵敏度几乎没有牺牲。在第二阶段，我们将增加软件和硬件组件，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，补充用于活细胞成像的其他显微镜模式。为了测试提出的显微镜在生物材料上的实用性，我们将继续研究活细胞内的结构动力学，特别是有丝分裂和减数分裂过程中含有微管的纺锤体，以及与生化信号转导途径的机械联系。 第二阶段 第一阶段本应利用以下组件的现有先进技术完成近乎实时的双折射延迟成像系统的实施：高速、灵敏的数码相机；允许同时收集多达四个偏振图像的四路分束器；以及超亮脉冲光源。这些组件将得到新的四扇区通用补偿器的补充，该补偿器采用在分束器内，并由新的偏振算法来补充，该算法可以在收集到图像时快速计算和显示延迟图像。 在第二阶段，我们将扩展这些能力，并增加工程和软件改进，以创建一个商业上可接受的、负担得起的和有用的仪器平台。新的能力将包括几乎同时荧光成像；光学修改，以提供新的成像选择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；以及三维光学切片。其他任务将用于在必要时更换第一阶段中使用的现成物品，无论是改进功能还是大幅降低成本，并进行系统工程，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改进。这些技术进步将在协作中部署。

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.