Digitally Enhanced Microscope for Crystal Detection

描述(由申请人提供)：我们建议研究、设计和制造一种复杂的、预生产的蛋白质晶体检测显微镜，Crystal Finder(Tm)显微镜工作站，结合消光偏振和定量双折射成像，用于数字增强显示晶体体积对比度(在此称为Detect-X(Tm)技术)。许多蛋白质结晶实验都是试错式的，通常每1000个实验中只有一个能提供晶体。通常，这些最初的撞击很难检测到，因为晶体可能很小，无色，被无定形沉淀物质掩盖。大多数用于分析蛋白质结晶结构的商用显微镜只提供晶体边缘对比度，这与高效筛选生长在脂类立方相中的无色膜蛋白质晶体不兼容。因此，迫切需要更可靠、更快速的方法来准确表征晶体材料。为解决这一问题，在该项目第一阶段进行的研究表明，在概念验证仪器中成功地实施了Detect-X(Tm)技术。这项技术允许根据晶体的整体属性(双折射)而不是其与背景的折射率差异(即晶体边缘和小平面)来检测晶体。这项技术可以准确检测到更多的蛋白质晶体，同时减少假阳性命中。具体目标一：阿巴拉契亚电子仪器公司(Fairlea，WV)将设计和制造采用Detect-X(Tm)技术的Crystal Finder(Tm)显微镜工作站，这是一种预生产的自动化偏振显微镜。具体目标II：Emerald BioSystems(华盛顿州班布里奇岛)将设计和实施用户友好的仪器控制界面和优化的Detect-X(Tm)软件。该软件将集成到现有的Crystal Miner(Tm)结晶跟踪软件和关系数据库中。具体目标III：该仪器将在使用数字增强晶体检测的结晶试验中对挑战蛋白质晶体的成像性能进行评估。将与竞争对手的商业蛋白质晶体成像仪器进行并列比较。预计Crystal Finder(Tm)显微镜工作站将极大地影响在结晶试验中成功识别难以检测的蛋白质晶体。Crystal Finder(Tm)显微镜工作站的独特功能和卓越性能为其商业成功奠定了基础。它有望为自动化高通量图像增强蛋白质结晶实验筛选设定标准，并将成为大型财团和中型研究实验室的首选仪器。 蛋白质结构是生物科学和医学的基石，为疾病和药物作用提供分子基础。这项提议中描述的技术进步，并被内置到蛋白质晶体检测显微镜工作站中，预计将对蛋白质结晶学社区和公众健康的需求做出重大贡献。预计它将加快解决蛋白质结构的速度，并将成为医学科学和药物发现进步的关键媒介。

DESCRIPTION (provided by applicant): We propose to investigate, design and manufacture a sophisticated, pre-production protein crystal detection microscope, the Crystal Finder(tm) microscope workstation, incorporating extinction polarization and quantitative birefringence imaging for digitally enhanced display of crystal bulk contrast (referred to herein as Detect-X(tm) technology). Many protein crystallization experiments are of the trial-and-error type, where frequently only one in 1,000 experiments provides a crystal hit. Often these initial hits are difficult to detect because crystals may be small, colorless and disguised by amorphous precipitate material. Most commercial microscopes used to analyze protein crystallization setups provide crystal edge contrast only, which is not compatible with efficient screening of colorless membrane protein crystals grown in lipidic cubic phases. Thus, there is a critical need for more reliable and rapid methods fort the accurate characterization of crystalline materials. To provide a solution to this problem, research conducted during Phase I of this project demonstrated the successful implementation of the Detect-X(tm) technology in a proof-of-concept instrument. This technology, allows detecting crystals by virtue of their bulk properties (birefringence) rather than their refractive index difference with the background (i.e. crystal edges and facets). This technology results in a higher number of accurately detected protein crystals, with a concomitant decrease in false positive hits. Specific Aim I: A pre-production automated polarization microscope, the Crystal Finder(tm) microscopic workstation with Detect-X(tm) technology will be designed and built by Appalachian Electronic Instruments (Fairlea, WV). Specific Aim II: A user friendly instrument control interface and optimized Detect- X(tm) software will be designed and implemented by Emerald BioSystems (Bainbridge Island, WA). This software will be integrated into existing Crystal Miner(tm) crystallization tracking software and relational database. Specific Aim III: The instrument will be evaluated for performance in imaging of challenging protein crystals in a crystallization trial using digitally enhanced crystal detection. A side-by-side comparison with competing commercial protein crystal imaging instruments will be carried out. It is anticipated that the Crystal Finder(tm) microscope workstation will dramatically impact the successful identification of difficult-to-detect protein crystals in crystallization trials. The unique features and superior performance of the Crystal Finder(tm) microscopic workstation provides the basis of its commercial success. It is expected to set the standards for automated high-throughput image-enhanced protein crystallization experiment screening and will be the instrument of choice for both large consortiums and medium-size research laboratories. Protein structure is a cornerstone in biological science and medicine providing a molecular basis for disease and drug action. The technology advancement described in this proposal, and built into a protein crystal detection microscope workstation, is expected to make a significant contribution to the needs of the protein crystallography community and public health. It is expected to accelerate the pace by which protein structures are solved, and will be a critical mediator of advancement in medical science and drug discovery.