System for generation of frozen-hydrated samples using the Focused Ion Beam

描述(由申请人提供)：我们申请的长期目标是开发硬件和程序，使研究人员能够更好地了解细胞的功能。我们的具体目标是提供一种系统，使研究人员能够更好地准备冷冻水化样品，以便通过在透射电子显微镜(TEM)中进行断层重建进行研究。目前使用冷冻-超微切割法制备冷冻水化样品的方法会产生具有组织变形的波状样品，需要投入大量的时间和耐心才能获得即使是低产率的结果。商用聚焦离子束(FIB)系统是一种很有前途的样品制备替代工具，但最新研究表明，目前与FIB一起使用的系统允许样品污染(主要是霜冻)，并且对于这项技术来说，样品处理硬件很差。我们将与沃兹沃斯中心NIH生物技术资源中心的生物复杂性可视化资源(RVBC)密切合作，开发一种新的样品夹具，展示该样品顺利地进出FIB，展示基本的FIB切割图案，并最终显示优越的断层图像。我们的目的是开发一种具有成本效益的系统并将其商业化，使这些技术能够广泛应用于生物研究界。公众评论：这项提案将开发工具，使生物学家能够以前所未有的分辨率和清晰度获得细胞结构和功能的图像。这些知识是我们生物功能基本知识的基础，并将直接影响医学研究和实践。

DESCRIPTION (provided by applicant): The long term objective of our application is to develop hardware and procedures that will allow researchers to better understand the function of the cell. Our specific aim is to provide a system that will allow researchers to better prepare frozen hydrated specimens for study via tomographic reconstruction in the transmission electron microscope(TEM) Current methods of preparing frozen hydrated specimens using cryo-ultramicrotomy produce wavy samples with tissue deformation and require a large investment of time and patience to get even low yield results. A commercial focused ion beam (FIB) system is a promising alternative tool for sample preparation, but the latest research suggests that currently available systems for use with the FIB allow sample contamination (primarily frost) and have poor sample handling hardware for this technique. In close collaboration with the Resource for the Visualization of Biological Complexity (RVBC) at the Wadsworth Center's NIH Biotechnological Resource, we will develop a new sample holder, demonstrate smooth transfer of this sample into and out of the FIB, demonstrate basic FIB cutting patterns and ultimately show superior tomograms. It is our intent to develop and commercialize a cost effective system that will enable these techniques to become widely available to the biology research community. Public comment: This proposal will develop tools that allow biologist to obtain images of the structure and function of the cell at unprecedented levels of resolution and thus clarity. This knowledge is fundamental to our basic knowledge of biological function, and will directly impact medical research and practice.