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个系统，这将使研究人员能够更好地准备冷冻水化标本的研究，通过在透射电子显微镜（TEM）的断层重建。目前的方法制备冷冻水化标本使用冷冻超微切片产生波浪状的样品与组织变形，需要大量的时间和耐心的投资，以获得即使是低产量的结果。商业聚焦离子束（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，目前可用的FIB系统允许样品污染（主要是霜冻），并且该技术的样品处理硬件较差。在与沃兹沃斯中心NIH生物技术资源的生物复杂性可视化资源（RVBC）密切合作中，我们将开发一种新的样本保持器，演示该样本平稳地转移到FIB中和从FIB中转移出来，演示基本的FIB切割模式，并最终显示上级断层图像。我们的目的是开发和商业化一个具有成本效益的系统，使这些技术成为生物研究界广泛使用。大众点评：该提案将开发工具，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。这些知识是我们对生物功能的基本知识的基础，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.