Detachable Nozzle Piezoelectric Dispense System for Cryo-TEM Sample Preparation

描述（由申请人提供）：项目摘要/摘要工程艺术（EA）提出了一种可拆卸喷嘴压电分配系统的发展，用于冷冻透射电子显微镜（Cryo-TEM）样品制备仪器。低温透射电镜是研究生物大分子天然水相结构的重要工具。制备用于冷冻透射电镜的“薄冰”冷冻水样品的具有挑战性和费力的过程几十年来基本保持不变。通常，将单个含水生物样品（~3¿l）手动涂在薄多孔电子显微镜（EM）网格上，然后将其吸光以去除多余的样品，然后将其浸入液体乙烷中进行速冻。这一过程有望在网格上留下足够的区域，这些区域具有适当的玻璃化“薄冰”，通常厚度为50至200nm，这是EM成像所需的。本文提出的可拆卸式压电喷嘴点胶系统是一项关键性创新，将为低温透射电镜工作流程带来生产率、质量和性能的突破。低温透射电镜（Cryo-TEM）工艺将实现以下主要优势：应用许多不同的样品（或样品条件）到每个冷冻透射电镜网格。2. 与目前的标准印迹方法相比，增加工艺控制和可接受薄冰的产量。3. 降低起始样本量要求和每个网格的消耗。4. 允许时间分辨研究混合/结合事件的多个样品。EA将设计、制造和优化一种多通道可拆卸喷嘴压电点胶系统，该系统能够将几种不同生物样品（或样品条件）的液滴（小于50皮升）分配到每个TEM网格上。在NRAMM（国家自动分子显微镜资源，Scripps, La Jolla CA）的定制冷冻- tem样品制备仪器（Spotiton仪器平台）上改装的三通道可拆卸喷嘴压电点胶系统，用于测试，优化和验证。

DESCRIPTION (provided by applicant): Project Summary / Abstract Engineering Arts (EA) proposes the development of a detachable nozzle piezoelectric dispense system for use in Cryo-TEM (Cold - Transmission Electron Microscopy) sample preparation instrumentation. Cryo-TEM is an important tool for studying the structure of biological macromolecules in their native aqueous state. The challenging and laborious process of preparing 'thin-ice' frozen aqueous samples for Cryo-TEM has remained essentially unchanged for decades. Typically a single aqueous biological sample (~3¿l) is manually applied onto a thin porous Electron Microscopy (EM) grid which is then blotted to remove excess sample and then plunged into liquid-ethane for flash freezing. This process hopefully leaves enough regions on the grid with the proper vitrified 'thin-ice', typically 50 to 200nm thickness, required for EM imaging. The detachable piezoelectric nozzle dispense system proposed here is a key innovation that will lead to productivity, quality and capability breakthroughs for Cryo-TEM workflow. The following key benifits for Cryo-TEM process will be realized: 1. Apply many different samples (or sample conditions) to each Cryo-TEM grid. 2. Increase process control and yields of acceptable thin-ice over current standard blotting methods. 3. Lower starting sample volume requirements and consumption per grid. 4. Allow time-resolved study of mixing / binding events of multiple samples. EA will design, fabricate and optimize a multichannel detachable nozzle piezoelectric dispense system that is capable of dispensing droplets (less than 50 picoliters) of several different biological samples (or sample conditions) onto each TEM grid. A three channel detachable nozzle piezoelectric dispense system retrofitted onto a custom Cryo-TEM sample preparation instrument (Spotiton instrument platform) at NRAMM (National Resource for Automated Molecular Microscopy, Scripps, La Jolla CA.) for testing, optimization and validation.