Ultra-stable liquid-nitrogen-free holder for high-resolution cryo-electron microscopy - Phase I

用于高分辨率冷冻电子显微镜的超稳定无液氮支架 - 第一阶段

• 批准号: 545135-2019
• 负责人: Sciaini, Germán
• 金额: $ 9.11万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Idea to Innovation
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=693393
• 关键词: Ultra; stable; liquid; nitrogen; free

One important technical problem in cryo-EM, when trying to attain atomically resolved 3D structures of frozen hydrated specimens, is the negative effect on the resolution due to vibrations and sample drift. Cryo-EM holders have so far relied on the application of a dewar that has to be filled with LN to cool down the specimen to temperatures below -145 C. However, the use of LN is often inconvenient. Water crystallization inside the dewar and relatively hot spots may induce vibration through LN bubbling, limiting the attainable spatial resolution. Significant user expertise is often required to circumvent these issues and perform single particle cryo-EM measurements with atomic spatial resolution, i.e. < 0.3 nm. In addition, LN spills limit the attainable single tilt angle required to recover 3D information in cryo-electron tomography studies.Waterloo researchers have designed a cryo-EM holder that does not require the use of LN, and therefore avoids the constant refilling step inherent with this method and all aforementioned issues associated with the use of LN. The design is capable of providing equal or lower sample temperatures than the current state of the art, with the additional benefit of minimal to no vibrations, no sample drift caused by LN evaporation, extended (basically unlimited) cooling periods, and any arbitrary single axis tilting.This technology has been assigned to the University of Waterloo (Waterloo) through its Commercialization Office (WatCo), which filed a US provisional patent (Petruk and Sciaini; USPTO 62/801,327, February 5th, 2019). The proposed cryogenic approach has the potential to become the method of choice in the performance of high-resolution cryo-EM work without the need of expensive cryo-TEMs with autoloaders.

当试图达到冷冻水合样品的原子解析的3D结构时，一个重要的技术问题是由于振动和样品漂移而导致的分辨率的负面影响。迄今为止，冷冻EM持有人依赖于必须用LN填充的Dewar的应用，以使样品冷却至-145 C以下的温度。但是，LN的使用通常不便。露水内部的水结晶和相对较高的斑点可能会通过LN冒泡引起振动，从而限制了可达到的空间分辨率。通常需要大量的用户专业知识来规避这些问题并使用原子空间分辨率进行单个粒子冷冻EM测量值，即<0.3 nm。此外，LN泄漏限制了在冷冻电子层析成像研究中恢复3D信息所需的可达到的单个倾斜角度。Waterloo研究人员设计了一个不需要使用LN的冷冻EM持有人，因此避免使用此方法固有的持续重新填充步骤，以及与LN使用相关的所有上述问题。该设计能够比目前的艺术状态提供相等或较低的样品温度，并提供最小化的额外好处，没有振动，没有LN蒸发引起的样品漂移，延长（基本上是无限的）冷却期以及任何任意的单轴倾斜以及任何任意的单轴倾斜。 Sciaini; USPTO 62/801,327，2019年2月5日）。拟议的低温方法有可能成为高分辨率低分机工作性能的选择方法，而无需使用自动加载器昂贵的冷冻TEM。