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.

当试图获得冷冻水合样品的原子分辨3D结构时，冷冻EM中的一个重要技术问题是由于振动和样品漂移对分辨率的负面影响。到目前为止，Cryo-EM支架依赖于杜瓦瓶的应用，杜瓦瓶必须充满LN以将样品冷却到-145 ℃以下。然而，LN的使用往往不方便。杜瓦瓶内的水结晶和相对热点可能会通过LN鼓泡引起振动，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执行具有原子空间分辨率（即< 0.3 nm）的单粒子低温EM测量。此外，LN溢出限制了在冷冻电子断层扫描研究中恢复3D信息所需的可达到的单个倾斜角。Waterloo研究人员设计了一种不需要使用LN的冷冻EM保持器，因此避免了该方法固有的恒定再填充步骤以及与使用LN相关的所有上述问题。该设计能够提供与现有技术相比相等或更低的样品温度，具有最小振动至无振动、没有由LN蒸发引起的样品漂移、扩展的温度范围、更高的温度范围和更低的温度范围等附加益处。（基本上是无限的）冷却周期，和任何任意单轴倾斜。这项技术已被分配给滑铁卢大学（滑铁卢）通过其商业化办公室（WatCo）提交了美国临时专利（Petruk和Sciaini; USPTO 62/801，327，2019年2月5日）。所提出的低温方法有可能成为高分辨率冷冻EM工作的性能的选择方法，而不需要昂贵的冷冻TEM与自动加载器。