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

在冷冻EM中，当试图获得冷冻水化样品的原子分辨3D结构时，一个重要的技术问题是由于振动和样品漂移而对分辨率造成的负面影响。到目前为止，冷冻-EM保持架依赖于杜瓦的应用，杜瓦必须填充液氮来将样品冷却到零下145摄氏度以下。然而，液氮的使用往往不方便。杜瓦内部的水结晶和相对热点可能会通过液氮气泡诱导振动，限制了可获得的空间分辨率。通常需要大量的用户专业知识来绕过这些问题，并以原子空间分辨率(即&lt；0.3 nm)进行单粒子低温电磁测量。此外，液氮泄漏限制了冷冻电子断层扫描研究中恢复3D信息所需的单一倾角。滑铁卢研究人员设计了一种不需要使用液氮的冷冻-EM支架，因此避免了这种方法固有的持续灌装步骤以及与使用液氮相关的所有上述问题。该设计能够提供与当前技术水平相同或更低的样品温度，另外的好处是最小或没有振动，没有液氮蒸发引起的样品漂移，延长(基本无限)的冷却期，以及任何任意的单轴倾斜。这项技术已通过其商业化办公室(Watco)分配给滑铁卢大学(滑铁卢大学)，该办公室申请了美国临时专利(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低温电磁工作的选择方法，而不需要昂贵的带有自动加载器的低温电磁显微镜。

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.