Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM.
Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM.
复制标题
DOI：
10.1016/j.jsb.2012.04.020
复制
发表时间：
2012-07
影响因子：
3
通讯作者：
Potter, Clinton S.
中科院分区：
文献类型：
--
作者：
Jain, Tilak;Sheehan, Patrick;Crum, John;Carragher, Bridget;Potter, Clinton S.
在过去三十年中，冷冻透射电镜已发展成为一种强大的技术，可对天然玻璃化状态的生物大分子进行高分辨率成像。然而，将标本玻璃化到 EM 网格上的技术本质上没有改变 - 将约 3 µL 样品施加到网格上，然后吸干并快速冷冻到液体乙烷中。通常需要进行多次试验才能获得适合冷冻 TEM 成像的合适薄（几百纳米或更小）玻璃化层，这会导致宝贵的样品和资源的浪费。虽然商用仪器提供一定程度的自动化来控制玻璃化过程，以提高质量和再现性，但获得满意的玻璃化标本仍然是冷冻 TEM 管道中的瓶颈。我们在这里描述了一种基于使用喷墨技术进行小体积（皮升至纳升）分配的全新电镜样品制备方法。第一个原型系统（Spotiton v0.5）展示了这种新的标本玻璃化方法的可行性。压电喷墨分配器与光学实时相机（100 Hz 帧速率）集成，可分析飞行中的皮升到纳升液滴轮廓以及网格上的扩散动态，从而提供一种优化过程时序的方法。使用 TEM 成像和生化分析，我们证明压电喷墨机制不会破坏大分子的结构或功能完整性。这些初步研究深入了解了需要进一步开发的因素和组成部分，以便使用这种新颖的方法实现稳健且可重复的标本玻璃化技术。
Over the last three decades, Cryo-TEM has developed into a powerful technique for high-resolution imaging of biological macromolecules in their native vitrified state. However, the technique for vitrifying specimens onto EM grids is essentially unchanged – application of ~ 3 µL sample to a grid, followed by blotting and rapid plunge freezing into liquid ethane. Several trials are often required to obtain suitable thin (few hundred nanometers or less) vitrified layers amenable for cryo-TEM imaging, which results in waste of precious sample and resources. While commercially available instruments provide some level of automation to control the vitrification process in an effort to increase quality and reproducibility, obtaining satisfactory vitrified specimens remains a bottleneck in the Cryo-TEM pipeline. We describe here a completely novel method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A first prototype system (Spotiton v0.5) demonstrates feasibility of this new approach for specimen vitrification. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process. Using TEM imaging and biochemical assays we demonstrate that the piezo-electric inkjet mechanism does not disrupt the structural or functional integrity of macromolecules. These preliminary studies provide insight into the factors and components that will need further development to enable a robust and repeatable technique for specimen vitrification using this novel approach.
登录
查看更多内容
影响因子：
--
作者：
Nagaraj, Vinay Janthakahalli;Eaton, Seron;Wiktor, Peter
通讯作者：
Wiktor, Peter
影响因子：
2
作者：
LEPAULT, J;BOOY, FP;DUBOCHET, J
通讯作者：
DUBOCHET, J
DOI：
10.1016/j.bbrc.2008.08.033
发表时间：
2008-10-31
影响因子：
3.1
作者：
Nagaraj, Vinay J.;Eaton, Seron;Wiktor, Peter
通讯作者：
Wiktor, Peter
影响因子：
3
作者：
White, HD;Thirumurugan, K;Trinick, J
通讯作者：
Trinick, J
影响因子：
2.2
作者：
BERRIMAN, J;UNWIN, N
通讯作者：
UNWIN, N