plunge freezing of electron microscopy (em) grids for cryotomography

用于原位细胞冷冻断层扫描的电子显微镜网格的制备

In situ cellular cryotomography is a powerful technique allowing for the study of biologically-relevant structures in cells without chemical fixation. By attaching cells to EM grids and plunge-freezing the grids in a cryogen, objects of interest are frozen in their natural cellular contexts without the formation of crystalline ice from intracellular water1,2. Both chemical fixation and crystalline ice formation can disrupt the structures of relevant molecules, such as proteins and lipids, reducing the biological accuracy of images obtained using these techniques3,4. In tomography, grids are imaged at incremental angles using electron microscopy, and these images are then used to construct three-dimensional representations of the target region imaged5. In situ cryotomography can be used alongside other microscopy techniques for integrative and correlative imaging, such as cryofluorescence imaging, soft x-ray tomography, and cryoFIB/SEM (cryogenic Focused Ion Beam/Scanning Electron Microscopy)6,7,8,9,10,11. Integration of multiple techniques allows for more information to be obtained about a structure or process than any single microscopy technique could achieve.
Despite all of the benefits of in situ cellular cryotomography, sample preparation can prove to be challenging for a variety of reasons. Due to their fragility, forceful manipulation of electron microscopy grids can lead to damage, with the thin carbon layer in particular being delicate and prone to tearing, reducing the imageable area of the grids. Electron microscopy grids are also difficult to manipulate due to their small size and are prone to becoming detached from the surface of the wells or microslide used to grow cells. Manipulation of the grids within the wells or microslides can prove difficult due to the geometry of these. Improper preparation of the grids (e.g., allowing them to float) can lead to low cell density and reducing the number of potential imaging areas, especially when cells are not prone to attach to the grids themselves. For direct cellular cryotomography, cells must&#160;spread very thin, which can be disrupted for many reasons, including improper temperatures or rough handling of the grids.
Through a variety of optimizations, the techniques presented in this article are meant to handle these most common pitfalls which arise during the preparation of electron microscopy grids for cryotomography. The use of 5/15 angled tweezers allows for the manipulation of grids within well plates or microslides. A fibronectin solution applied to both sides of the grids prior to plating makes floating grids less likely, which is beneficial in ensuring that grids have adequate cell density and that the grids are less likely to be damaged due to manipulation. By keeping the grids incubated at 37&#176;C until just before plunge freezing, we also ensure that the cells are kept in a comfortable environment to prevent the cells from retracting their thin edges. Blotting the grids from the back side also prevents damage to the cells from mechanical force. Altogether, these measures increase the success rate of sample preparation for in situ cellular cryotomography studies, increasing the accessibility of this imaging approach.

作者聚焦：优化增强型冷冻电子断层扫描的网格制备 

哺乳动物细胞 原位 冷冻断层扫描的样品制备

Watch this Scientific Journal Video about Plunge Freezing of Electron Microscopy EM Grids for Cryotomography at JoVE.com

Plunge Freezing of Electron Microscopy (EM) Grids for Cryotomography  

用于冷冻断层扫描的电子显微镜 （EM） 网格的浸入冷冻

在对含有细胞的电子显微镜网格进行深度冷冻之前，在倒置光学显微镜下检查网格。记录每个网格上的细胞密度，以调整浸入冷冻期间的印迹时间。在 37 摄氏度的空板中加热 2 至 3 毫升磷酸盐缓冲盐水或 PBS。对于金基准点制备，将 50 微升 10 纳米胶体金珠溶液移液到干净的 1.5 毫升管中。向其中加入两微升牛血清白蛋白，并通过反复微量移液匀浆。将试管以 15， 000 至 20， 000 G 离心 15 分钟。小心地除去上清液，不要使用微量移液管干扰沉淀。如前所述，将沉淀重新悬浮在50微升PBS中，并再次离心管。使用 10 微升吸头吸出 4 微升沉淀，并将其转移到干净的 1.5 毫升管中。将环境室设置在 95% 湿度和 30 摄氏度。使用 5 x 15 镊子，选择一个网格并用 PBS 清洗。将清洗过的网格转移到插入镊子上。固定网格后，取出 5 x 15 镊子并滑动 clamp 在插入镊子上。将网格插入插入冷冻机，同时保持夹子牢固。在网格的背面添加一微升的金基准点。然后将两到三微升的 PBS 添加到网格的碳侧。在浸入冷冻液态乙烷之前，利用自动印迹法对网格的背面进行印迹。转染 U2OS 细胞的冷冻相关光学和电子显微镜图像显示存在产生 HIV 的细胞。电子冷冻断层扫描图像显示多个HIV颗粒从U2OS细胞的质膜中萌芽。

plunge-freezing-of-electron-microscopy-em-grids-for-cryotomography

Research

JoVE Journal

Biochemistry

Plunge Freezing of Electron Microscopy (EM) Grids for Cryotomography

365 次观看. 在对含有细胞的电子显微镜网格进行深度冷冻之前，在倒置光学显微镜下检查网格。记录每个网格上的细胞密度，以调整浸入冷冻期间的印迹时间。在 37 摄氏度的空板中加热 2 至 3 毫升磷酸盐缓冲盐水或 PBS。对于金基准点制备，将 50 微升 10 纳米胶体金珠溶液移液到干净的 1.5 毫升管中。向其中加入两微升牛血清白蛋白，并通过反复微量移液匀浆。将试管以 15， 000 至 2...

视频: Plunge Freezing of Electron Microscopy (EM) Grids for Cryotomography  

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...