Name: Video: Plunge Freezing of Electron Microscopy (EM) Grids for Cryotomography
Uploaded: 2023-12-15T13:30:00
Duration: 2 min 45 s
Description: 362 Views. Plunge Freezing of Electron Microscopy (EM) Grids for Cryotomography

plunge freezing of electron microscopy (em) grids for cryotomography

Preparation of Electron Microscopy Grids for In Situ Cellular Cryotomography

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.

Author Spotlight: Optimizing Grid Preparation for Enhanced Cryoelectron Tomography 

Sample Preparation for In Situ Cryotomography of Mammalian Cells

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  

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Research

JoVE Journal

Biochemistry

362 Views. Plunge Freezing of Electron Microscopy (EM) Grids for Cryotomography

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

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 ...