Name: micropatterning transmission electron microscopy grids to direct cell positioning within whole-cell cryo-electron tomography workflows
Uploaded: 2021-09-13T13:00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows at JoVE.com

We thank Dr. Jill Wildonger, Dr. Sihui Z. Yang, and Mrs. Josephine W. Mitchell in the Department of Biochemistry, University of Wisconsin, Madison for generously sharing the elav-Gal4, UAS-CD8::GFP fly strain (Bloomington stock center, #5146). We would also like to thank Dr. Aur&#233;lien Duboin, Mr. Laurent Siquier, and Ms. Marie-Charlotte Manus from Alv&#233;ole and Mr. Serge Kaddoura from Nanoscale Labs for their generous support during this project. This work was supported in part by the University of Wisconsin, Madison, the Department of Biochemistry at the University of Wisconsin, Madison, and public health service grants R01 GM114561, R01 GM104540, R01 GM104540-03W1, and U24 GM139168 to E.R.W. and R01 AI150475 to P.W.S. from the NIH. A portion of this research was supported by NIH grant U24 GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. We are also grateful for the use of facilities and instrumentation at the Cryo-EM Research Center in the Department of Biochemistry at the University of Wisconsin, Madison.

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The authors have nothing to disclose.

Modern, advanced electron microscopes and software packages now support streamlined automated cryo-EM and cryo-ET data collection where hundreds to thousands of positions can be targeted and imaged within a few days32,33,34,35. One significant limiting factor for whole-cell cryo-ET workflows has been obtaining sufficient numbers of collectable targets per grid. Recently, a number of groups have developed protocols for micropatterning grids for cryo-EM, with one advantage being improved data collection efficiency16,17,18. Here a protocol is presented for using a commercially available micropatterning system to micropattern TEM grids for cryo-ET studies of primary Drosophila neurons and cultured human cell lines (uninfected or RSV-infected). This micropatterning system is versatile and many steps can be optimized and tailored to fit specific experimental goals. A user with TEM and fluorescence microscopy experience can quickly become skilled in grid preparation and micropatterning. With careful practice, good results should be achievable after a few iterations. Below, some of the options available, user considerations, potential benefits, and future applications of micropatterning for cryo-EM are discussed.
One of the important considerations for whole cell cryo-ET is EM grid selection. EM grids are composed of two parts: a mesh frame (or structural support) and the foil (or film), which is the continuous or holey film surface on which cells will grow. Copper mesh grids are commonly used for cryo-EM of proteins and isolated complexes. However, they are unsuitable for whole-cell cryo-ET due to the cytotoxicity of copper. Instead, a gold mesh is commonly used for cellular tomography. Other options include nickel or titanium, which may provide benefits over gold such as increased rigidity16. EM grids are available with different mesh dimensions to support a range of applications. Larger mesh sizes provide more room for cells to grow between grid bars and more areas that are amenable for tilt series collection, though at the cost of increased overall specimen fragility. The most commonly used foil is perforated or holey amorphous carbon, such as Quantifoils or C-flat grids. Biological targets can be imaged either through the holes in the carbon or through the electron-translucent carbon. Grids such as R 2/1 or R 2/2, where the holes are 2 &#181;m wide that are spaced 1 and 2 &#181;m apart respectively, provide a large number of holes and thus a large number of potential areas for data collection. However, some cells may grow and expand better on more uniform surfaces such as R 1.2/20 grids or continuous carbon. For downstream sample processing by focused-ion beam milling (cryo-FIB), the foil is removed through milling, reducing concerns over the continued presence of the underlying film. As with the mesh, foils from other materials are also available, with the patterning protocol presented here being equally suitable for SiO2 grids. Commonly used grids include gold Quantifoil, continuous carbon, or SiO2 film 200-mesh grids (~90 &#181;m spacing between grids bars) for whole-cell cryo-ET.
There are a number of considerations when designing a pattern. A majority of these decisions are guided by the cell type and purpose of the experiment. A good starting point is to choose a pattern that approximates the shape and dimensions of the cells in culture. Many studies have demonstrated significant effects of pattern shape on cell growth and cytoskeletal arrangement13,36,37. Special care should be taken during pattern design if this could alter the target of interest. Several patterns for each cell type were tested to determine which patterns promoted cellular adhesion and growth. The flexibility of the micropatterning system permits the testing of multiple patterns on a single grid and changing patterns for different grids within a single experiment. Larger patterns (~50-90 &#181;m), such as those used here, increase the likelihood that multiple cells adhere to a single region of the pattern and allow cells to expand and extend after adhesion. More constrained patterns (20-30 &#181;m) may be appropriate in experiments where cell isolation is more critical than cell expansion, such as for focused-ion beam milling (cryo-FIB) experiments. For tomography applications, one may need to consider the impact of the tilt-axis. If a pattern is positioned such that all cells grow parallel to one another in a single direction, it is possible that all of the cells will be perpendicular to the tilt-axis when loaded onto the microscope stage, resulting in a lower quality of data.
On unpatterned grids, cells often preferentially adhere to the grid bars, where they cannot be imaged by TEM. Even on patterned grids, cells are often observed to be positioned in the corners of grid squares partially on both the patterned carbon foil and grid bar. Recently, micropatterning was used to intentionally position part of the cell over the grid bar18. This could be considered for experiments where it is not critical to have the entire cell periphery on the foil. This can be especially important for cells that can grow larger than a single grid square, such as primary neurons growing over multiple days.
There are many tools that can be used to design a pattern. Here, the pattern was limited to less than 800 pixels in any dimension such that the pattern can be rotated to any angle and still fit within the maximum area that can patterned in a single projection by this micropatterning system. This allows the user to rotate the pattern to be properly oriented with the grid regardless of the orientation of the grid on the microscope. Here, the grid was divided into six patterning areas. Primarily, this allows focus adjustment between different regions of the grid. Gold grids, in particular, are very malleable and may not lay down completely flat on the glass. Proper focus is essential for clean, refined patterning results. By using segmented patterns, only minor adjustments to the pattern position need to be made if the grid shifts slightly during patterning process, though this is usually not an issue when using the PLPP gel with the PDMS stencils. Finally, the central four grid squares of the grid remained unpatterned. This supports a user being able to clearly identify the center of the grid, which is very useful for correlative-imaging experiments.
The patterning software for this micropatterning system, Leonardo, also has more advanced features such as stitching and the ability to import patterns as PDFs, which are beyond the scope of this protocol. This software also includes microstructure detection and automated pattern positioning that can be used on TEM grids. This feature is most useful when the grid is very flat and can be patterned without the need to adjust focus between different areas.
Selection of an ECM protein can have a significant impact on cell adhesion and expansion. Some cells are known to undergo physiological changes when grown on specific substrates38. Multiple ECM proteins and concentrations were tested for any new cell type based on prior work reported in the literature. Laminin, fibrinogen, fibronectin, and collagen are widely used for cultured cells and can be used as a starting point if other data is not available. However, other ECM proteins must also be considered if the commonly used ECM proteins fail to confer proper adherence properties for the cells. This was particularly true for primary Drosophila neurons, as a high-concentration of the plant lectin concanavalin A was necessary for proper cellular adherence. The compatibility of cellular adhesion and growth with the ECM can be tested by patterning on glass dishes or slides prior to transitioning to TEM grids. This pre-screening approach is time and cost-effective if a large number of combinations need to be examined. The inclusion of a fluorescently conjugated ECM protein is valuable for assessing the success and quality of patterning.
Cell seeding is one of the most important steps for whole cell cryo-ET, either with or without micropatterning6,16,39. For primary Drosophila or other neurons, which are fragile, unstable in suspension, and may be limited in quantity, single seeding approaches are preferred over monitored, sequential cell seeding. A single seeding step at an optimized cell density, as described in the protocol for Drosophila neurons, is a viable option for most cell types. However, it is also possible to seed cells onto the substrate at a lower initial concentration and add more cells in a monitored fashion as described here and in other literature18. This sequential seeding can provide more consistent results in some cases. Similar to standard cell culture, care should always be taken to maintain cell viability and minimize cell clumping during isolation.
When first starting with micropatterning, there are a few potential pitfalls that are detrimental to the final result. Careful grid handling and sterile technique, a uniform distribution of the PLPP gel, proper dose and focus during patterning, and maintenance of cell viability prior to seeding are among the most important considerations for success. A list of some of the potential issues as well as solutions were assembled in Table 1.
Micropatterned grids can be used to help position cells to establish a consistent cell density across the grid and to position regions of interest in areas suitable for tilt-series collection16,18. The placement and positioning of cells can be used as fiducial markers for correlation in cryo-CLEM experiments, reducing the need for fragile finder-grids and fluorescent fiducial markers. However, it should be noted that such fiducial markers may still be useful for sub-micrometer accuracy correlation29,40. Furthermore, an even distribution of isolated cells is also highly beneficial for focused-ion beam milling (cryo-FIB) experiments to maximize the number of cells from which lamella can be cut16.
The addition of micropatterning to cryo-EM workflows will result in measurable improvements in data throughput and potentially enable new experiments. As the technique is further adopted and developed, more advanced applications of micropatterning including ECM gradients, multiple ECM depositions, and microstructure assembly will further expand the capabilities of cryo-ET to study biological targets and processes in full cellular context.

With the development, expansion, and versatility of cryo-electron microscopy (cryo-EM), researchers have examined a wide range of biological samples in a near-native state from macromolecular (~1 nm) to high (~2 &#197;) resolution. Single-particle cryo-EM and electron diffraction techniques are best applied to purified macromolecules in solution or in a crystalline state, respectively1,2. Whereas cryo-electron tomography (cryo-ET) is uniquely suited for near-native structural and ultrastructural studies of large, heterologous objects such as bacteria, pleomorphic viruses, and eukaryotic cells3. In cryo-ET, three-dimensional (3D) information is obtained by physically tilting the sample on the microscope stage and acquiring a series of images through the sample at different angles. These images, or tilt-series, often cover a range of +60/-60 degrees in one-to-three-degree increments. The tilt-series can then be computationally reconstructed into a 3D volume, also known as a tomogram4.
All cryo-EM techniques require the sample to be embedded in a thin layer of amorphous, non-crystalline, vitreous ice. One of the most commonly used cryo-fixation techniques is plunge freezing, where the sample is applied to the EM grid, blotted, and rapidly plunged into liquid ethane or a mixture of liquid ethane and propane. This technique is sufficient for the vitrification of samples from &#60;100 nm to ~10 &#181;m in thickness, including cultured human cells, such as HeLa cells5,6. Thicker samples, such as mini-organoids or tissue biopsies, up to 200 &#181;m in thickness, can be vitrified by high-pressure freezing7. However, due to increased electron scattering of thicker samples, sample and ice thickness for cryo-ET is limited to ~0.5 - 1 &#181;m in 300 kV transmission electron microscopes. Therefore, whole-cell cryo-ET of many eukaryotic cells is limited to the cell periphery or extensions of cells unless additional sample preparation steps are used, such as cryo-sectioning8 or focused-ion beam milling9,10,11.
A limitation of many whole-cell cryo-ET imaging experiments is data collection throughput12. Unlike single-particle cryo-EM, where thousands of isolated particles can often be imaged from a single TEM grid square, cells are large, spread-out, and must be grown at low enough density to allow for the cells to be preserved in a thin layer of vitreous ice. Often the region of interest is limited to a particular feature or sub-area of the cell. Further limiting throughput is the propensity of cells to grow on areas that are not amenable for TEM imaging, such as on or near TEM grid bars. Due to unpredictable factors associated with cell culture on TEM grids, technological developments are needed to improve sample accessibility and throughput for data acquisition.
Substrate micropatterning with adherent extra-cellular matrix (ECM) proteins is a well-established technique for live-cell light microscopy to direct the growth of cells on rigid, durable, and optically transparent surfaces such as glass and other tissue culture substrates13,14. Micropatterning has also been performed on soft and/or three-dimensional (3D) surfaces. Such techniques have not only allowed for the precise positioning of cells; they have also supported the creation of multicellular networks, such as patterned neural cell circuits15. Bringing micropatterning to cryo-ET will not only increase throughput, but it can also open up new studies for exploring complex and dynamic cellular microenvironments.
Recently, several groups have begun using micropatterning techniques on TEM grids through multiple approaches16,17. Here, the use of a maskless photopatterning technique for TEM grids is described using the Alv&#233;ole PRIMO micropatterning system, which features high-resolution and contactless patterning. With this micropatterning system, an anti-fouling layer is applied on the top of the substrate, followed by the application of a photocatalyst and ablation of the anti-fouling layer in user-defined patterns with a UV laser. ECM proteins can then be added to the patterns for the appropriate cell culture. This method has been used by several groups for cryo-ET studies of retinal pigment epithelial-1 (RPE1), Madin-Darby canine kidney-II (MDCKII), human foreskin fibroblast (HFF), and endothelial cell lines16,17,18. This micropatterning system is compatible with multiple anti-fouling layer substrates as well as either a liquid or gel photocatalyst reagent. A variety of ECM proteins can be selected from and adapted for the specificity of the cell line, conferring versatility for the user.
Micropatterning has been successfully applied to a number of projects within the laboratory19. Here, a micropatterning protocol is presented, including specific adaptations to study cultured HeLa cells, respiratory syncytial virus (RSV)-infected BEAS-2B cells, and primary larval Drosophila melanogaster neurons20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1% (w/v) Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P8920-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m syringe filters PVDF membrane</td>
			<td>Genesee</td>
			<td>25-240</td>
			<td></td>
		</tr>
		<tr>
			<td>22x60-1 Glass cover slip</td>
			<td>Fisher</td>
			<td>12545F</td>
			<td></td>
		</tr>
		<tr>
			<td>5/15 Tweezers</td>
			<td>EMS (Dumont)</td>
			<td>0203-5/15-PO</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>ThermoFisher (Gibco)</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>BEAS-2B cells</td>
			<td>ATCC</td>
			<td>CRL-9609</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, bovine</td>
			<td>ThermoFisher (Gibco)</td>
			<td>A1064401</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin A, Alexa Fluor 350 Conjugate</td>
			<td>ThermoFisher (Invitrogen)</td>
			<td>C11254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Fisher (Lonza)</td>
			<td>BW12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH</td>
			<td>Fisher (Decon Labs)</td>
			<td>22-032-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen From Human Plasma, Alexa Fluor 647 Conjugate</td>
			<td>ThermoFisher (Invitrogen)</td>
			<td>F35200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Bovine Protein, Plasma</td>
			<td>ThermoFisher (Gibco)</td>
			<td>33010018</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>VWR</td>
			<td>0643-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Grid prep holder</td>
			<td>EMS</td>
			<td>71175-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher (SKC, Inc.)</td>
			<td>22600100</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher (ACROS Organics)</td>
			<td>AC172572500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher (Invitrogen)</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Fisher (Sigma Aldrich)</td>
			<td>NC0520015</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>MP Bio</td>
			<td>194844</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher (ACROS Organics)</td>
			<td>AC212595000</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica-DMi8</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Can be customized with camera, stage, and objective attachments</td>
		</tr>
		<tr>
			<td>Leonardo</td>
			<td>Alv&#233;ole</td>
			<td></td>
			<td>https://www.alveolelab.com/our-products/leonardo-photopatterning-software/</td>
		</tr>
		<tr>
			<td>Liberase Research Grade</td>
			<td>Fisher (Supply Solutions)</td>
			<td>50-100-3280</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>ThermoFisher (Invitrogen)</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Hammamatsu</td>
			<td>C13440-20CU</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized stage</td>
			<td>M&#228;rzh&#228;user Wetzlar</td>
			<td>00-24-599-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher (Fisher BioReagents)</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Fisher (ACROS Organics)</td>
			<td>AC207802500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher (Alfa Aesar)</td>
			<td>AAA1603736</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS stencils</td>
			<td>nanoscaleLABS</td>
			<td>PDMS_STENCILS_EM</td>
			<td>https://www.alveolelab.com/our-products/pdms-stencil-multiwell-plate/</td>
		</tr>
		<tr>
			<td>PEG-SVA</td>
			<td>nanoscaleLABS</td>
			<td>PEG-SVA-1GR</td>
			<td>mPEG-Succinimidyl Valerate, MW 5,000</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Fisher (Research Products International Corp)</td>
			<td>50-213-641</td>
			<td></td>
		</tr>
		<tr>
			<td>pH strips</td>
			<td>Fisher (Millipore Sigma)</td>
			<td>M1095350001</td>
			<td>pH probe can also be used</td>
		</tr>
		<tr>
			<td>PLPP gel</td>
			<td>nanoscaleLABS</td>
			<td>PLPP-GEL-300UL</td>
			<td>https://www.alveolelab.com/our-products/plpp-photoactivatable-reagent/</td>
		</tr>
		<tr>
			<td>PRIMO</td>
			<td>Alv&#233;ole</td>
			<td></td>
			<td>https://www.alveolelab.com/our-products/primo-micropatterning/</td>
		</tr>
		<tr>
			<td>pSynkRSV-I19F (BAC containing RSV A2-mK+ antigenomic cDNA )</td>
			<td>BEI Resources</td>
			<td>NR-36460</td>
			<td>https://www.beiresources.org/Catalog/BEIPlasmidVectors/NR-36460.aspx</td>
		</tr>
		<tr>
			<td>Quantifoil grids</td>
			<td>EMS (Quantifoil)</td>
			<td>Q2100AR1</td>
			<td>2 &#181;m holes spaced 1 &#181;m apart, other dimensions are available</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Fisher (Lonza)</td>
			<td>BW12-702F</td>
			<td></td>
		</tr>
		<tr>
			<td>RSV A2-mK+</td>
			<td>see entry for pSynkRSV-19F</td>
			<td>-</td>
			<td>Described in Hotard et al. [22]. Can be generated from pSynkRSV-ll9F</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Media</td>
			<td>ThermoFisher (Gibco)</td>
			<td>21720-024</td>
			<td></td>
		</tr>
		<tr>
			<td>SerialEM</td>
			<td>SerialEM (https://bio3d.colorado.edu/SerialEM/ )</td>
			<td></td>
			<td>https://bio3d.colorado.edu/SerialEM/</td>
		</tr>
		<tr>
			<td>Straight tweezers</td>
			<td>EMS (Dumont)</td>
			<td>72812-D</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Fisher (Fisher BioReagents)</td>
			<td>BP910-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Avantor</td>
			<td>4097-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma</td>
			<td>T8032-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Titan Krios electron microscope</td>
			<td>ThermoFisher</td>
			<td></td>
			<td>300kV, with direct electron detector camera and energy filter</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>ThermoFisher (Gibco)</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube Revolver/Rotator</td>
			<td>Fisher (Thermo Scientific)</td>
			<td>11676341</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS:mcD8:GFP Drosophila fly strain</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>5146</td>
			<td>http://flybase.org/reports/FBtp0002652.html</td>
		</tr>
	</tbody>
</table>

micropatterning transmission electron microscopy grids to direct cell positioning within whole-cell cryo-electron tomography workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

Watch this Scientific Journal Video about Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows at JoVE.com

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Research

JoVE Journal

Biochemistry

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of ...

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...

Coating CryoEM Grids with Monolayer Graphene to Improve Cryo-Sample Preparation

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids (Video) | JoVE

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...

Preparation of Electron Microscopy Grids for In Situ Cellular Cryotomography

Sample Preparation for In Situ Cryotomography of Mammalian Cells

Optimizing Grid Preparation for Enhanced Cryoelectron Tomography (Video) | JoVE

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

Fabrication of Streptavidin Affinity Grids for Cryo-EM Structural Studies

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation

Author Spotlight: Enhancing Cryo-EM Sample Preparation with Streptavidin-Biotin Approach

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, ...