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.

<ol>
	<li>Nogales, E., Scheres, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM:+A+unique+tool+for+the+visualization+of+macromolecular+complexity.">Cryo-EM: A unique tool for the visualization of macromolecular complexity.</a> Molecular Cell. 58 (4), 677-689 (2015).</li><li>Martynowycz, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+tomography+and+subtomogram+averaging.">Cryo-electron tomography and subtomogram averaging.</a> Methods in Enzymology. 579, 329-367 (2016).</li><li>B&#228;uerlein, F. J., Pastor-Pareja, J. C., Fern&#225;ndez-Busnadiego, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+tomography+of+native+Drosophila+tissues+vitrified+by+plunge+freezing.">Cryo-electron tomography of native Drosophila tissues vitrified by plunge freezing.</a> bioRxiv. , 437159 (2021).</li><li>Hampton, C. 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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><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 &amp;micro;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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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&amp;eacute;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&amp;auml;rzh&amp;auml;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>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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&amp;eacute;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 &amp;micro;m holes spaced 1 &amp;micro;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&#039;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 ...

micropatterning-transmission-electron-microscopy-grids-to-direct-cell

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.5K Views.  University of Wisconsin, Madison. Ons onderzoeksteam ontwikkelt technologieën en protocollen voor cryo-elektronenmicroscopie en cryo-elektronentomografie. We hebben protocollen ontwikkeld voor het micropatriëren van TEM-rasters om celgroei en positionering voor cryo-ET-experimenten te sturen. Hele-cel cryo-elektronentomografie wordt gebruikt om resolutiestructuren op nanometerniveau van macromoleculaire complexen te produceren in cellen die bewaard zijn gebleven in een inheemse bevroren gehydrateerde toestand.Micropa...

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

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Immunology and Infection

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Bioengineering

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

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 easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples 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 ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

Biology

Preparation of Electron Microscopy Grids for In Situ Cellular 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.

Author Spotlight: Optimizing Grid Preparation for Enhanced Cryoelectron Tomography 

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 ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...

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

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