Name: Cryo-Electron Tomography Workflow for Thick Tissues Demonstrated in Mouse Hippocampus (Video) | JoVE Journal
Uploaded: 2026-03-27T14:30:00.000+00:00
Duration: 10 min 6 s
Description: Learn cryo-electron tomography for in situ structural analysis of thick tissues using mouse hippocampus. This standardized workflow integrates high-pressure freezing, cryo-FIB milling, and automated imaging to enable high-resolution visualization of native cellular architecture.

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5 minutes

The authors thank the staff of the Cryo-EM Center at the Southern University of Science and Technology for their technical support. This project was supported by the National Science and Technology Innovation (Brain Science and Brain-like Intelligence Technology &#8211; National Science and Technology Major Project, Grant No. 2022ZD0211905) and the National Natural Science Foundation of China (Grant No. 32200998 and No. 32161133022).

All procedures involving animal tissue complied with SUSTech IACUC (Protocol SUSTech&#8209;JY202501019). The regents and the equipment used are listed in the Table of Materials.
1. Sample support preparation
<ol>
	<li>HPF carrier preparation (see Figure 1A)
	<ol>
		<li>Obtain a pair of 6 mm copper HPF carriers (type B). Ensure one of the carriers has a flat surface.</li>
		<li>Wet the flat surface of the carrier and proceed to grind. First, use 8000-grit sandpaper until the original machining marks are completely removed. Then, switch to 12000-grit sandpaper and continue grinding until scratches from the previous step are largely eliminated, resulting in a uniformly smooth finish. Figure 2 shows typical images of the carrier surface during the grinding and polishing process.</li>
		<li>Polish the ground surface using a soft metallographic cloth and W0.5 diamond paste: first, apply the paste to the cloth, then polish the surface in a circular motion for 1&#8211;2 min until a smooth, highly polished finish is achieved. Rinse the carrier thoroughly with ethanol, followed by distilled water to remove all abrasive residues.</li>
		<li>Submerge the polished carriers in 1-hexadecene for a minimum of 4 h (or overnight) prior to their use. 
		NOTE: Polished and treated carriers can be stored submerged in 1-hexadecene. 
		CAUTION: 1&#8209;Hexadecene is flammable and harmful if inhaled; work in a fume hood with PPE.</li>
	</ol>
	</li>
	<li>TEM support grid preparation (see Figure 1B)
	<ol>
		<li>Use 100-mesh copper grids with a continuous Formvar/carbon support film.</li>
		<li>Sputter-coat both sides of the grids with gold using a sputter coater: set the current to 50 mA and coat each side for 120 s to achieve a layer of approximately 6 nm per side.</li>
	</ol>
	</li>
	<li>Copper foil preparation (see Figure 1C)
	<ol>
		<li>Obtain high-purity copper foil with a thickness of 1 &#181;m. 
		NOTE: The thermal conductivity of copper varies with purity, and lower purity results in lower thermal conductivity. Therefore, high-purity copper foil (Cu &#8805; 99.9%) is required.</li>
		<li>Punch the copper foil into discs with a diameter of 3 mm using a circular hole punch.</li>
	</ol>
	</li>
</ol>
2. High-pressure freezing
<ol>
	<li>Place a 10 &#181;L droplet of 10% sucrose solution on a clean glass surface.</li>
	<li>Rapidly dissect the hippocampus from a freshly euthanized mouse. Excise small tissue pieces of approximately 0.5&#8211;1 mm3 using a sharp razor blade (see Figure 1D).</li>
	<li>Transfer the tissue onto the copper support TEM Grids (100 mesh) within the HPF carrier using a toothpick or fine brush. Use 2-methylpentane&#160;to gently maneuver the tissue into place while minimizing mechanical stress on the grid. 
	CAUTION: 2-Methylpentane is extremely flammable. Handle it in a fume hood with no ignition sources.</li>
	<li>Cover the tissue and grid with a 3-mm-diameter copper foil disc. Complete the assembly by placing the second HPF carrier (flat face down) on top to form the sealed &#8220;waffle&#8221; sandwich (see Figure 1E). 
	NOTE: The copper foil serves a critical mechanical function in the waffle assembly. It sandwiches the tissue between the TEM grid and the foil itself. During assembly, this configuration applies gentle pressure, helping to form a thinner, more uniform tissue slice ideal for vitrification. Furthermore, the foil prevents tissue loss during handling and helps distribute pressure during high-pressure freezing, reducing the risk of grid breakage. After freezing, the foil also facilitates easier separation of the carriers from the vitrified sample grid. However, this preparation method can introduce mechanical compression damage and may not be suitable for samples sensitive to pressure-induced artifacts. For such samples, alternative cutting techniques that achieve appropriate thickness without compression, such as vibratome sectioning or sample punching, should be considered.</li>
	<li>Immediately initiate the high-pressure freezing procedure in accordance with the manufacturer&#39;s operational protocol. Set the target pressure to ~2100 bar and ensure the total time from tissue dissection to freezing does not exceed 90 s. 
	&#8203;NOTE: The entire procedure from dissection to freezing should be completed within 90&#160;s to minimize structural alterations associated with prolonged ex&#160;vivo time. The tissue thickness range for brains is about 20&#8211;40 &#181;m.</li>
</ol>
3. FIB milling and lift-out
NOTE: Perform the following steps using a dual-beam microscope equipped with a cryo-stage. Maintain the sample temperature below -170 &#176;C throughout.
<ol>
	<li>Preparation of the receiver grid
	<ol>
		<li>Obtain a new 300-mesh gold grid to serve as the final receiver grid for the lift-out procedure.</li>
		<li>Under a stereomicroscope at room temperature, gently clip the grid into a compatible cryo-FIB AutoGrid ring.</li>
		<li>Store the assembled AutoGrid directly in a dry oven maintained at approximately 40 &#176;C to minimize frost accumulation prior to its use. 
		NOTE: During clipping ensure the grid lies flat and centered to prevent bending or misalignment.</li>
	</ol>
	</li>
	<li>Retrieval and mounting of the vitrified tissue grid
	<ol>
		<li>While continuously immersing the assembly in liquid nitrogen (LN&#8322;), carefully separate the vitrified TEM grid (containing the tissue and the copper foil) from the surrounding &#8220;waffle&#8221; assembly components. 
		NOTE: The copper foil spacer typically makes the grid easy to separate from the carriers.</li>
		<li>On a pre-cooled cryo-loading workstation, mount the isolated tissue grid into a second cryo-FIB AutoGrid ring under LN&#8322; protection.</li>
	</ol>
	</li>
	<li>Co-loading grids onto the cryo-transfer shuttle
	<ol>
		<li>Load both AutoGrids into the designated slots of the cryo-transfer shuttle.</li>
		<li>Orient each AutoGrid so that the milling notch faces outward and is vertical.</li>
		<li>Keep the loaded shuttle under LN&#8322; immersion or pre-cooled until transfer into the FIB/SEM microscope.</li>
	</ol>
	</li>
	<li>Localization and conductive coating in the FIB/SEM
	<ol>
		<li>After cryo-transfer into the microscope, navigate to the tissue grid at low SEM magnification.</li>
		<li>Apply a conductive and protective coating to the entire grid surface.
		<ol>
			<li>Sputter-coat the grid surface with platinum (Pt) at 30 mA for 15 s.</li>
			<li>Deposit a 0.5&#8211;1.0 &#181;m thick organometallic Pt cap using the Gas Injection System (GIS).</li>
			<li>Apply a final sputter-coated Pt layer to the grid surface at 30 mA for 15 s. 
			NOTE: The GIS cap is critical for reducing curtaining artifacts.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tissue block isolation and lift-out (see Figure 3)
	<ol>
		<li>Site selection and orientation
		<ol>
			<li>Navigate to the selected region of interest (ROI) on the tissue grid using the SEM electron beam image.</li>
			<li>Orient the sample for front-side milling. A typical stage position is set to 110&#176; rotation and 7&#176; tilt, and this position presents the sample surface at an angle suitable for ion beam access. 
			NOTE: In the absence of fluorescent markers a pre-screening strategy can be employed.&#160;This strategy aims to locate the target tissue region and exclude areas containing cryoprotectant or other non-tissue components. After selecting a region apparently free of surface ice, mill a small test trench using a higher beam current (e.g., 15 nA at 30 kV) at the standard front-side orientation and polish using a lower beam current (e.g., 1 nA at 30 kV). Switch to the electron beam to examine the cross section: the tissue exhibits excellent contrast under the electron beam, a characteristic that is particularly pronounced in membranous structures and regions rich in heavy elements; in contrast, the cryoprotectant typically shows uniform contrast under the electron beam. This allows for rapid verification before proceeding with the full lift-out procedure.</li>
		</ol>
		</li>
		<li>Trench milling to isolate the block (Figure 3A)
		<ol>
			<li>Define a rectangular trench surrounding the target tissue block using the ion beam operated at 30 kV.</li>
			<li>Begin milling at a certain distance from the block using a relatively high current of 15 nA to achieve rapid material removal.</li>
			<li>As the trench walls approach the block to within a few micrometers, reduce the ion beam current to 7 nA. Reducing the current achieves more precise control and minimizes ion beam damage and heating of the biological material in the region of interest.</li>
			<li>Leave one side of the block attached to the bulk material as a &#8220;tab&#8221; to hold it in place temporarily.</li>
		</ol>
		</li>
		<li>Optional undercut milling for thick blocks (Figure 3B)
		<ol>
			<li>For tissue blocks with an estimated thickness greater than 20 &#181;m, perform an undercut milling step from the reverse side to facilitate the final lift-out.</li>
			<li>Rotate the stage (e.g., to -70&#176;) and adjust the stage tilt (e.g., to 23&#176;) to access the bottom of the block.</li>
			<li>Mill the underside connection using the FIB at 30 kV and a current of 3 nA.</li>
		</ol>
		</li>
		<li>Attachment to the micromanipulator needle (Figure 3C)
		<ol>
			<li>To attach the sample to the needle perform sequential cross-section milling patterns. 
			NOTE: The gold block can be extracted from the bar of a 300-mesh gold grid.</li>
		</ol>
		</li>
		<li>Lift-out of the tissue block (Figure 3D)
		<ol>
			<li>Sever the remaining &#8220;tab&#8221; connecting the block to the bulk sample using the FIB (30 kV, 1 nA).</li>
			<li>Carefully maneuver the micromanipulator needle to lift the now-isolated tissue block so that it is completely clear of the trench and the surrounding original grid. Then, retract the needle and transport the block to the receiver grid.</li>
		</ol>
		</li>
		<li>Block transfer, welding, and re-coating
		<ol>
			<li>Navigate the needle to a pre-cooled 300-mesh gold receiver grid.</li>
			<li>Lower the block onto a chosen grid square. Bring the tissue block into contact with the film on the grid, then lift the block vertically to break the contact. Mill a ~3&#8211;4 &#181;m thick slice from the block onto the grid film using the ion beam (30 kV, 1 nA) (Figure 3E). 
			NOTE: If ice debris is present, pre-clean the attachment site with a rectangular pattern (30 kV, 50 pA). Make sure the block makes contact with the grid film to reduce electrostatic charge, which may randomly displace the slice.</li>
			<li>Weld the slice securely by milling using cleaning cross-section patterns on both sides of the junction (30 kV, 30 pA) (Figure 3F).</li>
			<li>Reapply the conductive/protective coating as described in Step 3.4.2 (Figure 3G). 
			NOTE: For this GIS plating step the duration can be reduced depending on block size.</li>
		</ol>
		</li>
		<li>Lamella thinning
		<ol>
			<li>Rotate the stage (e.g., to -70&#176;) and set an initial stage tilt (e.g., 15&#176;) (Figure 3H). 
			NOTE: The sample holder features a pre&#8209;tilt of 45&#176;.</li>
			<li>Define the final lamella position within a smooth area of the tissue block. 
			NOTE: Confirm that the selected area is free of ice contamination.</li>
			<li>Perform systematic milling using the ion beam currents specified in Table 1.&#160;Progress is made from higher to lower currents as the target plane is approached (Figure 3I,J). Optionally, when within 1&#8211;2 &#181;m of the lamella, a stress-relief trench may be milled approximately 1.5 &#181;m away to prevent bending or curling of the lamella30,31. 
			&#8203;NOTE: When the remaining material is within ~1 &#181;m of the target plane limit the thickness removed per pass.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Cryo-ET data collection
<ol>
	<li>Following successful lamella preparation, carefully load the grid into a transmission electron microscope (TEM) equipped with a cryo-holder, an energy filter, and tomography software (e.g., SerialEM).</li>
	<li>Acquire an atlas map of the entire grid (e.g., Figure 4A; typical magnification is 155&#215;).</li>
	<li>Navigate to the lamella position and acquire a montage map (e.g., Figure 4B; typical magnification is 3600&#215; with a defocus of -100 &#181;m).</li>
	<li>Set the nominal recording magnification and verify that the pixel size is appropriate for the intended resolution; in the present study, a pixel size of 2.109 &#197; was used. Adjust the corresponding illumination parameters, including spot size, C2 aperture diameter, and illumination area, to achieve an optimized dose rate for the direct electron detector.</li>
	<li>Configure the focus and trial illumination conditions to match those set for the Record parameter.</li>
	<li>Conduct data collection using the PaceTomo scripts32, following the steps below:
	<ol>
		<li>Pre-select targets on the montage map using the &#34;add points&#34; function in the navigator, ensuring the first feature exhibits sufficient contrast.</li>
		<li>Select targets using the PACEtomo_selectTargets.py script32, adjusting the featured area at the preview magnification if necessary. Typical regions of interest are displayed in&#160;Figure 4C.</li>
		<li>Acquire tilt series using a dose-symmetric scheme (e.g., 0&#176;, +3&#176;, -3&#176;, +6&#176;, -6&#176;... up to &#177;60&#176;&#8211;66&#176;) with an increment of 2&#176; or 3&#176;. Set acquisition parameters in the PACEtomo.py script: the starting tilt angle is determined by the milling angle specified in Step 3.7.1 (i.e., 8&#176;), with its sign dependent on the sample loading orientation in the cryo-EM; the minimum and maximum tilt angles refer to absolute values; and the step size corresponds to the tilt increment (e.g., 3&#176;). 
		NOTE: This starting tilt angle is designed to position the lamella horizontally and perpendicular to the electron beam. If the milling angle is unknown, tilt the image to display the lamella at its largest apparent height; this provides a sufficiently accurate alignment.</li>
		<li>Maintain a cumulative electron dose of 100&#8211;150 e&#8315;/&#197;2&#160;distributed across all tilts. If available, use an energy filter with a slit width of 10 eV.</li>
		<li>Initiate data acquisition. Automated focusing and tracking are implemented at each tilt angle. The typical acquisition time for a single tilt series is 5&#8211;8 min. 
		NOTE: A troubleshooting guide is provided in Table 2.</li>
	</ol>
	</li>
</ol>
5. Data processing
<ol>
	<li>Process Tilt-Series Motion Correction, CTF Estimation, Alignment, and 3D Tomogram Reconstruction images using AreTomo 333&#160;to perform three core steps: (1) motion correction of raw image frames, (2) estimation of the contrast transfer function (CTF) for each tilt image, and (3) alignment of the full tilt-series.
	<ol>
		<li>Following alignment reconstruct three-dimensional (3D) tomograms via the weighted back-projection algorithm.&#160;Required inputs for this step included: raw image frames, mdoc metadata files (which document tilt-series acquisition parameters), and the pre-determined rotation axis of the sample.</li>
	</ol>
	</li>
	<li>Missing wedge correction
	<ol>
		<li>After reconstruction subject the generated tomograms to missing wedge correction using IsoNet34.</li>
	</ol>
	</li>
	<li>Particle picking and curating
	<ol>
		<li>Conduct particle picking in EMAN 2.935 using the template matching method.&#160;After automated picking, false positives (i.e., non-target regions or erroneously selected regions) were manually excluded to ensure the quality of the selected particle dataset.</li>
	</ol>
	</li>
	<li>Subtomogram averaging
	<ol>
		<li>Import the tilt series and their corresponding particle coordinates into RELION 4.0. Perform subtomogram averaging following the standard workflow outlined in the RELION 4.0 documentation&#160;(https://relion.readthedocs.io/en/release-4.0/STA_tutorial/Introduction.html), which ensures adherence to established best practices for reproducibility.</li>
	</ol>
	</li>
</ol>

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<li>Nguyen, H. T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serialized+on-grid+lift-in+sectioning+for+tomography+%28solist%29+enables+a+biopsy+at+the+nanoscale%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Serialized on-grid lift-in sectioning for tomography (solist) enables a biopsy at the nanoscale.</a> Nat Methods. 21 (9), 1693-1701 (2024).</li>
<li>Tang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Step-by-step+procedure+for+an+optimized+serial+lift-out+cryo-focused+ion+beam+milling+technique+in+tissue+analysis%5BTitle%5D)+AND+%22Biophys+Rep%22%5BJournal%5D)">Step-by-step procedure for an optimized serial lift-out cryo-focused ion beam milling technique in tissue analysis.</a> Biophys Rep. 11, 1-13 (2025).</li>
<li>Wolff, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mind+the+gap%3A+Micro-expansion+joints+drastically+decrease+the+bending+of+fib-milled+cryo-lamellae%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">Mind the gap: Micro-expansion joints drastically decrease the bending of fib-milled cryo-lamellae.</a> J Struct Biol. 208 (3), 107389(2019).</li>
<li>Berger, C., Watson, H., Naismith, J. H., Dumoux, M., Grange, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenon+plasma+focused+ion+beam+lamella+fabrication+on+high-pressure+frozen+specimens+for+structural+cell+biology%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Xenon plasma focused ion beam lamella fabrication on high-pressure frozen specimens for structural cell biology.</a> Nat Commun. 16 (1), 2286(2025).</li>
<li>Eisenstein, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parallel+cryo+electron+tomography+on+in+situ+lamellae%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Parallel cryo electron tomography on in situ lamellae.</a> Nat Methods. 20 (1), 131-138 (2022).</li>
<li>Peck, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aretomolive%3A+Automated+reconstruction+of+comprehensively-corrected+and+denoised+cryo-electron+tomograms+in+real-time+and+at+high+throughput%5BTitle%5D)+AND+%22Preprint.+bioRxiv%22%5BJournal%5D)">Aretomolive: Automated reconstruction of comprehensively-corrected and denoised cryo-electron tomograms in real-time and at high throughput.</a> Preprint. bioRxiv. , (2025).</li>
<li>Liu, Y. -T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isotropic+reconstruction+for+electron+tomography+with+deep+learning%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Isotropic reconstruction for electron tomography with deep learning.</a> Nat Commun. 13 (1), 6482(2022).</li>
<li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+complete+data+processing+workflow+for+cryo-ET+and+subtomogram+averaging%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">A complete data processing workflow for cryo-ET and subtomogram averaging.</a> Nat Methods. 16 (11), 1161-1168 (2019).</li>
<li>Yang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+reduction+of+fib+damage+on+cryo-lamella+by+lowering+energy+of+ion+beam+revealed+by+a+quantitative+analysis%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">The reduction of fib damage on cryo-lamella by lowering energy of ion beam revealed by a quantitative analysis.</a> Structure. 31 (10), 1275-1281.e4 (2023).</li>
<li>Tacke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+streamlined+workflow+for+automated+cryo+focused+ion+beam+milling%5BTitle%5D)+AND+%22J+Struct+Biol%22%5BJournal%5D)">A streamlined workflow for automated cryo focused ion beam milling.</a> J Struct Biol. 213 (3), 107743(2021).</li>
<li>Boltje, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cryogenic%2C+coincident+fluorescence%2C+electron%2C+and+ion+beam+microscope%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">A cryogenic, coincident fluorescence, electron, and ion beam microscope.</a> Elife. 11, e82891(2022).</li>
<li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+fluorescence+microscopy+%28IFLM%29+for+cryo-fib-milling+and+in-situ+cryo-ET%5BTitle%5D)+AND+%22Preprint.+bioRxiv%22%5BJournal%5D)">Integrated fluorescence microscopy (IFLM) for cryo-fib-milling and in-situ cryo-ET.</a> Preprint. bioRxiv. , (2023).</li>
<li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eli+trifocal+microscope%3A+A+precise+system+to+prepare+target+cryo-lamellae+for+in+situ+cryo-ET+study%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Eli trifocal microscope: A precise system to prepare target cryo-lamellae for in situ cryo-ET study.</a> Nat Methods. 20 (2), 276-283 (2023).</li>
<li>Medeiros, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+workflow+and+instrumentation+for+cryo-focused+ion+beam+milling+of+samples+for+electron+cryotomography%5BTitle%5D)+AND+%22Ultramicroscopy%22%5BJournal%5D)">Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography.</a> Ultramicroscopy. 190, 1-11 (2018).</li>
<li>Goetz, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modular+platform+for+automated+cryo-fib+workflows%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">A modular platform for automated cryo-fib workflows.</a> Elife. 10, e70506(2021).</li>
<li>Eisenstein, F., Fukuda, Y., Danev, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Smart+parallel+automated+cryo-electron+tomography%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Smart parallel automated cryo-electron tomography.</a> Nat Methods. 21 (9), 1612-1615 (2024).</li>
<li>Swulius, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+learning-based+segmentation+of+cryo-electron+tomograms%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Deep learning-based segmentation of cryo-electron tomograms.</a> J Vis Exp. (189), e64435(2022).</li>
</ol>

The authors have nothing to disclose.

The workflow presented herein establishes a generalized, optimized, and reproducible pipeline for in situ cryo-electron tomography of native, thick tissues. Its primary contribution lies not in the invention of singular techniques, but in the systematic integration, refinement, and detailed standardization of the entire process, from robust vitrification using a modified waffle-HPF method to a precisely calibrated cryo-FIB lift-out and thinning protocol. This synthesis addresses the critical reproducibility and ...

Cryo-electron tomography (cryo-ET) has emerged as a pivotal technique for visualizing macromolecular complexes and cellular architectures in their native, hydrated state1,2,3,4,5. By reconstructing three-dimensional volumes from tilt-series of projection images, cryo-ET bridges the gap between cellular context and near-atomic resolution, offering unprecedented insights into structural biology in situ6. While cryo&#8209;electron tomography workflows for cellular specimens7 (including cryo&#8209;FIB&#8209;thinned cells in suspensions and adherent cells) are now well&#8209;established, their direct extension to thick, architecturally intact tissues remains highly challenging8,9,10. This is especially true for high&#8209;water&#8209;content tissues such as brain11, where reliable vitrification and the preparation of electron&#8209;transparent lamellae (typically &#60;200&#160;nm) continue to pose persistent bottlenecks12.
Achieving vitrification in tissues is challenging due to their high water content and limited heat transfer. While plunge freezing is effective for thin samples (&#8804;20 &#181;m)13, it fails for most tissues. High-pressure freezing (HPF) suppresses ice nucleation by applying high pressure, enabling vitrification of samples up to ~200 &#181;m in theory. However, the efficiency of conventional HPF is often suboptimal for vitrifying tissues. To improve outcomes, recent efforts have employed strategies such as adding cryoprotectants14 and performing pre-sectioning15,16 (e.g., using a vibratome) to reduce the sample thickness mechanically prior to freezing11,17. While these approaches can enhance vitrification quality, they often entail prolonged ex vivo handling and the use of chemical additives that risk altering the native structures. To address these limitations, this study modified the &#8220;waffle&#8221; carrier strategy originally developed by Kelley et al.18 by incorporating a critical optimization: the brain tissue is physically compressed using two polished copper carriers prior to freezing, which pre-thins the sample while simultaneously securing it between a filmed grid and a copper foil. This design not only promotes rapid and uniform vitrification but also significantly enhances mechanical stability, thus effectively preventing grid breakage and sample loss during subsequent handling, establishing a robust foundation for obtaining intact, high-quality frozen tissue specimens.
Following vitrification, thinning remains a major barrier to both throughput and quality. As an early and widely adopted technique, cryo-ultramicrotomy enables numerous foundational in situ structural studies. Nevertheless, it frequently introduces compression and cutting artifacts19,20 that compromise structural integrity. In contrast, cryo-focused ion beam (cryo-FIB) milling21 offers superior precision and minimal distortion7,22. Initially, cryo&#8209;FIB milling was applied directly to vitrified samples on grids to produce lamellae23; however, this&#160;in situ&#160;approach is primarily effective for thin specimens24,25 (e.g., cultured cells or peripheral tissue regions) and cannot access deeper structures within thick, bulky tissues. To overcome this depth limitation, lift-out strategies26 were developed, enabling the site-specific extraction of regions of interest (ROIs) from the interior of samples. Subsequent methodological advances, including serial lift-out27 and on-grid serialized sectioning (SOLIST)28, further enhanced throughput, lamellar stability, and precision of 3D correlative targeting in complex tissues. More recently, innovations have continued to advance the field, including the six&#8209;sided attachment method by Tang et al.29, which enhances mechanical stabilization during lift&#8209;out and thinning, as well as novel on&#8209;grid milling approaches by Benjamin C. Creekmore et al.11 that enable deep&#8209;structure access while preserving native tissue context. Despite these advances, protocols still vary in efficiency, reproducibility, and adaptability across different tissue types. A critical gap persists in the establishment of a standardized, easily replicable, and broadly applicable workflow that integrates these optimizations into a coherent pipeline for diverse thick tissues.
This gap is addressed here by synthesizing key technical details from established methodologies and refining them into a generalized, end-to-end pipeline. This protocol not only employs the enhanced HPF-waffle method for robust vitrification but also incorporates a series of optimized steps in cryo-FIB lift-out and thinning, such as systematic over-tilt compensation, standardized conductive coating, and calibrated milling sequences, that collectively enhance lamella uniformity, reduce artifacts (e.g., curtaining), and improve throughput. Unlike protocols tailored to specific cell types or model organisms, this workflow is designed with generalizability at its core, providing a versatile framework adaptable to a wide range of thick, complex tissues, including those that are relatively soft and prone to compression, beyond the demonstrated mouse hippocampus.
Demonstrated in the challenging model of the mouse hippocampus, notable for its susceptibility to ice damage and structural complexity, this integrated and generalized workflow reliably produces high-quality lamellae (120&#8211;200 nm) and supports efficient tilt-series acquisition (5&#8211;8 min/series). This consolidation of best practices into a coherent, accessible, and widely applicable protocol provides a reproducible framework that lowers the technical barrier to implementing cryo-ET in thick tissue research across diverse biological and biomedical contexts.

<table><tbody><tr><td>1-Hexadecene</td><td>Sigma-Aldrich</td><td>H2131</td><td>Chemicals &amp;amp; Reagents</td></tr><tr><td>2-Methylpentane</td><td>Sigma-Aldrich</td><td>M65807</td><td>Chemicals &amp;amp; Reagents</td></tr><tr><td>Clip-ring</td><td>Thermo Fisher Scientific</td><td>1131750;1188953</td><td>Consumables</td></tr><tr><td>Copper foil</td><td>Fengcheng Metal Materials</td><td>1 &amp;micro;m thickness</td><td>Consumables</td></tr><tr><td>Copper support TEM Grids (100 mesh)</td><td>Beijing Zhongjingkeyi Technology Co., Ltd</td><td>BZ10021b</td><td>Consumables</td></tr><tr><td>Cryo-EM</td><td>Thermo Fisher Scientific</td><td>Krios G4</td><td>Equipment&amp;nbsp;</td></tr><tr><td>Cryo-FIB</td><td>Thermo Fisher Scientific</td><td>Aquilos 2</td><td>Equipment&amp;nbsp;</td></tr><tr><td>Cryo-FIB Autogrid</td><td>Thermo Fisher Scientific</td><td>1205101</td><td>Consumables</td></tr><tr><td>Grids (UltrAufoil R 1.2/1.3 300 mesh)</td><td>Quantifoil</td><td>AltrAufoil</td><td>Consumables</td></tr><tr><td>High-Pressure Freezer</td><td>Leica Microsystems</td><td>EM ICE</td><td>Equipment&amp;nbsp;</td></tr><tr><td>HPF carrier, copper</td><td>Leica Microsystems</td><td>type B (6 mm)</td><td>Consumables</td></tr><tr><td>Ion Sputter Coater</td><td>Quorum</td><td>Q150RS plus</td><td>Equipment&amp;nbsp;</td></tr><tr><td>Metallographic Polishing Paste</td><td>Lab-testec</td><td>W0.5</td><td>Consumables</td></tr><tr><td>Polishing cloth</td><td>GORAL</td><td>B00809</td><td>Consumables</td></tr><tr><td>Sandpaper</td><td>Eagle Brand Sandpaper</td><td>8000-grit, 12000-grit</td><td>Consumables</td></tr><tr><td>Stereomicroscope</td><td>Leica Microsystems</td><td>S9i</td><td>&lt;a name=&quot;RANGE!D17&quot;&gt;Equipment&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>200-334-9</td><td>Chemicals &amp;amp; Reagents</td></tr><tr><td>Tilt-Series Acquisition Software</td><td>https://bio3d.colorado.edu/SerialEM/</td><td>SerialEM 4.0.9</td><td>Software</td></tr><tr><td>Tilt-Series Alignment &amp;amp; Reconstruction Software</td><td>https://github.com/czimaginginstitute/AreTomo3&amp;nbsp;</td><td>AreTomo 3</td><td>Software</td></tr><tr><td>Tilt-Series Acquisition Software</td><td>https://github.com/eisfabian/PACEtomo</td><td>PaceTomo</td><td>Software</td></tr><tr><td>Tilt-Series Alignment &amp;amp; Reconstruction Software</td><td>https://github.com/IsoNet-cryoET/IsoNet</td><td>IsoNet</td><td>Software</td></tr><tr><td>Tilt-Series Alignment &amp;amp; Reconstruction Software</td><td>https://github.com/cryoem/eman2</td><td>EMAN2</td><td>Software</td></tr><tr><td>Tilt-Series Alignment &amp;amp; Reconstruction Software</td><td>https://relion.readthedocs.io/en/release-4.0/index.html</td><td>Relion 4</td><td>Software</td></tr></tbody></table>

cryo-electron tomography workflow for thick tissues demonstrated in mouse hippocampus

Cryo-Electron Tomography Workflow for Thick Tissues Demonstrated in Mouse Hippocampus

Cryo&#8209;electron tomography (cryo&#8209;ET) enables 3D visualization of native cellular ultrastructure, but its application to thick tissues is limited by the lack of reliable vitrification and low&#8209;throughput lamella preparation. Here, an end&#8209;to&#8209;end workflow is presented that integrates high&#8209;pressure freezing (HPF; ~2100 bar; dissection&#8209;to&#8209;freezing time &#8804;90 s) using a &#8220;waffle&#8221; assembly with copper foil to pre&#8209;thin tissue, cryo&#8209;FIB lift&#8209;out, and controlled thinning to 120&#8211;200 nm lamellae with over&#8209;tilt compensation. Conductive coating is standardized (sputter&#8209;Pt 30 mA/15 s; GIS cap 0.5&#8211;1.0 &#181;m) to minimize curtaining. Automated, dose&#8209;symmetric data acquisition (0&#176;, &#177;3&#176;, &#177;6&#176;&#8230; to &#177;60&#8211;66&#176;; step size of 2&#8211;3&#176;; total dose 100&#8211;150 e&#8315;/&#197;2) is implemented via PACEtomo scripts, with the starting tilt angle derived from the milling angle and sample orientation. Processing with AreTomo 3 provides functions for motion correction, CTF estimation, alignment, and WBP reconstruction in near&#8209;real time; IsoNet may optionally reduce missing&#8209;wedge anisotropy. Subtomogram averaging (RELION 4.0) follows a pseudo&#8209;subtomogram strategy with resolution reporting based on an FSC cutoff of 0.143. Demonstrated on mouse hippocampus, the workflow routinely produces well&#8209;vitrified samples and uniform lamellae (target thickness 150 &#177; 40 nm) and achieves high&#8209;throughput tilt&#8209;series collection (typically 5&#8211;8 min/tilt series). This protocol offers a reproducible route for studying in situ structural biology directly from complex tissue environments.

Successful application of this protocol enables the production of high-quality, vitrified tissue lamellae and the subsequent reconstruction of tomograms that reveal native cellular ultrastructure. The following outcomes, demonstrated using mouse hippocampal tissue, are representative of a successful experiment.
Tissue blocks processed by high-pressure freezing with the "waffle" method exhibited a uniform and electron-transparent appearance (Figure 4B,C</st...

This protocol standardizes cryo-electron tomography (cryo&#8209;ET) for thick tissues by integrating high-pressure freezing (HPF) with the waffle method, cryogenic focus ion beam (cryo&#8209;FIB) lift&#8209;out, and dose&#8209;symmetric, parallel acquisition, yielding uniformly thin lamellae and vitrified specimens for reproducible in situ analysis in the mouse hippocampus.

Cryo&#8209;electron tomography (cryo&#8209;ET) enables 3D visualization of native cellular ultrastructure, but its application to thick tissues is limited ...

My research focuses on establishing a standardized cryo-ET workflow to enable in-situ structural analysis of thick tissues like the mouse hippocampus. Existing methods lack reliability for thick tissues, so this protocol integrates optimized steps for standardized analysis. To begin, place a 10 L droplet of 10%sucrose solution on a clean glass surface.Rapidly dissect the hippocampus from a freshly euthanized mouse. Using a sharp razor blade, excise small tissue pieces of 0.5 to 1 cubic millimeters. Using a toothpick or fine brush, transfer the tissue onto the prepared copper support TEM grids within the high-pressure freezing carrier.Use 2-methylpentane to keep the tissue moist and adjust its positioning on the grid, while minimizing mechanical stress to avoid grid damage. Then, cover the tissue and grid with a 3-millimeter diameter copper foil disc. To complete the assembly, place the second high-pressure freezing carrier, flat-side down, on top to form a sealed waffle sandwich.And check the freezing parameters to ensure there is no abnormality in the instrument. Ensure the total time from tissue dissection to freezing does not exceed 90 seconds. Obtain a new 300-mesh gold grid to serve as the final receiver grid for the lift-out procedure.Under a stereo microscope at room temperature, gently clip the grid into a compatible cryo-FIB AutoGrid ring. Then, store the assembled AutoGrid directly in a dry oven, maintained at approximately 40 degrees Celsius, to minimize frost accumulation before use. While continuously immersing the assembly in liquid nitrogen, carefully separate the vitrified TEM grid from the surrounding waffle assembly components.Using pre-cooled fine-tipped tweezers, gently pry the two high-pressure freezing carriers apart. Next, on a pre-cooled cryo-loading workstation, mount the isolated tissue grid into a second cryo-FIB AutoGrid ring under the protection of liquid nitrogen. Load both AutoGrids into the designated slots of the cryo-transfer shuttle sample holder.Orient each AutoGrid so that the milling notch faces outward and is positioned vertically to enable a lower milling angle. Keep the fully loaded shuttle immersed in liquid nitrogen or maintained in a pre-cooled state until transfer into the FIB-SEM. Load the sample stage into the instrument using the sample rod.After cryo-transfer into the microscope, navigate to the tissue grid at low SEM magnification. Then, sputter coat the grid surface with platinum at 30 milliampere for 15 seconds to provide conductive protection. Deposit a 0.5 to 1.0 micrometer thick organometallic platinum cap using the gas injection system for approximately 60 seconds.Apply a final sputter coated platinum layer to the grid surface at 30 milliampere for 15 seconds. Using the SEM electron beam image, navigate to the selected region of interest on the tissue grid. To orient the sample for front-side milling, adjust the stage to approximately 110 degrees rotation and 7 degrees tilt to present the sample surface at an angle suitable for ion beam access.For trench milling, define a rectangular trench surrounding the target tissue block using the ion beam operated at 30 kilovolts. Begin milling at a distance from the block using a relatively high current of 15 nanoampere to achieve rapid material removal. As the trench walls approach the block to within a few micrometers, reduce the ion beam current to 7 nanoampere to achieve more precise milling control.Leave one side of the block attached to the bulk material to create a temporary holding tab. For tissue blocks with an estimated thickness greater than 20 micrometers, perform an undercut milling step from the reverse side to facilitate the final lift out. Rotate the stage and adjust the tilt to access the bottom of the block.Mill the underside connection using the focused ion beam at 30 kilovolts and a current of 3 nanoampere. Mill the bottom of the metal block to fit tightly against the sample block. Precision mill the contact surface between the sample and the gold block to make the contact tighter.To attach the sample to the needle, ensure that the gold block is in direct contact with the target sample. Using the focused ion beam at 30 kilovolts and 0.5 nanoampere, perform 8 to 12 sequential cross-section milling patterns at the interface to weld the gold block to the sample. To lift out the tissue block, sever the remaining tab connecting the block to the bulk sample using the focused ion beam.Carefully maneuver the micromanipulator needle to lift the isolated tissue block completely clear of the trench and the surrounding grid. Retract the needle and transport the block to the receiver grid. Navigate the micromanipulator needle to a pre-cooled 300-mesh gold receiver grid.Lower the block onto a chosen grid square. Mill away small ice crystals on the grid to make the tissue block and grid adhere more tightly. Lower the tissue block so that it just contacts the grid.Mill a 3 to 4 micrometer-thick slice from the block onto the grid film using the ion beam. Next, weld the slice securely by milling, using cleaning cross-section patterns on both sides of the junction, and reapply the conductive and protective coating to the grid surface as demonstrated previously. For lamella thinning, after setting the stage tilt, define the final lamella position within a smooth area of the tissue block.Perform systematic milling using ion beam currents and over-tilt compensations specified in the presented table. When within 1 to 2 micrometers of the lamella, optionally mill a stress-relief trench approximately 1.5 micrometers away to prevent bending or curling of the lamella. After lamella preparation, acquire images using cryo-electron microscopy.Tissue blocks processed by high-pressure freezing with the waffle method exhibited a uniform and electron-transparent appearance. Reconstructed tomograms resolved mitochondrial structures within the cellular environment. Macromolecular complexes, such as ribosomes, were clearly resolved in the cytoplasm.Cytoskeletal and membrane-associated cellular features and nuclear pore complexes were also visible in the reconstructed tomograms. Synaptic structures and features, including calcium stores and myelin sheath structures, were clearly seen in the tomograms. This protocol enables researchers to study native cellular outer structure directly within complex tissue environments.The most critical challenge in this cryo-ET protocol is achieving uniform, artifact-free vitrification of thick tissue samples. Following this procedure, researchers can localize regions of interest using correlative light and electron microscopy or labeling techniques, then perform sub-tomogram averaging and structure analysis to resolve macromolecular complexes.

cryo-electron-tomography-workflow-for-thick-tissues-demonstrated

Research

JoVE Journal

Neuroscience

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse.  Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices.  By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis.

Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

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.

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

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

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

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the Saccharomyces cerevisiae.

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

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.

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 µm.

Visualization of Organelles In Situ by Cryo-STEM Tomography

This workflow enables lamella production targeting fluorescently labeled biological structures that are small (&lt;1 μm in axial extent) and rare (1 copy per cell) using a cryogenic tri-coincident imaging platform. This platform integrates fluorescence microscopy, focused ion beam milling, and scanning electron microscopy at a single focal position and enables simultaneous fluorescence microscopy while milling.

Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells

Learn cryo-electron tomography for in situ structural analysis of thick tissues using mouse hippocampus. This standardized workflow integrates high-pressure freezing, cryo-FIB milling, and automated imaging to enable high-resolution visualization of native cellular architecture.

Cryo-Electron Tomography Workflow for Thick Tissues Demonstrated in Mouse Hippocampus

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