We thank Inga Wolf for supporting the IT infrastructure, Florian Beck for computational support, and Oda H. Schi&#248;tz for the critical reading of the manuscript. Funding was provided in part through an Alexander von Humboldt returners fellowship to Philipp S. Erdmann and an EMBO Long-term Fellowship ALTF 764-2014 to Florian Wilfling. Anna Bieber was supported by a Boehringer Ingelheim Fonds Ph.D. fellowship.

<ol>
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The authors declare no conflicts of interest.

1. Critical steps in the protocol
Optimization of cell culture and grid plunging parameters is fundamental for this workflow. At the beginning of a project, it is worth investing time to optimize tagging strategies, the distribution of cells and fiducial beads, and test different grid preparation and blotting parameters. Working with an optimally plunge-frozen sample will significantly facilitate downstream processing.
As for any TEM experiment, vitreous samples are required. For large mammalian cells such as HeLa, 1-2 cells per grid square are preferable, but cells may still be vitreous at higher density. Optionally, vitrification can be improved in mammalian cells (e.g., HEK293, HeLa) by incubating them with 2.5-10% (v/v) glycerol added to the culture medium 10 min before plunging23. If available, grid patterning may be used to ensure perfect placement and distribution of the cells, thereby improving vitrification and later correlation24.
While specific cells can be selected during the workflow, too few cells that show the biological feature of interest will significantly reduce overall throughput. To improve correlation in POI-positive cells, sufficiently bright fluorophores should be used. This is especially important at endogenous expression levels. We found that under cryo-conditions, mVenus often performed better than EGFP due to its increased brightness25 and the hypsochromic shift, which keeps it suitable for standard GFP filter setups under cryo-conditions26. For non-point-like target structures, the trade-off between wavelength and localization accuracy (Abbe diffraction limit) should also be considered.
Efficient 3D-correlation also requires that grids are mechanically stable and are handled with great care. While standard gold or copper grids with carbon support may be used, the success rate may significantly be increased by using more rigid SiO2 films depending on the project. However, it has not yet been conclusively determined whether (a) mechanical stability or (b) matching thermal expansion coefficients (substrate vs. film) to reduce cryo-wrinkling27, is the most crucial factor for successful 3D correlation. Moreover, for picking up fragile Au grids, polydimethylsiloxane-coated dishes may be used5.
In addition to ensuring sample stability, a careful choice of FLM imaging parameters is necessary for obtaining high-quality fluorescence stacks that are suitable for optimal targeting during FIB milling. In this regard, testing different denoising28 or deconvolution techniques on the FLM data is also advised, as it may considerably improve the localization of fiducials and cellular signals. When correlating the fluorescence signal to FIB-SEM images, a good sampling of fiducial beads is important. They should be well distributed around the cells and possibly at different z&#160;heights. It is also good practice to validate the consistency of the correlation by checking the predicted vs. actual positions of beads that were deliberately left out of the fiducial model but can clearly be correlated by eye. 3DCT&#39;s RMSE values should also always be considered to check the registration consistency.
Since the deposition of milled material and residual water from the FIB-SEM chamber (i.e., recontamination) increases the effective lamella thickness by adding amorphous material to both sides of it, keeping fine-milled lamellas in the microscope for a prolonged time generally reduces TEM data quality due to additional electron scattering events. Accordingly, milling is most often performed in a two-step fashion: first, all positions are milled roughly (i.e., to about 800 nm), and then finely (to ~150-250 nm), and the grid is immediately unloaded after the last lamella has been completed. Better correlation success may, however, be achieved by processing the positions of interest in a site-wise manner, hence performing rough and fine milling on the same lamella directly after one another since this leaves no time for bending or deformation. This, however, reduces the maximum number of lamellas that can be produced per grid depending on the recontamination rate of the system. For a rate of 20 nm/h, 4-6 lamellas are produced within 1-1.5 h.
Movement of the entire grid or the rough-milled lamellas &#62;300 nm will result in poor or unsuccessful correlation (see also limitations discussed below). It should therefore be checked regularly, e.g., by comparing IB images before, during, and after FIB milling. Sites that show significant movement (&#62;300 nm) should be discarded. Optimize the sample preparation (i.e., choice of grid type, cell density, and plunging parameters; see protocol section 1) and milling strategy to avoid these movements. Lamella bending can significantly be reduced by site-wise milling as described in step 3.6 and reducing the lamella width. As mentioned before, while stress relief cuts15 have been designed to reduce lamella bending, they often result in a concerted movement of the de-coupled lamella, thereby effectively preventing correlation. Integrated FLM systems may be used to solve this problem.
2. Modifications and troublehooting of the method
It is highly advised to perform a thorough characterization of the sample in live-cell imaging before going to cryo-conditions. Optimizing the cellular samples, treatment schemes, and knowing what kind of signal to expect before entering the cryo-workflow can substantially improve its success rate.
In the workflow presented here, a stand-alone fluorescence microscope with a cryo-stage is used to image the samples, followed by a transfer of the grids into the focused ion beam microscope. However, it has been tested on systems where a fluorescence microscope is integrated into the FIB-SEM chamber, and therefore no sample transfer is required to acquire fluorescence images29,30,31. Using such integrated systems, positions of interest can be imaged during and after FIB milling to check for the presence of the target fluorescence signal without increasing the risk of contaminating the final lamellas. It is, however, important to keep in mind the optical parameters of the used microscopes, as, e.g., a low NA objective will limit the precision with which fiducial beads and target signals can be localized. Nonetheless, integrated FLM setups will help to also better deal with slight deformations of grids and lamellas, as FLM stacks can continuously be updated and compared to up-to-date SEM and IB views.
As an alternative to fluorescence imaging of the lamella between FIB milling and TEM data acquisition, post-TEM correlation can be used to verify correct placement and milling of the lamellas5,6.
During all steps of the correlative workflow, but especially during TEM, it is recommended to create an overlay of the projected fluorescence data on the FIB-SEM/TEM images. Such classical CLEM views help understand more intuitively which part of the cells is contained within the lamellas. This also serves as a useful sanity check to verify the accuracy of the correlation.
3. Limitations of the method
The 3D-correlative FIB approach requires samples that can be supplied with fiducial beads. Accordingly, this method is presently restricted to plunge-frozen grids. For high-pressure (HPF) frozen (tissue) samples, presently, only 2D-2D correlations can be performed. Potentially, internal fiducial markers (e.g., organelles, stained lipid droplets) could be a solution to this problem32,33. The final correlation success rate depends on many factors, including the sample quality, the fluorescence microscopy setup, the lamella thickness, and the size of the targeted structure. The correlation accuracy using the described 3D registration approach is estimated to be in the range of 200-300 nm on the final IB image, roughly corresponding to the typical thickness of FIB-milled lamellas7. Accordingly, cellular structures much smaller than this will be hard to target at present. Additionally, excessive movement at the milling site (&#62;300 nm) also reduces the accuracy of the correlation, an issue that can potentially be addressed with FLM setups integrated into FIB/SEM instruments. Lamellas that show strong deformation or bending during milling should, in any case, be excluded from the downstream workflow.
Overall, cryo-fluorescence imaging is currently limited by the Abbe diffraction criterion. With more routine application (and commercialization) of super-resolved cryo-FLM methods, more accurate targeting of cellular structures might become possible, especially when integrated into the FIB/SEM for on-the-fly operation.
4. Significance of the method
Especially in comparison to non-targeted and post-correlation techniques, the 3D-correlated FIB milling approach allows the selection of suitable positions before the time- and resource-consuming TEM step. It, thereby, enables more efficient data collection and project planning. Moreover, the correlated fluorescence data adds a layer of information that can be crucial for interpreting the tomograms and for integrating the cryo-ET results in multi-scale projects, especially when dealing with non-structured protein assemblies or those too small for template matching and subtomogram averaging.
5. Importance and potential future applications
In combination with advanced workflows such as cryo-lift out of HPF samples34,35, cryo-FIB-SEM volume36 and super-resolution fluorescence imaging26,37,38,39, 3D-targeted lamella preparation offers the prospect of not only dissecting biological processes in isolated cells but also to make tissue and patient samples accessible to FIB milling and cryo-electron tomography. As such, it will allow dissection of pathological processes at high resolution and thus be an integral building block toward a biopsy at the nanoscale.

Focused ion beam milling allows the preparation of thin biological samples from cryo-fixed specimens without the problems commonly associated with mechanical sectionings such as knife marks and compression artifacts1. When paired with cryo-electron tomography, FIB milling enables high-resolution biological studies of the cellular morphology and determination of the structure of macromolecular complexes directly from within cells at sub-nanometer resolution2,3,4. While abundant species, such as ribosomes, are readily found in randomly cut FIB lamellas, many cellular processes rely on the colocalization of several complexes or are localized to specific sites within the cell. Consequently, efficient targeting is required to not lose the biological feature of interest during the milling process and be limited to random hits. A correlative approach that combines data from the scanning electron microscope (SEM)-FIB and a cryo-fluorescence light microscope (FLM) is therefore necessary. While it is possible to omit the initial correlation and combine FLM and cryo-ET data only after TEM acquisition5,6, fluorescence-guided focused ion beam milling enables an accurate selection of the milling area beforehand, thereby resulting in more efficient data acquisition. Since its conception7, the application of 3D-correlated FIB milling in biological studies had been limited until we recently reported identifying a new liquid-liquid phase-separated (LLPS) compartment in yeast using this technique8.
Described here is a generalized 3D cryo-correlated light and electron microscopy (CLEM) protocol, which can be used to study a broad variety of samples ranging from bacteria to yeast and mammalian cells. While the experiments were performed using a certain set of instruments, the individual steps are not bound to specific hardware and can easily be transferred to other systems as an extension to existing protocols3,5. A list of tested equipment and suggested settings are provided in the Table of Materials and Table 1. The four key steps of the pipeline are (1) sample preparation, (2) localization of features of interest by cryo-fluorescence microscopy, (3) 3D-correlated focused ion beam milling, and (4) localization of the targeted structures for cryo-ET data acquisition on the lamellas in the cryo-transmission electron microscope (Figure 1).

<table><tbody><tr><td>Autogrids</td><td>Thermo Fisher Scientific / Homemade</td><td>1036173 (no cutout), 1205101 (with cutout)</td><td></td></tr><tr><td>C-rings</td><td>Thermo Fisher Scientific</td><td>1036171</td><td></td></tr><tr><td>Corrsight with cryo module</td><td>Thermo Fisher Scientific</td><td></td><td>FLM Alternative 1</td></tr><tr><td>Dynabeads MyOne COOH</td><td>Thermo Fisher Scientific</td><td>65011</td><td>recommended 1 &amp;micro;m fiducial beads</td></tr><tr><td>EM Grids R1/4 SiO2</td><td>Quantifoil</td><td>N1-S13nAu20-01</td><td></td></tr><tr><td>Falcon 4 camera w. post-column Selectris X energy filter</td><td>Thermo Fisher Scientific</td><td></td><td>Camera/Filter Alternative 1</td></tr><tr><td>FIB Aquilos 1</td><td>Thermo Fisher Scientific</td><td></td><td>FIB Alternative 1</td></tr><tr><td>FIB Aquilos 2</td><td>Thermo Fisher Scientific</td><td></td><td>FIB Alternative 2</td></tr><tr><td>FIB Quanta 3D FEG</td><td>Thermo Fisher Scientific</td><td></td><td>FIB Alternative 3</td></tr><tr><td>FIB Scios</td><td>Thermo Fisher Scientific</td><td></td><td>FIB Alternative 4</td></tr><tr><td>K2 summit camera w. post-column energy filter 968 Quantum K2</td><td>Gatan</td><td></td><td>Camera/Filter Alternative 2</td></tr><tr><td>Leica TCS SP8 with cryo module</td><td>Thermo Fisher Scientific</td><td></td><td>FLM Alternative 2</td></tr><tr><td>Plasma Cleaner PDC-3XG</td><td>Harrick</td><td></td><td></td></tr><tr><td>Teflon Sheet (0.25 mm)</td><td>plastx24.de</td><td>11645</td><td>Cut to same dimensions as filter paper</td></tr><tr><td>TEM Titan Krios XFEG 300 kV Gi2</td><td>Thermo Fisher Scientific</td><td></td><td>TEM Alternative 1</td></tr><tr><td>TEM Titan Krios XFEG 300 kV Gi4</td><td>Thermo Fisher Scientific</td><td></td><td>TEM Alternative 2</td></tr><tr><td>THUNDER Imager&amp;nbsp; EM Cryo CLEM</td><td>Thermo Fisher Scientific</td><td></td><td>FLM Alternative 3</td></tr><tr><td>Vitrobot Mark IV</td><td>Thermo Fisher Scientific</td><td></td><td>alternativevly, use manaual plunger</td></tr><tr><td>Whatman filter paper</td><td>Sigma Aldrich</td><td>10311807</td><td>55 mm diamater; needs to be cut to fit the Vitrobot</td></tr></tbody></table>

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).

The protocol provides a walk-through of the pipeline used to discover the EH domain-containing and endocytosis protein 1 (Ede1)-dependent endocytic protein deposit (END) and its degradation and trapping in autophagic bodies8. The END is a liquid-liquid phase-separated compartment in S. cerevisiae, which buffers a variety of proteins involved in clathrin-mediated endocytosis (CME) after failed endocytic events. One of its main components is Ede1, which doubles as a CME component and as a selective autophagy receptor for the degradation of this new LLPS compartment. Accordingly, an EGFP fusion of Ede1 (EGFP-Ede1) under the control of the alcohol dehydrogenase (ADH) promoter was used to visualize ENDs since Ede1 overexpression interferes with the early stages of endocytosis and therefore constitutively induces LLPS.
On a plunge-frozen grid with EGFP-Ede1 overexpressing yeast cells and 1 &#181;m fiducial markers, five positions were selected for FLM stack acquisition in the GFP channel (Figure 6A; TFS Corrsight; confocal mode, 300 nm focus step size, 10 &#181;m range). The grid was transferred to the FIB instrument (Quanta 3D FEG), and the grid squares for which FLM stacks had been acquired were identified by performing a 2D-2D correlation of the fluorescence and SEM grid overviews (compare step 3.2).
For each of the chosen squares, ion beam images were taken at a low current (10 pA, 1200x magnification), and corresponding fiducial positions were registered in 3DCT. After selection of positions with the biological feature of interest and fitting their 3D position within the FLM stack, the found transformation was applied to putative END positions, and the sites for lamella preparation were selected (Figure 6B). A FIB beam inclined 11&#176; relative to the grid surface was used in the examples shown here (45&#176; FIB shuttle pre-tilt; 18&#176; stage tilt). Positions of interest were transferred, and FIB patterns were drawn manually (Figure 6D) by measuring the distance of the predicted positions relative to prominent landmarks in the FIB image (e.g., holes, ice contaminations, fiducial beads). The accuracy of the registration was evaluated by deliberately leaving out beads that could clearly be identified in the FLM and IB image, and then comparing their actual and predicted positions in the ion beam view (e.g., diamond in Figure 6B,C). The correlation for the square shown in Figure 6C was found to be accurate (i.e., the predicted position of FLM bead positions perfectly coincided with their corresponding IB location and 3DCT reported sub-pixel RMSE values for the registration). Thus, lamellas were cut at the predicted positions (Site B) and fine-milled to a thickness of ~200 nm (final pattern offset).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62886/62886fig06.jpg" /> 
Figure 6: Representative results for 3D-correlative targeting of endocytic protein deposits (END) in yeast. (A) SEM overview of the grid before milling. The colored boxes indicate grid squares for which fluorescence stacks were taken beforehand. (B-C) 3D correlation in a grid square. After registering several corresponding fiducial beads (colored boxes) in the FLM data (B, shown here as maximum intensity projection) and the ion beam image (C), the accuracy of the 3D registration was verified by predicting the position of the bead indicated with the diamond. Next, the positions of the target signal (red circles) were predicted in the ion beam view for two potential milling sites. (D) Zoom-in of Site B showing the predicted positions of three target puncta (red circles) and the initial milling patterns (yellow boxes). A fourth fluorescence punctum was predicted to be much lower than the other puncta and therefore not targeted during milling (gray circle). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62886/62886fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After successful FIB milling and transfer of the grid to the cryo-transmission electron microscope (Titan Krios operated at 300 kV and equipped with a Gatan K2 direct electron detector and Bioquantum energy filter), a grid overview was recorded in SerialEM and used to locate squares with lamellas. For each lamella, overview images were acquired, and the FLM data was registered in 3DCT (3D-2D) using corresponding fiducial beads. Positions of the biological features of interest (Figure 7A) were then predicted using the transformation calculated from the fiducial beads. Lamella overviews recorded at higher magnification were stitched, and sites of interest correlated using clearly visible landmarks (e.g., fiducial beads). Alternatively, classical CLEM overviews can be produced in various softwares10,12.
Based on the correlation, four potential sites for tomogram acquisition were found for the lamella shown in Figure 7A. However, this also includes a position that was not targeted during the 3D-correlated FIB-milling (compare Figure 6D; gray circle) and a position blocked by ice contamination (Figure 7; gray boxes). Accordingly, tomograms could only be recorded for two positions (Figure 7B). Overall, a correlation success of ~75%, i.e., lamellas that survived the transfer to the TEM and END structures were found at the predicted sites, was achieved (12 correlated sites). After tomogram reconstruction, segmentation and template matching, individual END structures can be visualized within their native context (Figure 7C,D). This includes the fenestrated endoplasmic reticulum (ER) surrounding the END, lipid droplets occasionally making contact, and ribosomes, which are excluded from the LLPS compartment. Taken together, this shows how 3D-correlative FIB milling can provide molecular-level information of rare biological processes from intact cells.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62886/62886fig07.jpg" /> 
Figure 7: Representative results for visualizing the END with cryo-ET. (A) The low magnification TEM overview of the milling site shown in Figure 6 can readily be correlated with the FLM maximum intensity projection (Figure 6B) to localize biological features of interest (red crosses). (B) In a second step, a higher magnification (stitched) view can be correlated, and positions for tomogram acquisition (yellow boxes) are set up. Locations resulting from the out-of-plane signal (gray box, compare Figure 6D) were ignored. (C-D) Using this 3D-correlative FIB approach, the endocytic protein deposit (END) can be visualized in its native environment. Structures such as the endoplasmic reticulum (ER), ribosomes, membranes, and lipid droplets can be identified and visualized. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62886/62886fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plasma Cleaner Settings</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Harrick Plasma Cleaner PDG-3XG :</td>
			<td colspan="3">Radio Frequency setiing: &#34;HI&#34;, 30 s; N2 plasma</td>
		</tr>
		<tr>
			<td>Plunger Settings</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>TFS Vitrobot Mk IV:</td>
			<td colspan="3">100% humidity; blotforce = 8; blottime = 10 s; wait time 0 s; (this should work for most suspension and adherent cells)</td>
		</tr>
		<tr>
			<td>FIB GIS Positions and Timings</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Quanta 3D FEG:</td>
			<td colspan="3">Tilt = 0, Rotation = -180,&#160; Z position = 13.5, Temperature setpoint =26.15&#176; , Time = 8 s</td>
		</tr>
		<tr>
			<td>TFS Scios:</td>
			<td colspan="3">Tilt = 0, Rotation = -180, Z position = 9.8, Temperature setpoint =28&#176; C, Time = 7 s</td>
		</tr>
		<tr>
			<td>TFS Aquilos 1:</td>
			<td colspan="3">Software predefined position, Temperature setpoint = 28&#176; , Time = 7 s</td>
		</tr>
		<tr>
			<td>TFS Aquilos 2:</td>
			<td colspan="3">Software predefined position, Temperature setpoint = 28&#176;, Time = 7 s</td>
		</tr>
		<tr>
			<td>FIB Sputter Coater Settings</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Quorum System:</td>
			<td colspan="3">In Quorum prep chamber: 10 mA, 40 s</td>
		</tr>
		<tr>
			<td>TFS Scios:</td>
			<td colspan="3">10 W, 500 V, 250 mA, 0.2 mbar, 15 s</td>
		</tr>
		<tr>
			<td>TFS Aquilos 1:</td>
			<td colspan="3">1kV, 10 mA, 10 Pa, 15 s</td>
		</tr>
		<tr>
			<td>TFS Aquilos 2:</td>
			<td colspan="3">1kV, 10 mA, 10 Pa, 15 s</td>
		</tr>
		<tr>
			<td>Tomogram Acquisition</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Titan Krios Gi2</td>
			<td colspan="3">K2 camera, Gatan Bioquantum energy filter</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">20 eV slit; dose symmetric tilt scheme (Hagen) with 2&#176; steps; start at +10&#176; (lamella pre-tilt!) to +70&#176;and -50&#176;</td>
		</tr>
		<tr>
			<td>Titan Krios Gi4</td>
			<td colspan="3">Falcon 4; Selectris X energy filter</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">10 eV slit; dose symmetric tilt scheme (Hagen) with 2&#176; steps; start at +10&#176; (lamella pre-tilt!) to +70&#176; and -50&#176;</td>
		</tr>
		<tr>
			<td>FLM Acquisition</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Corrsight (Confocal Mode)</td>
			<td colspan="3">Objective: Zeiss EC Plan-Neofluar 40&#215;/0.9 NA Pol; Stack acquisition parameters: x-y pixel size = 161.25 nm, z step size = 300 nm.</td>
		</tr>
		<tr>
			<td>Leica SP8 Cryo-Confocal</td>
			<td colspan="3">Objective: Leica HCX PL APO 50x / 0.90 CLEM; Stack acquisition parameters: x-y pixel size = 84 nm, z step size = 300 nm.</td>
		</tr>
	</tbody>
</table>
Table 1: List of tested equipment and suggested settings.

Watch this Scientific Journal Video about Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography at JoVE.com

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

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, ...

sample-preparation-3d-correlative-focused-ion-beam-milling-for-high

Nanyang Technological University

Research

JoVE Journal

Biology

3.2K Views.  Max Planck Institute of Biochemistry. 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.

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

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Neuroscience

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 ...

Biochemistry

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

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae 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&#160;Saccharomyces cerevisiae.

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

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

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 ...

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.

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

Summary

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Chapters in this video

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

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

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

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

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

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

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition