Name: a 3d cartographic description of the cell by cryo soft x-ray tomography
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This project has received funding from the European Commission Horizon 2020 iNEXT-Discovery project and the European Union&#39;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement No 75439.

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The authors declare no conflict of interest.

Sample preparation is a critical step to obtain high quality soft X-ray tomograms, as their quality directly depends on the quality of the sample vitrification and the ice layer thickness in which the cell is embedded. Projections with high signal-to-noise ratio will be collected in regions with thin ice layer, allowing to minimize the radiation dose required to achieve the highest possible resolution. In addition, the cell confluency will also affect the final tomogram quality, since one should avoid having neighboring cells entering the FoV upon rotation. Finally, the right dispersion of Au fiducial markers will determine the accuracy of the projection alignment and then ultimately determine the quality of the final 3D reconstructed volume. Note that a proper spread of Au fiducials on the grid enables automatization of the projection alignment step, without which a high expertise is needed for such a critical step.
The protocol herein only depicts one possible sample preparation strategy, which has similarities with the ones used in cryo electron tomography (cryo-ET). In both cases, protocols improving the demanding sample preparation for better reproducibility will be fundamental for the success of these techniques, and efforts are being made toward this goal29. It is worth mentioning that in addition to imaging isolated cells, sections of tissue can also be visualized provided the transmission signal through the section will be enough at high tilt angles. Typically, this will imply sections of few microns (below 10 &#181;m).
To image a specific structure or event inside a cell, one needs to make sure this particular feature is inside the FoV of the tilt series. As the FoV in cryo-SXT is limited to 10 x 10 &#181;m2 to 15 x 15 &#181;m2 depending on the lens and accounting for a pixel oversampling of the resolution to at least a factor of 2, it is often smaller than the full cell extension (see the red squares indicated in Figure 5). Therefore, the ROI must be found and properly labeled. This is usually done by means of fluorescent tags and visible light correlative approaches. 2D strategies combining epifluorescence are straightforward as the soft X-ray transmission microscope has an integrated on-line visible light fluorescence microscope, but other approaches for high resolution 2D or 3D fluorescence signal are also available4,12,13,15,16. In those cases, the grid needs to be imaged first in specific instruments such as super resolution microscopes. Note that the most efficient correlative approaches are those involving data collection at cryogenic conditions. This is because the time lapse between room temperature (RT), visible light fluorescence imaging, and sample vitrification, for instance, will hinder catching the right cellular event on time; in addition, the vitrification procedure might detach the cell of interest that has been imaged at RT from the grid. Even if most correlative imaging approaches might imply that the sample grids have to be manipulated and transported from one instrument to the other, and despite the increased risk of grid contamination or damage this poses, the reward is clear: to be able to pinpoint specific events or molecules within the cellular landscape.
When whole cell imaging is required, stitching different tomograms is possible provided the total dose applied does not exceed the radiation damage limit. Usually, the deposited dose for collecting few tomograms on the same cell is well below the limit at the achievable resolution (109 Gy) and, therefore, no specific strategy is required to lower the dose, although this is sample- and experiment-dependent. In the case of intensive data collection such as spectro-tomography, minimizing the dose would indeed be required and convenient data collection and specific processing strategies would need to be applied.
Cryo-SXT has several limitations, which should be mentioned here. The first one is the well-known missing wedge, which is intrinsic to using flat sample supports. Capillary sample supports allowing 180-degree rotation have been used in the past and are still used at some facilities, but they also present drawbacks such as an impoverished contrast due to the glass absorption and the restriction of using cells in suspension. A way to diminish the effect of the missing wedge is by performing dual tilt tomography. This is indeed possible at the Mistral beamline nowadays. The second limitation is set by the Fresnel zone plate lens used in such microscopes. This lens sets the ultimate resolution achievable and the depth of field (DoF), both being tightly related. This implies that increasing the resolution will diminish the DoF while the thickness of the cell will often be larger. For example, a 40 nm lens will have in theory a DoF of 3 &#181;m and a resolution of 24.4 nm half pitch. The compromise between resolution and DoF is therefore strategic and the choice of the lens will depend on the type of the cell30,31. Finally, operational TXMs worldwide are far from being ideal microscopes and efforts are being made to improve the optical systems to reach the theoretical expectations. Finally, the visualization and segmentation of the reconstructed volumes can be carried out with specific software tools25,32,33,34.
In summary, cryo-SXT allows imaging cells quantitatively at medium resolution (25-30 nm half pitch) and in statistical numbers (few tens of tomograms per day). This allows obtaining the organization, distribution, and dimension of organelles at specific conditions, for instance, during pathogen infection or diseases, at precise time points or after particular treatments. It is, therefore, a useful complementary biological imaging technique to the more common electron and visible light microscopies, each of them tackling a specific range of sample dimensions and resolution. Cryo-SXT is frequently used in correlative approaches involving visible light fluorescence, but other cryo correlative strategies are also possible.

Cryo-SXT can play a central role in biological imaging research as it provides 3D medium resolution (25-30 nm half pitch) volumes of hydrated whole cells1,2,3,4,5,6. In the water window energy range, between the carbon and the oxygen absorption K edges (4.4-2.3 nm), carbon-rich cellular structures absorb 10 times more than the oxygen-rich medium that permeates and surrounds them. In this energy range, vitrified cells up to 10 &#181;m thickness can be imaged without the need for sectioning or staining, leading to quantitative high absorption contrast projections, which, combined with sample rotation capabilities, allow for the tomographic reconstruction of the cellular structure. Cryo-SXT fills a niche in terms of specimen dimensions and spatial resolution that is not easily accessible by any other imaging technique.
In brief, the absorption contrast of cryo-SXT is quantitative, as the attenuation of the photons through the specimen of thickness t obeys the Beer-Lambert law as follows: <img alt="Equation 1" src="/files/ftp_upload/62190/62190eq01.jpg" style="margin:-.4em 0;" />, where I0 represents the incident intensity and &#181;l&#160;the linear absorption coefficient, which depends on the wavelength &#955; and the imaginary part&#160;&#946; of the refractive index of the specimen (<img alt="Equation 2" src="/files/ftp_upload/62190/62190eq02.jpg" style="margin:-.4em 0;" />). The attenuation is a function of the biochemical composition and the thickness of the structures being imaged, with each biochemical component having a specific X-ray linear absorption coefficient &#181;l (LAC). This means that each tomography voxel value depends on the chemical elements and their concentration in that voxel7. This allows for the natural discrimination of different organelles such as nuclei, nucleoli, lipid bodies or mitochondria, or different compaction states of chromatin solely based on their inherent LAC values reconstructed2,8,9.
In addition, cryo-SXT is a high throughput technique with tomograms being collected in few minutes. This specifically enables mesoscale imaging of cell populations that can be captured at key time points such as division, differentiation, and apoptosis, but also at different response states such as those induced by chemical exposure to specific drug therapies or to pathogenic infections. Data collected at those key points will deliver 3D description of the system with a faithful record of the spatial organization of the different cellular organelles at those specific moments.
Usually, cryo-SXT is used in combination with other techniques following correlative approaches that allow locating specific features, events, or macromolecules within the 3D cellular environment4,10,11,12,13,14,15,16, or hard X-ray fluorescence data17,18. Correlative approaches at cryogenic conditions are of paramount importance in order to obtain the most complete and valuable picture of the system of interest. A succinct summary of the typical workflow at the Mistral (Alba) and B24 (Diamond) cryo-SXT beamlines is sketched in Figure 1.
Moreover, taking advantage of the wavelength tuning capability at synchrotron facilities, spectroscopic information can be obtained in addition to the structural one using the specific differential absorption of particular elements contained in the sample. An example of this would be the location of calcium in the study of biomineralization processes in cells19,20,21. By taking 2D images at different photon energies (spectra) or tomograms below and at&#160;the x-ray absorption edge of interest, the pixels or voxels containing the selected element can be identified. Spectra also permits differentiating chemical states (i.e., the evolution of amorphous calcium to hydroxyapatite as in the previous biomineralization example20). Quantification of different elements is possible in 2D and 3D. Spectroscopic imaging of vitrified cells is typically done in the water window, but is also possible at other energy ranges if the water content is low enough or if other sample preparation protocols, including dehydration, are used22. A detailed spectroscopy step-by-step protocol is beyond the focus of the protocol herein.
In what follows, the protocol focuses on briefly summarizing the major sample preparation steps, although each system might need individual refinement, followed by a detailed step-by-step data collection procedure for cryo soft X-ray tomography.

<table><tbody><tr><td>Amira</td><td>Thermo Fisher</td><td></td><td>Software for segmentation</td></tr><tr><td>Au Holey Carbon Films&amp;nbsp; finder grids</td><td>(Quantifoil Micro Tools Gmb</td><td>&amp;nbsp;R 2/2 Au G200F1</td><td>Au Holey Carbon Films finder grids</td></tr><tr><td>Au nanoparticles</td><td>&amp;nbsp;BBI Group, Cardiff, UK</td><td>Au nanoparticles 100nm</td><td>100 nm Au nanoparticles (NPs) at Mistral (Alba)</td></tr><tr><td>Au nanoparticles</td><td>&amp;nbsp;BBI Group, Cardiff, UK</td><td>Au nanoparticles 250nm</td><td>250 nm Au nanoparticles (NPs) at B24 (Diamond)</td></tr><tr><td>Axio Scope A1</td><td>Zeiss</td><td>430035 9060</td><td>Fluorescence microscope</td></tr><tr><td>Blotting No.1 filter paper</td><td>Whatman</td><td>WHA10010155</td><td>Blotting filter</td></tr><tr><td>Bsoft</td><td></td><td></td><td>Software for projection alignment, reconstruction and visualization (Heymann et al., 2008)</td></tr><tr><td>Chimera</td><td></td><td></td><td>Software for segmentation (Pettersen et al. 2004)</td></tr><tr><td>Cryo-EM Glow Discharge Set</td><td>PELCO easiGlow</td><td>91000S</td><td>Glow Discharge Cleaning System</td></tr><tr><td>Cu Holey Carbon Films&amp;nbsp; finder grids</td><td>(Quantifoil Micro Tools Gmb</td><td>&amp;nbsp;R 2/2Cu G200F1</td><td>Cu Holey Carbon Films finder grids</td></tr><tr><td>Fetal calf serum</td><td>Sigma</td><td>F9665</td><td>Heat Inactivated, sterile-filtered, suitable for cell culture</td></tr><tr><td>ImageJ</td><td></td><td></td><td>Software for image processing and analysis in Java (NIH &amp;amp; LOCI University of Wisconsin)</td></tr><tr><td>IMOD</td><td></td><td></td><td>Software for projection alignment, reconstruction and visualization (Kremer et al., 1996)</td></tr><tr><td>Leica EM GP Grid Plunger</td><td>Leica</td><td>16706401</td><td>Automatic Plunge Freezer EM</td></tr><tr><td>LINKAM cryo-stage</td><td>Linkam Scientific Instruments</td><td>CMS 196</td><td>Cryo-Correlative Microscopy Stage</td></tr><tr><td>MIB</td><td></td><td></td><td>Software for segmentation (Belevich et al. 2016)</td></tr><tr><td>P100 Petri dish</td><td>Sigma</td><td>P6106</td><td>Treated for cell culture and sterile</td></tr><tr><td>P60 Petri dish</td><td>Sigma</td><td>D8054</td><td>Treated for cell culture and sterile</td></tr><tr><td>Polylysin</td><td>Sigma</td><td>P4707</td><td>Poly-L-lysine solution 0.01%, sterile-filtered</td></tr><tr><td>Soft X-Ray microscope 0.25-1.2keV</td><td>Xradia</td><td>NCT-SB</td><td>Transmission soft X-Ray microscope</td></tr><tr><td>SURVOS</td><td></td><td></td><td>Software for segmentation&amp;nbsp; (Luengo et al. 2017)</td></tr><tr><td>Tomo3d</td><td></td><td></td><td>Software for reconstruction (SIRT, WBP) (Agulleiro et al. 2011)</td></tr><tr><td>TomoJ</td><td></td><td></td><td>Software for reconstruction (ART) (Messaoudi et al., 2007)</td></tr><tr><td>XM Data Explorer</td><td>Zeiss</td><td></td><td>TXM software</td></tr></tbody></table>

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.

Preparing samples for cryo-SXT can be challenging. Even within the same sample grid, it is possible to have areas that are ideal, and areas that are non-ideal, as can be seen in Figure 5, which shows two squares from the same Au finder grid. The ideal sample should have single cells at the center of a square mesh, embedded in a thin layer of ice and surrounded by well-dispersed Au fiducial markers used for the alignment of tilt projections prior to tomography reconstruction. Figure 5A shows a fibroblast-like cell (NIH 3T3) that complies with many of these criteria. A single slice from a 3D reconstruction using ART27 of the area marked with the red box indicating the field of view (FoV) is shown in Figure 5B. Many different organelles such as mitochondria (M), endoplasmic reticulum (ER), vesicles (V) and the nucleus (N) can be distinguished thanks to the quantitative reconstruction of the LACs. In addition, the signal-to-noise ratio of the reconstruction is very high allowing to achieve high contrast of the cellular features. On the other hand, Figure 5C shows a square with higher cell density. Because of this, the blotting is usually less efficient, leading to a thicker ice layer, or even vitrification issues. In some cases, this can already be observed when screening the grid using epifluorescence mapping prior to the X-ray imaging, and those grids should be avoided at any cost. In Figure 5C, a crack within the grid and the vitrified ice can be observed going through the entire mesh square (marked by the red arrows). Any imaging near cracks should be avoided due to probable instability of the grid when exposed to the beam. In addition, cracks can be a sign of thick ice, as was the case in this area. A tilt series was recorded in the area marked with the red box. In Figure 5D, a single slice from the corresponding 3D reconstruction is shown. Even though some larger structures can be recognized, fine details are lost within the noise and grainy texture due to the poor vitrification quality of the thick ice, as can be seen specifically, for instance, on the upper mitochondria pointed by the arrow.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62190/62190fig1v2.jpg" /> 
Figure 1: Workflow. Schematic workflow followed prior to cryo-SXT data collection. <a href="https://www.jove.com/files/ftp_upload/62190/62190fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62190/62190fig02.jpg" /> 
Figure 2: Growing cells on grids. (A) Cells growing in a P100 Petri dish with a confluence around 80%-90%. (B) P60 Petri dish with several grids after seeding the cells. (C) Cells growing on top of a grid after 24 h. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62190/62190fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62190/62190fig03.jpg" /> 
Figure 3: Loading grids on the sample holders and into the transfer chamber. (A) Workstation filled with liquid nitrogen with the shuttle and the cryoboxes ready for loading the grids. (B) Sample holder inserted into the loader with the grid loaded. (C) Shuttle with the sample holder in position 3 without the cover. (D) Workstation with the transfer chamber attached. <a href="https://www.jove.com/files/ftp_upload/62190/62190fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62190/62190fig04.jpg" /> 
Figure 4: Loading samples into the TXM (A) Attaching the transfer chamber to the TXM. (B) Shuttle inside the TXM. (C) TXM robot arm inserting the sample holder into the sample stage. <a href="https://www.jove.com/files/ftp_upload/62190/62190fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62190/62190fig05.jpg" /> 
Figure 5: Example of cryo soft X-ray tomograms. Upper row: ideal sample, (A) 2D mosaic view of a grid square showing an isolated cell at the center. (B) One slice from the reconstructed 3D volume showing the marked area with the red box (A). Compared to (D) the image is much smoother and more details are visible. Lower row: non-ideal samples, (C) 2D mosaic view of a grid square showing too high cell confluency and cracks in the ice and grid foil (red arrows). (D) One slice from the reconstructed 3D volume showing the area marked with the red box in (C). The poor or suboptimal vitrification can be identified by the grainy texture of the image. N: Nucleus; M: Mitochondria; ER: Endoplasmic Reticulum; MV: Multivesicular bodies; V: Vacuole; Scale bars: A &#38; C 20 &#181;m; B &#38; D 2 &#181;m.V<a href="https://www.jove.com/files/ftp_upload/62190/62190fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography at JoVE.com

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

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

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Research

JoVE Journal

Biology

3.8K Views.  Alba Light Source, Cerdanyola del Valles, Spain. 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.

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

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

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

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

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.

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

Neuroscience

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 ...

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 ...

Biochemistry

Electron Cryotomography of Bacterial Cells

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

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

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

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. Various automated approaches have been proposed, many of which work well for high-contrast datasets where the features of interest can be easily detected and are clearly separated from one another. Our inner ear stereocilia cryo-electron tomographic datasets are characterized by a dense array of hexagonally packed actin filaments that are frequently cross-connected. These features make automated segmentation very challenging, further aggravated by the high-noise environment of cryo-electron tomograms and the high complexity of the densely packed features. Using prior knowledge about the actin bundle organization, we have placed layers of a highly simplified ball-and-stick actin model to first obtain a global fit to the density map, followed by regional and local adjustments of the model. We show that volumetric model building not only allows us to deal with the high complexity, but also provides precise measurements and statistics about the actin bundle. Volumetric models also serve as anchoring points for local segmentation, such as in the case of the actin-actin cross connectors. Volumetric model building, particularly when further augmented by computer-based automated fitting approaches, can be a powerful alternative when conventional automated segmentation approaches are not successful.

Here, we present a protocol to place simplified volumetric models into noisy, complex, tomographic 3D volumes. This allows the fast segmentation of actin filament densities, detection of systematic filament bending and of gaps in hair bundle filaments, as well as convenient quantification of volumetric model properties, such as distances.

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. ...

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

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

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

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

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...

Visualization of Organelles In Situ by Cryo-STEM Tomography

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Visualization of Organelles In Situ by Cryo-STEM Tomography

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 &#181;m.

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

Summary

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Chapters in this video

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

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

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

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

Electron Cryotomography of Bacterial Cells

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms

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

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Visualization of Organelles In Situ by Cryo-STEM Tomography