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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amira</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>Software for segmentation</td>
		</tr>
		<tr>
			<td>Au Holey Carbon Films&#160; finder grids</td>
			<td>(Quantifoil Micro Tools Gmb</td>
			<td>&#160;R 2/2 Au G200F1</td>
			<td>Au Holey Carbon Films finder grids</td>
		</tr>
		<tr>
			<td>Au nanoparticles</td>
			<td>&#160;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>&#160;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&#160; finder grids</td>
			<td>(Quantifoil Micro Tools Gmb</td>
			<td>&#160;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 &#38; 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&#160; (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

3.7K 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

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

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 ...

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 ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique cristae-rich double membrane ultrastructure of this organelle is elegantly arranged to carry out multiple functions by partitioning biomolecules. Mitochondrial ultrastructure is intimately linked with various functions; however, the fine details of these structure-function relationships are only beginning to be described. Here, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle. Serial-section electron tomography may be adapted to study any cellular structure in three-dimensions.

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

In this protocol, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle.

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...