The authors thank Tammo Reisewitz for critical reading of the manuscript, and both Mako Hayashi and Sayuri Hagiwara for careful cultivation and observation of cyanobacteria, Yoshitaka Kimori for image processing, Chihong Song, Naoyuki Miyazaki, and Miyoko Nagayoshi for helping with segmentation of the structures. This work was supported by the Collaborative Study Program of the National Institute for Physiological Sciences (NIPS) to Y.K.

1. Synchronous Culture of Cyanobacteria
<ol>
	<li>Culture S. elongatus PCC 7942 on sterilized BG 11 plate (in a 9 cm sterile plastic petri dish) containing 1.5% (w/v) agar and 0.3% (w/v) sodium thiosulfate8.</li>
	<li>Place the plates in a growth chamber at 23 &#176;C with a light intensity of 50 &#181;E/m2/s and subject to 12 h light/12 h dark cycles.</li>
	<li>Transfer the cells onto fresh BG11 agar plates once a week. 
	NOTE: The cultures on the agar will appear as green bands of actively proliferating cells after one week under this culture condition.</li>
	<li>Take green clumps of cells with a flame sterilized wire loop and streak the cells onto a fresh BG11 agar plate. Do this on a clean bench.</li>
</ol>
2. Monitoring by Fluorescence Microscopy
<ol>
	<li>Use cells cultured on the agar plate for 6 days to observe DNA compaction. Collect cells from the plate at the end of the light period by pouring 1 mL of 0.2 M sucrose solution over the cells. Repeat pouring the solution onto the cells so that most of the cells are collected. Transfer the suspended-cell solution into a micro tube for DNA staining.</li>
	<li>Add DNA staining dye (e.g. Hoechst 33342) solution to 500 &#181;L of the suspended cell solution in a micro tube to a final concentration of 1 &#181;g/mL. Then, keep the tube in the dark for 10 min.</li>
	<li>Centrifuge for 1 min at 2,000 x g to sediment the cells. Discard the supernatant and add 10 &#181;L of 0.2 M sucrose solution to obtain a dense cell suspension.</li>
	<li>Transfer 1 &#181;L of the solution containing stained cells to a slide glass, put a cover slip and observe with a fluorescence microscope equipped with a UV filter using an objective lens with a magnification of 100X and immersion oil.</li>
	<li>Confirm that the DNA compaction is observed at this point in most cells, then prepare the sample for the next freezing step.</li>
</ol>
3. Sample Freezing for Cryo-HVET
<ol>
	<li>Set up a plunge-freezing device. Fill the tank with liquid nitrogen and start cooling the cryo-chamber after connecting the tank and the chamber with a Teflon tube.</li>
	<li>Fill liquid ethane into a small cupper pot inside the chamber after cooling down the chamber to liquid nitrogen temperature. Wear glasses during operation, because liquid ethane is explosive.</li>
	<li>Glow discharge the carbon-side of a holey carbon-coated EM grid (holey grid) for 30 s at 50 mA using a plasma ion bombarder.</li>
	<li>Apply 1 &#181;L of BSA gold tracer (15 nm) to the holey grid as a fiducial marker.</li>
	<li>Apply a 2.5 &#181;L aliquot of cells in the DNA compaction stage to the holey grid. Blot off the excess solution with a filter paper. Plunge the grid into liquid ethane using a plunger in the plunge freezing device immediately.</li>
	<li>Store the frozen grids in liquid nitrogen storage until they are examined.</li>
</ol>
4. Cryo-HVET
<ol>
	<li>Set up the HVEM at a high voltage of 1 MV.</li>
	<li>Mount the frozen grid in a cryo-specimen holder for HVEM precooled to -150 &#176;C with liquid nitrogen inside the cryo-workstation, and load it into the HVEM. Be careful to avoid contamination by ice.</li>
	<li>Select an imaging area at a lower magnification of 1,000X. Adjust the eucentric z-axis height.</li>
	<li>Tilt the specimen stage to -60&#176; and remove the backlash of the tilting rotation.</li>
	<li>Adjust the focus near the target location at a magnification of 10,000X. Set an under focus of 6 to 10 &#181;m by deviation from the focused image. For imaging, set the dose to 2 e-/&#197;-2 or less on the specimen beforehand.</li>
	<li>Measure the electron dose by the current density on the image screen in HVEM. Take an image on an electron film or by digital camera at the same magnification as in the focusing process.</li>
	<li>Collect tilt images manually by the same procedure as in (4.5) from -60&#176; to +60&#176; at a tilt angle increment of 2&#176; to 4&#176;. 
	NOTE: In many modern electron microscopes, the acquisition of the tilt series is automated by the combination of a digital camera. In that case, follow the instruction manual. For negative films, develop the films for 12 min at 20&#176;C in a developer tank using a full-strength developer and fix in a fixer for 10 min. Digitize the films at a resolution of 4,000 dpi (0.635 nm/pixel on the image) using a flatbed scanner. In the case of a digital camera, subject the collected images directly to the next image processing step. If necessary, reduce the image size by a median filter using two to four binning (size reducing factor) and appropriate software (e.g. ImageJ).</li>
</ol>
5. Tomographic Reconstruction
<ol>
	<li>Make a stack image file from the individual tilt images using the command &#34;tif2mrc&#34; or &#34;newstack&#34; in IMOD software9.</li>
	<li>Start eTomo GUI software in IMOD and set image parameters: pixel size, fiducial diameter, image rotation, etc. Then create scripts.</li>
	<li>Execute individual programs according to the software listed in tables of materials, where the tilt series is aligned using fiducial markers (with a mean residual error less than 0.5). Finally, reconstruct a 3D tomogram using the SIRT algorithm in IMOD.</li>
	<li>Extract a region of interest (ROI) from the tomogram and denoise using a denoise filter: an anisotropic diffusion filter in IMOD, a bilateral filter in EMAN10, or a mathematical morphology filter11, etc., with appropriate parameters to enhance the contrast.</li>
</ol>
6. Segmentation of the Feature of Interest
NOTE: The procedure described below is specific to the software used (see Materials Table) but other software packages can be used instead. Refer to their user guide.
<ol>
	<li>In the 3D Viewer window, open the tomogram file on Amira software and generate an OrthoSlice.</li>
	<li>In the Segmentation Editor window, create a segmentation file by selecting a new &#34;label field&#34;.</li>
	<li>Manually trace the border of the feature of interest (FOI). Follow the FOI through all tomogram slices. For the second FOI, create a new &#34;material&#34; and repeat the same operation.</li>
	<li>Generate a surface rendering by selecting the &#34;SurfaceGen&#34; menu. To visualize the segmented volume, select the &#34;SurfaceView&#34; menu. To move, rotate, and zoom in the 3D volume, use the tools in the 3D Viewer window.</li>
	<li>For automatic segmentation, use the Magic Wand Tool. Click on an object, and adjust the sliders in Display and Masking to cover the range of values so that the object is fully selected by its features.</li>
</ol>

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The authors declare no competing financial interests.

We have presented a sequence of protocols for visualizing transient DNA compaction in cyanobacteria. The basic concept is similar to that of correlative light and electron microscopy (CLEM)12. In addition, in this method, live cyanobacteria were monitored by fluorescence microscopy, rapidly frozen on EM grids, and directly visualized with high voltage cryo-electron tomography. As a first application, the detailed structure of DNA compacted bacterial cells was successfully visualized in 3D. Currently, this procedure is specific to this subject, but it will be applied more extensively, with modified methodology in some cases. Here, the advantages, the limitations, and the future possibilities of this method are discussed.
One of the advantages of this method is the 3D visualization of the whole cell. 1 MV HVEM successfully visualized the dynamic structure of the subcellular organelles in the DNA compacted cells. However, the fine structure inside normal cells could not be distinguished due to low image contrast. Increasing inelastic and multiple scattering in thick specimens blurs the image13. Zero-loss and most-probable-loss image filtering by an energy filter can improve image contrast by reducing inelastic scattering14,15, but it will not work for specimens thicker than iMFP. The zero-loss and most-probable-loss peaks drastically decrease with specimen thickness. It is particularly difficult to obtain a sufficient signal-to-noise ratio for the electron sensitive ice-embedded specimens. Murata et al. have shown that 1MV scanning transmission microscopy (STEM) gives higher image contrast than a bright field image in plastic embedded yeast cells with 5 &#181;m thickness, where the image contrast is primarily given by amplitude contrast13. However, it is expected that the effect of knock-on damage on higher accelerated electrons creates another limitation to the irradiation dose for damage-sensitive cryo-specimens16. The application of Volta and Zernike phase plates17,18 for HVEM may be able to reduce knock-on damage by reducing the total dose in the future. Another limitation to using HVEM for thick specimens comes from the fact that the user facilities that provide HVEM are scarce worldwide.
Using an alternative methodology to observe thick specimens, cryo-STEM tomography at 300 kV has demonstrated high contrast images on frozen-hydrated specimens with thickness in excess of several hundred nanometers19. To retrieve phase contrast in cryo-STEM, electron pthychographic microscopy has also been introduced, in which the phase plate in the condenser lens transposes a phase-modulated diffraction to a pixelated 2D detector20. The images are retrieved by calculation from multiple diffractions. For direct and fast 3D cryo-imaging of large native frozen samples, cryo-FIB-SEM can also be used21, where serial sectioning with a focused ion beam and block face imaging is applied for imaging fully hydrated frozen specimens. Although these technologies expand the viewing range of biological specimens, it is difficult to find the target location of the bacteria, e.g. labeled bacteria, because the target is completely under the ice and cannot be identified before trimming.
DNA compaction produces a distinct structure in cyanobacteria. DNA compacted cells are readily distinguished even without being stained due to a large density bias within the cells that is not present in normal cells. However, in order to visualize more local events within the cell, it is necessary to transfer the fluorescently labeled ROI into the electron microscope. For correlative light and electron microscopy (CLEM), light microscope images and electron microscope images are generally correlated using fluorescent latex beads or quantum dots on EM finder grids12. The labelling particles must be of high electron density in addition to fluorescence. They can accurately and reliably correlate the positions between the two images. Furthermore, by confirming the labeled area with cryo-light microscopy, complete overlap of ROI can be achieved between the two microscopes. When characterizing more detailed structural events in DNA compaction, these particles and cryo-light microscope will be an indispensable tool for a more robust and accurate correlation in the future.
This article shows how to characterize the transient structure of DNA compaction in cyanobacteria by a combination of synchronous culture, fluorescence microscopy and high voltage cryo-electron tomography. This protocol focuses on the observation of compacted DNA. By combining this method with other new technologies mentioned above, it will be possible to investigate the process of DNA compaction in further detail, and suitably modified methods are widely applicable to other dynamic structural events in bacteria.

DNA compaction is a dramatic cytoplasmic event that has been identified in some cyanobacteria. When Synechococcus elongatus was cultured under 12 h each light/dark cycle, DNA appeared condensed at the end of the light period, which was clearly different from its appearance at the other time points1. It has been suggested that this process is controlled by a circadian clock based on the Kai proteins2. Seki et al. have reported that the DNA stained with Hoechst 33342 was compacted in S. elongatus cells toward the end of the light period and showed a wavy rod-shape under a fluorescence microscope. The compacted DNA then separated into two at the center of the rod as the cell divided, and finally returned to a normal uniform distribution in each daughter cell3. However, its transient nature and large size for electron microscopy impeded structural analysis. Murata et al. combined several methods, including synchronous culture, fluorescence microscopy, rapid freezing, and high voltage electron cryo-tomography (cryo-HVET), and succeeded in identifying the structure of transient DNA compaction, including the kinetics of polyphosphate bodies (PPBs)4. The manuscript provides visual explanation of such a difficult material in detail by combining the experimental procedures.
S. elongatus has a capsule shape, a length of 2 to 5 &#181;m, a width of about 0.5 &#181;m, and the perfect DNA compaction appears in living cells only for a very short time. Therefore, the structural changes occurring in the cyanobacterial DNA compaction were unknown in detail. In order to investigate these structures by electron microscopy, it is necessary to overcome two main technical problems. One is the observation of such a thick specimen of the whole bacterium at near native conditions, and the other is the rapid fixation of a dynamic structure. As for the first problem, the inelastic mean free path (iMFP) of electrons depends on the accelerating voltage of the electron microscope5. In a transmission electron microscope (TEM) of 300 kV, it is less than 350 nm. For example, when an ice-embedded cyanobacterium (specimen thickness &#8776; 600 nm) is observed in 200kV TEM (iMFP &#8776; 250 nm), the structures inside cell are difficult to observe. By contrast, 1 MV TEM (iMFP &#8776; 500 nm) can give and image of the cytoplasmic structure throughout the cell (Figure 1). In this protocol, as one part of the solution, a high voltage electron microscope (HVEM) at an accelerating voltage of 1 MV was employed. However, facilities that implement HVEM are limited worldwide. Possible alternative solutions are also discussed in the Discussion section. The second problem was solved by cryo-electron microscopy (cryo-EM). This is a powerful tool for visualizing dynamic structures at near native conditions, where the specimen is rapidly frozen in liquid ethane using a rapid freezing device, and the frozen moment is directly observed with a minimum of modifications6. Combining with tomography, a snapshot of three-dimensional (3D) structures can be reconstructed from the tilt series7. In this experiment, DNA compaction was reproduced in S. elongatus using synchronous culture under 12 h each light/dark cycle, and the timing of the freezing of the specimen was determined by monitoring under a fluorescence microscope.
The approaches described here are widely applicable to the study of dynamic structures in bacterial cells, e.g. cell division, chromosome segregation and phage infection, and have a potential to open new avenues in microbiological research.

<table><tbody><tr><td>Hoechst 33342 solution</td><td>Dojindo</td><td>346-07951</td><td>1mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Agar Powder (for plant culture)</td><td>Wako</td><td>016-11875</td><td></td></tr><tr><td>Boric acid</td><td>Wako</td><td>027-02192</td><td>99.5%</td></tr><tr><td>Manganese chloride tetrahydrate</td><td>Wako</td><td>139-00722</td><td>99%</td></tr><tr><td>Zinc sulfate heptahydrate</td><td>Wako</td><td>264-00405</td><td>99.5%</td></tr><tr><td>Sodium molybdate</td><td>Wako</td><td>196-02472</td><td>99%</td></tr><tr><td>Copper sulfate pentahydrate</td><td>Wako</td><td>039-04412</td><td>99.5%</td></tr><tr><td>Cobalt nitrate hexahydrate</td><td>Wako</td><td>031-03752</td><td>99.5%</td></tr><tr><td>Sodium nitrate</td><td>Wako</td><td>191-02542</td><td>99%</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Wako</td><td>131-00405</td><td>99.5%</td></tr><tr><td>Calcium chloride dehydrate</td><td>Wako</td><td>031-00435</td><td>99.5%</td></tr><tr><td>Citric acid</td><td>Wako</td><td>036-05522</td><td>98%</td></tr><tr><td>EDTA-2Na</td><td>Dojindo</td><td>343-01861</td><td>99.5%</td></tr><tr><td>Sodium carbonate</td><td>Wako</td><td>197-01581</td><td>99.8%</td></tr><tr><td>Potassium phosphate dibasic</td><td>Wako</td><td>164-04295</td><td>99%</td></tr><tr><td>TES (Good&amp;rsquo;s buffer)</td><td>Dojindo</td><td>344-02653</td><td>99%</td></tr><tr><td>Ferric ammonium citrate</td><td>Wako</td><td>092-00802</td><td>1st Grade</td></tr><tr><td>Sodium thiosulfate pentahydrate</td><td>Wako</td><td>197-03585</td><td>99%</td></tr><tr><td>BSA gold tracer 15nm</td><td>Aurion</td><td>215.133</td><td></td></tr><tr><td>Quantifoil EM grid</td><td>Quantifoil MicroTools</td><td>R3.5/1 Copper grid</td><td></td></tr><tr><td>Electron films</td><td>Kodak</td><td>SO-163</td><td></td></tr><tr><td>Developer</td><td>Kodak</td><td>D19</td><td></td></tr><tr><td>Fixer</td><td>Kodak</td><td>Rapid fixer</td><td>Solution</td></tr><tr><td>Filter paper</td><td>Whatman</td><td>Grade 1</td><td></td></tr><tr><td>Growth chamber</td><td>NKsystem</td><td>LH-100SP</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Nikon</td><td>ECLIPSE 50i</td><td></td></tr><tr><td>High voltage TEM</td><td>HItachi</td><td>H1250M</td><td></td></tr><tr><td>Cryo-specimen holder for HVEM</td><td>Gatan</td><td></td><td></td></tr><tr><td>plunge-freezing device</td><td>Leica</td><td>EM CPC</td><td></td></tr><tr><td>Plasma Ion bombarder</td><td>Vacuum device</td><td>PIB-10</td><td></td></tr><tr><td>Liquid nitrogen storage</td><td>Taylor-Wharton</td><td>25LDB</td><td></td></tr><tr><td>Developing tank</td><td>Dosaka EM</td><td>TB-3-75</td><td></td></tr><tr><td>flatbed scanner</td><td>Nikon</td><td>Coolscan 9000ED</td><td></td></tr><tr><td>Segmentation software</td><td>FEI</td><td>Amira</td><td>https://www.fei.com/software/amira</td></tr><tr><td>Tomographic Reconstruction software</td><td>eTOMO</td><td></td><td>http://bio3d.colorado.edu/imod</td></tr></tbody></table>

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.

In a precise synchronous culture under 12 h each light/dark cycle, DNA labeled with Hoechst shows a normal uniform distribution in the dark condition (Figure 2a). However, it progressively compacts within the cell during the light period, and appears as a wavy rod-like structure (Figure 2b) at the end of the light period. Finally, the rod divides at the center (arrows in Figure 2c) and its two parts are distributed into the daughter cells (Figure 2d). After cell division, the compacted DNA disappears immediately, and the DNA returns to a normal uniform distribution.
When an aliquot of cells containing the cells in the final stage of DNA compaction were immediately transferred onto a holey grid and rapid frozen in liquid ethane, and the frozen grid was observed by 1 MV cryo-HVEM, the internal structures of cyanobacteria including DNA, thylakoid membrane layers, cell walls, and PPBs, appeared as in a snapshot at the moment of freezing (Figure 3a). Many cells showed distinct DNA compaction in the cells (white arrows in Figure 3a), and could be easily distinguished from normal cells (white arrowheads in Figure 3b). Some exhibited a constriction at the center of the cells as expected before cell division (yellow arrow in Figure 3a).
In 3D tomograms, major organelles of the cell could be segmented; cell wall, thylakoid membrane layers, DNA, and PPBs could be distinguished (Figure 4). In particular, the compacted DNA was separated by a distinct gap in the cytoplasm where the DNA was surrounded by low density material and the thylakoid membrane layers were distorted along the wavy rod of compacted DNA. Dynamic behavior of PPBs was newly observed: in DNA compacted cells, many small PPBs were seen to adhere to DNA, whereas they are large and fewer in normal cells. In addition, most of the PPBs appeared as pairs, and some DNA appeared to be in a process of separation from the PPBs. This suggested that PPBs themselves are divided into two by DNA duplication and function as suppliers of phosphate for DNA synthesis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57197/57197fig1.jpg" /> 
Figure 1. Cryo-EM images of ice-embedded normal cyanobacteria, S. elongatus PCC 7942 at different accelerating voltages. a) 200kV and b) 1000kV. Scale bar = 500 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57197/57197fig1large.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/57197/57197fig2.jpg" /> 
Figure 2: Fluorescence microscopy of cyanobacterium S. elongatus cells. After synchronous culture under 12 hour each light/dark cycles, cells were stained with Hoechst 33342. (a) Cells after 2 h of onset of the dark state show uniform DNA labeling. The DNA in many cells gradually condensed during the light period. It ultimately formed a thick wavy rod (b) typical of DNA compaction. After this stage, the compacted DNA structures quickly divided at their centers (arrowheads) during cell division (c), and the two fragments separated into the daughter cells (d). The daughter cells returned to uniform DNA labeling again in the dark period. Scale bar = 2 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57197/57197fig2large.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/57197/57197fig3.jpg" /> 
Figure 3: Cryo-HVEM image of cyanobacteria embedded in amorphous ice. (a) shows a raw image at 0&#176; tilt. Images were taken at the end of the light period. Some cells show wavy rod-shaped DNA bodies similar to eukaryotic condensed chromosomes (white arrows). In normal cells, cyanobacteria exhibit a discernable DNA structure within the cytoplasm (white arrowheads). Some exhibit a constriction at the center of the cells as expected before cell division (yellow arrow). (b) xy, xz, yz-slices of a 3D tomogram. Yellow dotted lines show intersections of slices. Scale bar = 2 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57197/57197fig3large.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/57197/57197fig4.jpg" /> 
Figure 4: Ultrastructure of cells containing compacted DNA. The major components are segmented: cell wall (bright yellow), thylakoid membranes (red), and DNA (blue). (a) shows a z-slice of a 3D tomogram. (b) all segments. The constriction indicating cell separation appears in the center of the cell (arrow). PPBs are modeled as yellow or orange spheres; each orange sphere represents the counterpart of the closest yellow sphere. Scale bar = 500 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57197/57197fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography at JoVE.com

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

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 ...

visualization-dna-compaction-cyanobacteria-high-voltage-cryo-electron

Nanyang Technological University

Research

JoVE Journal

Biology

9.1K Views.  National Institute for Physiological Sciences. Dit protocol wordt beschreven hoe de voorbijgaande verdichting van het DNA in cyanobacteriën visualiseren. Synchrone teelt, toezicht door fluorescentie microscopie, snelle invriezen, en hoge spanning cryo-Elektronentomografie worden gebruikt. Een protocol voor deze methodologieën wordt gepresenteerd, en toekomstige toepassingen en ontwikkelingen worden besproken.

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

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 ...

Bioengineering

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 ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

Immunology and Infection

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. ...

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

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

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

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

Cryo-Electron 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.

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

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