Name: visualization of organelles in situ by cryo-stem tomography
Uploaded: 2023-06-23T12:20:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Visualization of Organelles In Situ by Cryo-STEM Tomography at JoVE.com

We are extremely thankful for the continuous and constant support from the author and maintainer of the SerialEM software package, David Mastronade, and G&#252;nther Resch. P.K. was funded by the Austrian Science Fund (FWF) through a Schr&#246;dinger Fellowship J4449-B. For the purpose of open access, the authors have applied a CC-BY public copyright license to any Author Accepted Manuscript version arising from this submission. M.E. and S.G.W. acknowledge funding from the Israel Science Foundation, grant no.1696/18, and from the European Union Horizon 2020 Twinning project, ImpaCT (grant no.857203). M.E. acknowledges funding from the ERC in the CryoSTEM project (grant no. 101055413). M.E. is the incumbent of the Sam and Ayala Zacks Professorial Chair and head of the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging. The laboratory of M.E. has benefited from the historical generosity of the Harold Perlman family. We also acknowledge the funding by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.

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The authors declare no conflicts of interest.

The protocol should assist life science microscopists who are interested in obtaining a 3D view of intracellular organelles in regions of the cell that are not accessible to conventional TEM tomography. The same protocol may be also used for STEM tomography of plastic sections, with relaxed constraints on the exposure. The protocol should be regarded as a starting point rather than a set of hard rules. Indeed, the power of STEM is its flexibility; there is no one right way to operate it.
We emphasize that STEM, per se, refers only to the scanned probe and does not define the image formation. Contrast depends primarily on the configuration of detectors. The more straightforward methods employ detectors with axial symmetry, either a disk centered on the optic axis or an annulus surrounding it. In general, when the illumination impinges on the detector directly, we record a BF image where the (electron-scattering) specimen appears dark; conversely, when the detector collects only scattered electrons, we record a DF image where the dense specimen appears bright. When suitable hardware is available, SerialEM can acquire and record both these signals simultaneously. Still more sophisticated configurations are available, ranging from detectors with multiple segments to fully pixelated cameras. Phase contrast imaging can be achieved, for example, by evaluating the difference between off-axis detector elements32,33. Collecting the entire 2D scattering (diffraction) pattern per pixel defines the method known as 4D STEM34, which enables the reconstruction of multiple image contrasts from the same original data. Four-dimensional STEM enables electron ptychography, which provides another means to obtain phase contrast35.
We have focused on the particular STEM modality that we consider to be most useful for the study of organelles and intact cells or micron-thick cell sections. This entails specifically the use of BF imaging with a small semi-convergence angle in the illumination and a relatively large collection angle at the detector. The small convergence provides a large depth of field so that the entire sample remains in focus throughout the tilt series19. In practice, with a good microscope stage, it can also eliminate the need for focus adjustment during acquisition. The price is a compromise in resolution, as described in section 3 of the protocol. We have suggested 4k images with a probe of ~2 nm, which with 1 nm pixel spacing reaches a field of view of 4 &#181;m. However, the reader is strongly encouraged to experiment. The second consideration is on the side of the detector. When the illumination disk underfills the on-axis BF detector, phase contrast is suppressed and scattering (amplitude) contrast dominates; this condition has been called incoherent bright field contrast36. The question is by what fraction to underfill, and the answer depends on the sample. A very thin sample will show best contrast with the detector completely filled (i.e., the collection angle matching that of the illumination), but a thicker sample will scatter all the intensity away, leaving a noisy signal with poor contrast. A useful rule of thumb is that the thicker the sample, the larger the outer cutoff angle of the BF detector should be21. The detector size and position are of course fixed, so the diffraction disk size is adjusted using the camera length, as described in section 1. If the detector amplifier settings are such that the signal fills the dynamic range but does not saturate under direct illumination (1.12.3), then the camera length can be increased until a reasonable signal intensity and contrast are reached. Again, the reader is encouraged to experiment. The art, so to speak, is in the angles.
Another parameter, not discussed in the protocol, is the microscope acceleration voltage. Interaction of the illuminating electrons with the specimen will be stronger at a lower voltage. With all else equal, we can expect higher contrast at lower voltage. On the other hand, it is the onset of multiple scattering within the specimen that limits the useable specimen thickness, so a higher voltage permits the use of thicker samples. These effects are rather subtle, however. Our experiences to date with 200 kV and 300 kV accelerations are similar.
Considering what can be expected of STEM in terms of resolution, this again depends on the specimen and the detector configuration. Using a single particle analysis approach, metal ions on ferritin could be localized by annular dark field STEM to a precision of a few angstrom37. More recently, sub-nanometer resolution was achieved for virus and protein samples using images obtained by integrated differential phase contrast (iDPC) STEM32 as well as ptychography35. For unique objects in thick cellular specimens, and with the methods described here, such high resolution is not realistic. The optimal resolution is the probe diameter, which relates to the semi-convergence angle as described. Other factors will degrade resolution, particularly a coarse pixel sampling to reach a large field of view, misalignments in the tilt series, and spreading of the probe beam in transmission. Images compare well with plastic section tomography. With the setup described here, for example, it should be easy to see the hollow core of the microtubules, but not the individual protofilaments.
To summarize, STEM methods and hardware are both in a development phase. We can expect that innovations in imaging will impact tomography as well, leading STEM into domains that have not been accessible by other techniques. We expect that a convergence with correlative cryogenic fluorescence imaging based on modern optical super-resolution methods will be especially fruitful. The scale of organelles, 100-1,000 nm, is an ideal target for these emerging methods.

Three-dimensional (3D) visualization of organelles is a paramount task in modern cell biology. Given the scales involved, ranging from tens of nanometers for secretory vesicles to many microns for the cell nucleus, it is challenging to find a single microscopy technique to fit all applications. While modern fluorescence microscopy can span much of the range in terms of resolution, only the labeled molecules appear. The cellular theater remains the realm of electron microscopy. Conventional methods of chemical fixation, plastic embedding, and staining with heavy metals are strongly invasive, so the results may depend on the details of sample preparation. Cryo-EM, on the other hand, is constrained by the need to vitrify the aqueous medium; ice crystals that form diffract the electron illumination, causing contrast artifacts of higher contrast than the organic material of interest.
The past decade has seen a proliferation of EM imaging techniques developed or adapted for cellular studies1. High-pressure freezing combined with iterative focused ion beam (FIB) milling and serial surface imaging using the scanning electron microscope (i.e., FIB-SEM) is currently the method of choice for large specimens2. Cryogenic soft X-ray tomography (cryo-SXT) is suitable for samples of several microns in size, limited by the characteristic absorption length of the soft X-rays in water3,4,5. This scale includes many intact cell types, and the quantitative nature of the X-ray absorption contrast adds an aspect of concentration measurement6 or spectroscopy7. When combined with subtomogram averaging, cryo-transmission electron tomography (cryo-ET), based on phase contrast transmission electron microscopy (TEM), offers the highest resolution for macromolecules or complexes8,9,10. However, it is rare that intact organelles are so regular that they can be averaged whole. Moreover, the conventional mode of wide-field TEM is limited for specimen thickness to a few hundred nanometers by the combination of inelastic scattering (involving energy loss) in the specimen and chromatic aberration in the magnetic objective lens11,12. The large energy spread dictates the use of an energy filter to remove the resulting out-of-focus haze, but the sensitive specimen still suffers radiation damage while the image signal becomes exponentially weaker with increasing thickness.
The alternative imaging mode, scanning transmission EM (STEM), circumvents the need for energy filtering and retains the inelastically scattered electrons for image formation, albeit currently at a lower resolution than for TEM tomography (Figure 1). In fact, no real image is formed. Instead, as in a scanning EM, measurements are made point by point, and the image is assembled by the computer. The magnification is determined only by the size of the scan steps without changing the lens currents. When properly configured, the useful range of specimen thickness for cryo-STEM tomography (CSTET) can reach 1.5 or even 2 &#181;m, though the comfort zone, where the useful signal intensity remains a significant fraction of the illumination, is around 600-900 nm11,13. This is sufficient to see a large fraction of the cytoplasm, and occasionally an edge of the cell nucleus. In practice, we find that vitrification by the standard method of plunging into cryogenic fluid imposes&#160;a more severe constraint on thickness than STEM imaging. The goal of this video article is to facilitate the incorporation of CSTET into the tool chest for cell and organelle imaging in research labs and microscopy facilities.
The first challenge is that microscope operations in CSTET are not yet standardized for life science applications in the way that they have been for cryo-TEM tomography. STEM hardware has rarely (if ever) been targeted to the cryo-EM market. However, this is changing with the newest generation of microscopes, and many existing tools can be retrofitted. STEM as a technique has taken off and largely taken over in the materials sciences, where there is also budding interest in cryogenic and low-dose methods14,15. The materials science literature abounds with acronyms BF-STEM, ADF-STEM, HAADF-STEM, 4D-STEM, DPC-STEM, etc., which add to the confusion. We offer here a recommended starting point that, in our collective experience at the Weizmann Institute of Science, provides the most general protocol for useful results based on bright field (BF) STEM imaging16. In no way does it exhaust or even explore the range of possibilities, but it will serve as a basis for further enhancements. While we emphasize cryo-STEM, most of the protocol is equally relevant for room-temperature STEM tomography of plastic-embedded sections.
The essence of STEM is to scan the specimen with a focused electron probe (Figure 1), the illumination cone, and to record signals from the diffraction (scattering) plane in transmission, pixel by pixel, to produce 2D images17,18. Amorphous specimens, including most cellular materials, will produce a diffuse scattering pattern in transmission. The simplest practical STEM configuration is to place a circular detector to record the central disk (i.e., the probe illumination that would be transmitted without a specimen). The specimen scatters electrons away from this illumination cone to the extent that the signal decreases. This produces a BF image-the specimen appears dark on a bright background. An annular detector may also (or instead) be used to detect the scattering from the specimen outside the illumination cone. With the specimen removed, there is no signal. When a specimen is in place, objects appear bright on a dark background in the dark field (DF) image. The nomenclature for STEM (BF, annular dark field [ADF], high-angle annular dark field [HAADF], etc.) refers mainly to the ranges of collection angles for the detectors.
The convergence angle of the illumination represents an essential adaptation of STEM to cellular tomography. When the top priority is high resolution, the convergence angle should be as large as possible. (This is similar to confocal laser scanning microscopy; the resolution is determined by the probe diameter, which scales as the wavelength divided by the numerical aperture. Note that we refer to the half-angle or semi-convergence angle for EM.) When the priority is the depth of field, on the other hand, a compromise in resolution affords a great advantage, as the focused beam remains roughly parallel for a distance equal to twice the wavelength divided by semi-angle squared. Ideally, the entire cell volume remains in focus19. For example, at 300 keV, the electron deBroglie wavelength is 0.002 nm, so a convergence of 1 mrad yields a resolution of 2 nm and a depth of field of 4 microns. Under these conditions, tomography can be performed even without focusing during the data collection process, but only once at the beginning of the acquisition. A conventional tomography-capable STEM can reach a semi-convergence angle of 7 or 8 mrad; therefore, in principle, we could reach a resolution in the order of 0.25 nm, but then with a focal depth of only 62 nm. This is clearly too thin for cellular imaging. More advanced microscopes with three condenser lenses offer continuous adjustment of the semi-convergence angle over a considerable range. With the more traditional two-condenser configuration, the convergence is fixed discretely by the condenser (C2) aperture.
For robust, plastic-embedded samples, one can record a focal series at each tilt and combine them for high resolution20, but for cryogenic specimens, the radiation budget is too severely constrained. Finally, in weighing the advantages of BF or DF imaging, for thick specimens, one should consider the effects of multiple elastic scattering in the specimen. The BF signal is less corrupted by multiple scattering and shows a higher resolution for thick specimens16,21.
A useful rule of thumb has been to set collection angles several times larger than the convergence. The thicker the specimen, the larger the collection disk should be. Too small a disk will provide a low signal intensity; too large a disk will result in poor image contrast, as only the highest-angle scattering will contribute. The collection angles should be optimized for a given sample. The detector angles as a function of (diffraction) camera length must be calibrated independently. They may be displayed conveniently by the microscope software. In practice, a factor of two to five in the ratio of collection to illumination semi-angles, &#952; to &#945;, respectively (Figure 1), is a recommended starting point for CSTET of cellular specimens.
The following protocol describes STEM tomography operation using the popular SerialEM software for microscope control22,23. SerialEM is not tied to a specific manufacturer, and it is widely used in TEM tomography. Most of the operations in setting up for tomography can be carried over directly from TEM. The SerialEM strategy is to model the scanning system as a camera. This enables the simple crossover from TEM to STEM. One should keep in mind, though, that parameters such as magnification and binning are entirely artificial. The important parameters are the field of view in microns, the number of pixels in the field of view, and the exposure time. The pixel spacing, or sampling, is the linear field divided by the number of pixels, while the dwell time is the number of pixels divided by the exposure time.
The minimum configuration for STEM and CSTET involves three features on the microscope: a scan generator, a STEM detector, and tomography control software. The protocol refers to the nomenclature of FEI/Thermo Fisher Scientific (TFS), but the concepts are generic. The proprietary software of TFS has been described in JoVE for TEM24, and the STEM operation is very similar.
We assume that the microscope has been aligned in advance by the service team or experienced staff and that a column alignment can be called up by loading a file. Minor adjustments are called direct alignments and can be stored in so-called FEG registers (TFS microscopes). Direct alignments include rotation center, pivot points, diffraction alignment, and compensation for condenser astigmatism. Adjustments have to be performed iteratively. Note that TFS microscopes implement distinct nanoprobe (nP) and microprobe (&#181;P) modes; for a given condenser aperture, these provide a relatively narrow or wide field of view with parallel illumination in TEM and a more or less convergent (tightly focused) electron beam in STEM, respectively. Other manufacturers use different schemes to cover the range of convergence angles.
Before starting, the field of view, L, and the sampling (pixel width), l, should be chosen, depending on the sample under study. For example, for l = 1 nm/pixel, a 4,000 x 4,000 pixel image that will cover a field of view 4 &#181;m2 should be chosen. The resolution will be, at best, twice the spatial sampling, so 2 nm, and the probe diameter, d, should match that. Calibration of the probe angle is beyond the scope of this protocol, so we assume that a table or a screen reading is available. The probe diameter is approximately the electron wavelength divided by the semi-convergence angle (in radians): d = &#955;/&#952;. The wavelength, &#955;, is 0.002 nm for 300 keV and 0.0025 nm for 200 keV electrons, so &#952;&#160;of 1 mrad will provide a spot diameter of 2 or 2.5 nm, respectively.
The protocol is presented in a progression of increasing complexity. The first task is to produce a STEM image, which depends on the microscope manufacturer&#39;s software, and then a tilt series, for which we use SerialEM. Many readers will undoubtedly be familiar with SerialEM, so the more complicated tasks will come naturally. There is no need to follow the procedures strictly. Developments relating to automation may be implemented directly for STEM as well as for TEM. Experienced users will likely invert the protocol, beginning with correlative registration of fluorescence maps and continuing to set up batch tomography. Further details can be found in the extensive documentation and tutorial libraries for SerialEM itself, including a recent JoVE article on the latest developments in automation25.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;SerialEM</td>
			<td>University of Colorado</td>
			<td>Veriosn 4.0</td>
			<td>SerialEM is a free software platform for microscope control and data acquisition.&#160;</td>
		</tr>
		<tr>
			<td>STEM-capable transmission electron microscope</td>
			<td></td>
			<td></td>
			<td>The protocol was written based on experience with several microscopes of Thermo Fisher Scientific: Titan Krios, Talos Arctica, and Tecnai T20-F. In principle it should be applicable to other manufacturers as well.</td>
		</tr>
	</tbody>
</table>

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.

A full grid montage prepared in STEM shows the areas with cells of interest (Figure 4). Note that the image is in dark field, so empty holes appear dark. Cells appear partly bright, where the electrons are scattered toward the HAADF detector. At the thickest parts, typically the centers or near the grid bars, the contrast goes dark again. This is due to multiple scattering, whereby the electrons reach angles not captured by the detector. In practice, these areas will be too thick for tomography.
The next stage involves maps of intermediate magnification (Figure 10). These are often called medium montage maps, or square maps, referring to the grid squares rather than the shape. Even at the lowest microprobe STEM mode, they will also be acquired as montage images. Clicking on the item in the Navigator window loads the map image into the main window. Specific points for tomogram acquisition are chosen within this area. Use the Preview mode to locate the area at the recording magnification. Keep in mind that there may be a lateral shift between Preview and Record, depending on the scan speeds. A single tilt series may be recorded here. Mitochondria, for example, may often be identified in the Preview and Record image (Figure 12).
For batch tomography, the stage will travel around the grid and may not return to precisely the same location. Anchor maps are used to ensure that the desired Record areas are re-identified reliably by matching the Preview image to a lower magnification View, even if the stage movement has shifted. PyEM23,24 offers a faster alternative that is certainly recommended, but is not yet part of the standard installation.
In the CLEM approach, a fluorescence map may be used to identify the areas of interest for tomography. The fluorescence is acquired externally and must be registered onto the full grid montage. It is useful to have the grid bars and holes visible, so a merged fluorescence + bright field composite may be prepared before import, for example in GIMP (https://www.gimp.org) or ImageJ31 software (take care that ImageJ inverts the vertical axis). When the dynamic range of the fluorescence is large, it may be difficult to create such a merge without saturation. In this case, the two maps may be imported separately and then registered together as described (Figure 13). This way, a point on the fluorescence map in the Navigator window, followed by &#34;Go To XY&#34;, brings the stage to the corresponding position on the grid as mounted in the TEM. Take care that small inconsistencies in the creation of the montage (or the fluorescence map, if it too is a montage) will lead to small displacements; therefore, for optimal precision, the fluorescence should be re-registered specifically to the square map. It is often sufficient to do this registration by eye, on the basis of holes in the support film or features shared with the bright field light image.
Reconstruction of the tilt series results in a 3D volume (Figure 14). This example has a pixel size of 2.042 nm, which results in a field of view of ~4 &#181;m. Calcium phosphate deposits (orange arrowheads) stand out, because of the higher atomic number compared to the surrounding. Microtubules (red arrowheads) can be traced throughout the whole field of view. Also, the inner and outer mitochondrial membranes (green arrowheads) can be clearly seen. Actin can be observed as bundles or as individual filaments. To achieve a more isotropic resolution, the reconstruction may be processed by deconvolution.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65052/65052fig01.jpg" /> 
Figure 1: Comparison of TEM versus STEM. In TEM, the field of view is illuminated by a near-parallel beam, and the objective lens forms a magnified image on the camera. For cryo-TEM, the objective aperture is opened to pass the scattered electron waves (dashed red line), which, with defocus, produce phase contrast by interference with the unscattered (green) waves. STEM, on the other hand, rasters a focused beam across the specimen and collects the scattered electrons in a pixel-by-pixel manner. Multiple detectors may collect electrons scattered to different angles. This figure has been modified from30. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig01large.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/65052/65052fig02.jpg" /> 
Figure 2:&#160;Example of image distortion on the left side due to the high scan rate. Note the distortion marked by yellow arrowheads, as well as, the distorted oval-shaped holes in the Quantifoil. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig02large.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/65052/65052fig03.jpg" /> 
Figure 3: Montage setup dialogue for the whole grid montage. Choose the magnification and the number of pieces in X and Y so that the total area reaches around 2,000 &#181;m to get a map for most of the grid. Also choose Move stage instead of shifting image and Use Montage Mapping, not Record parameters. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig03large.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/65052/65052fig04.jpg" /> 
Figure 4: Full low magnification GridMap recorded in STEM mode. Broken areas appear completely black in the dark field image. Dark gray areas are likely too thick and poorly vitrified. Good candidate areas for tomography are white or light gray in this scan. Scale bar = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig04large.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/65052/65052fig05.jpg" /> 
Figure 5: Shift to marker process. A recognizable object is marked in the low resolution map by Add Points (38 in this image). Note the shift between the low resolution map (green circle) and the medium resolution map (orange circle). Then, the object is marked by the marker (green cross, red circle), and the Shift to Marker dialog is started. The chosen option moves all maps at 245x by the same amount. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65052/65052fig06.jpg" /> 
Figure 6: Tilt series setup dialogue for a STEM tilt series. The dose-symmetric scheme is used from -60&#176; to +60&#176; in steps of 2&#176;. Autofocus is skipped due to the high depth of focus when using a low convergence angle. No need to refine eucentricity as this has been done before. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65052/65052fig07.jpg" /> 
Figure 7: File properties dialogue for medium resolution maps. Save as an MRC stack file, choose integers, and save extra information in a .mdoc metadata file. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65052/65052fig08.jpg" /> 
Figure 8: Montage setup dialogue for saving medium resolution maps. For a square on a 200 mesh grid, which is 90 &#181;m wide, a total area of at least 90 &#181;m x 90 &#181;m is advisable. Choose Move stage instead of shifting image and Use View parameters in Low Dose mode. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65052/65052fig09.jpg" /> 
Figure 9:&#160;Recording medium resolution map. Acquire at Items dialogue for recording medium resolution maps. Choose Mapping, Acquire and save image or montage, and tick Make Navigator map. In the Tasks before or after Primary, choose Rough and Fine Eucentricity. When unassisted, optionally choose No message box when error occurs and Close column valves at end. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65052/65052fig10.jpg" /> 
Figure 10:&#160;Image of medium resolution map. Medium resolution map (Square Map) recorded in STEM mode by a montage of 3 x 3. The two medium resolution Anchor Maps for data collection are indicated by the blue squares. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65052/65052fig11.jpg" /> 
Figure 11:&#160;Batch tilt series data. Acquire at Items dialogue for collecting batch tilt series. For tilt series data collection, choose Final Data and Acquire tilt series. At Tasks before or after Primary, check Manage Dewars/Vacuum (choose appropriate settings in the Setup menu), Realign to Item, Fine Eucentricity, and Run Script before Action. In the General options, check No message box when error occurs and Close column valves at end (see 5.14). <a href="https://www.jove.com/files/ftp_upload/65052/65052fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/65052/65052fig12.jpg" /> 
Figure 12: 0&#176; tilt of a STEM tilt series of a mitochondrion. Orange arrowheads point to calcium phosphate deposits (matrix granules), green arrowheads to cristae, and red arrowheads to 15 nm gold fiducial markers. Scale bar = 500 nm. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/65052/65052fig13.jpg" /> 
Figure 13: Registered cryo-CLEM maps. (A) Medium resolution STEM map (Square map, 26-A) is registered to the (B) low resolution STEM Map, (C) BF channel cryo-FM Map, and (D) GFP channel cryo-FM map. The nine registration points (labels 4-21) are shown in the (E) Navigator. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/65052/65052fig14.jpg" /> 
Figure 14: Volume rendering. Volume rendering of (A) 60 nm and a (B) 40 nm thick sections through a SIRT-like (30 cycles) filtered tomogram at different depths. The pixel size is 2.048 nm, which results in a field of view of approximately 4 &#181;m. Please note the inverted density compared to Figure 12, meaning that high-intensity features are bright. Orange, white, red, green, and light blue arrowheads point to calcium phosphate deposits, ribosomes, microtubules, the inner and outer mitochondrial membrane, and actin filaments, respectively. Scale bar = 500 nm. <a href="https://www.jove.com/files/ftp_upload/65052/65052fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualization of Organelles In Situ by Cryo-STEM Tomography at JoVE.com

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 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 ...

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