The FIB-SEM and A.S. (the position of the FIB-SEM operator) are funded by the Cluster of Excellence and Deutsche Forschungsgemeinschaft (DFG) Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB). We thank the lab of Thomas M&#252;ller-Reichert for providing the C. elegans samples. We thank Ulrich Weikert for participating in the movie.

All experiments including samples from animals described here have been approved by the Institutional Animal Care and the Lower Saxony State Office for Customer Protection and Food Safety (LAVES).
1. High-pressure freezing and freeze substitution
<ol>
	<li>Dissect the nervus tibialis from a mouse (e.g., C57Bl6N, 12 weeks, female)9.</li>
	<li>Immerse the nervus tibialis in a droplet of 20% polyvinylpyrrolidone (1 g of PVP in 5 mL) in phosphate buffered saline (PBS) and cut pieces of 2 mm length. Then, place it in a metal sample carrier (Type A, 0.2 mm depth) using fine forceps. Add another flat carrier (Type B) coated with a thin layer of hexadecane as a lid and insert this assembly into the specimen cartridge.</li>
	<li>Transfer several C. elegans suspended in a droplet of 20% bovine serum albumin (BSA) in M9 (see Table of Materials) with a disposable plastic pipette into the metal carrier (Type A). Then, add a second carrier as lid on top (Type B) and assemble the cartridge.</li>
	<li>For both of the samples, proceed with the following: high-pressure freeze the sample carrier assemblies one-by-one by loading them into the three-piece cartridge in the loading station of the high-pressure freezer. Press the process button according to the manufacturer&#8217;s operating instructions. 
	NOTE: Both types of samples can be processed together in the subsequent freeze substitution.</li>
	<li>Place the samples into cryovials containing 2 mL of frozen 0.1% tannic acid in acetone. Perform the transfer in a bath of liquid nitrogen without spilling liquid nitrogen into the vials. Transfer the cryovials into an automatic freeze substitution unit10 set at -90 &#176;C and start the program. 
	NOTE: The samples are kept at -90 &#176;C for 100 h. Tannic acid before osmification is used as mordant to increase the uptake of osmium tetroxide11.</li>
	<li>Manually wash the samples 3x for 30 min with 2 mL of acetone at -90 &#176;C in the freeze substitution unit.</li>
	<li>Leave the samples in 2 mL of 2% osmium tetroxide, 0.1% uranyl acetate in acetone for continued staining in the automatic freeze substitution unit for 7 h at -90 &#176;C. 
	CAUTION: Osmium tetroxide is toxic and should be handled under a fume hood, and protective equipment should be worn. 
	NOTE: The freeze substitution unit automatically raises the temperature to -20 &#176;C at 5 &#176;C/h. The samples are kept at -20 &#176;C for 16 h in the freeze substitution unit. The temperature will be raised automatically to 4 &#176;C at 10 &#176;C/h.</li>
	<li>Exchange the osmium tetroxide solution with pure acetone when the temperature has reached 4 &#176;C. Remove the cryovials from the freeze substitution unit.</li>
</ol>
2. Microwave-assisted processing
NOTE: Perform all of the following steps at room temperature12 using a temperature control unit to keep the temperature stable (see Table of Materials).
<ol>
	<li>Leave the caps of the reaction tubes open during processing in the microwave.</li>
	<li>Take the metal sample carriers out of the cryovials and remove the samples from the metal carrier using fine forceps or a needle, while keeping the samples submersed in acetone in a watch glass dish (150 mm).</li>
	<li>Transfer the samples into 2 mL reaction tubes using fine forceps or a pipette and wash with 1 mL of acetone four times for 40 s at 250 W. 
	CAUTION: Thiocarbohydrazide is toxic and should be handled under a fume hood, protective equipment should be worn.</li>
	<li>Stain samples with 1 mL of 1% thiocarbohydrazide in acetone for 14 min at 150 W to increase the contrast. To perform the staining, pipette the thiocarbohydrazide solution into the reaction tube, place the tube in the microwave, and set the program to a power level of 150 W (with the vacuum function turned on) for 2 min on/2 min off, iterating for 14 min total. Start the microwave. 
	NOTE: Thiocarbohydrazide reacts with the osmium tetroxide that was applied during the freeze substitution. It forms a bridge, allowing more osmium tetroxide to be deposited onto the original osmium tetroxide sites13.</li>
	<li>Wash the samples 4x with 1 mL of acetone for 40 s at 250 W. Pipette the acetone into a reaction tube, place the tube into the microwave, and select 250 W for 40 s. Start the microwave.</li>
	<li>Stain in 1 mL of 2% osmium tetroxide in acetone for 14 min at 150 W. Pipette the osmium tetroxide solution into a reaction tube, place the tube into the microwave, and set the program to a power level of 150 W (with the vacuum function turned on) for 2 min on/2 min off, iterating for 14 min total. Start the microwave.</li>
	<li>Wash the samples 4x with 1 mL of acetone for 40 s at 250 W (as done in step 2.5).</li>
	<li>Infiltrate with 1 mL increasing concentrations of resin in acetone (see Table of Materials) of 25%, 50%, 75%, 90%, 100%, and 100% for 3 min each at 250 W. Pipette the resin into a reaction tube, place the tube into the microwave, and set the program to a power level of 250 W (with the vacuum function turned on) for 3 min. Start the microwave.</li>
</ol>
3. Minimal resin embedding8
<ol>
	<li>Take the nervus tibialis and C. elegans out of the reaction tube with a toothpick and place them on a piece of plastic film (see Table of Materials).</li>
	<li>Use the toothpick to gently push the sample around on the plastic film until no remaining resin is detected. Use a halogen lamp for heating (see Table of Materials) so the resin becomes less viscous and is able to be removed more easily. Place the halogen lamp close enough to heat up the resin (being careful not to burn hands).</li>
	<li>Polymerize the samples for at least 48 h at 60 &#176;C on top of the plastic film in the oven.</li>
</ol>
4. Preparation for FIB-SEM
<ol>
	<li>Cut out the polymerized samples together with the plastic film using a razor blade at a size fitting the SEM stub and attach them to the SEM stubs using a conductive silver resin (see Table of Materials). Polymerize again at 60 &#176;C for at least 4 h or overnight.</li>
	<li>Sputter coat the samples on the SEM stub with gold for 1 min at 35 mA using a sputter coater (platinum/palladium can also be used; a 10 nm thick coating is sufficient) and place them inside the FIB-SEM.</li>
</ol>
5. Data acquisition inside the FIB-SEM
<ol>
	<li>Image the samples with the secondary electron detector using the basic software driving the microscope (see Table of Materials). The sample should be oriented so that the region of interest (e.g., middle part of the nervus tibialis or the C. elegans) is in the field of view. 
	NOTE: To perform the data acquisition, tilt the stage to a position so that the sample faces the ion beam at a 90&#176; angle at an appropriate working distance (5 mm) in the so-called coincidence point of the ion and electron beam. Using our instrument, this is achieved at a stage tilt of 54&#176; and 5 mm working distance.</li>
	<li>To set up the correct position of the sample, center a common feature at a 0&#176; tilt while imaging with the secondary electron detector. Slowly tilt to 54&#176; (5&#176;, 20&#176;, 54&#176;), recentering the same object by moving the M-axis.</li>
	<li>Find the coincidence point by centering a feature while imaging with the secondary electron detector at 54&#176;, followed by moving the feature to the central position in the FIB mode. Perform this by displaying the crosshairs in the SEM software to have an indication of the center. Then first move the selected sample feature to the center of the image area by using the center point function of the SEM software. Then center the feature along the y-axis by moving the stage in z. Any final offset between FIB and SEM is corrected with the SEM beam shift.</li>
	<li>Open the advanced software (see Table of Materials) for recording 3D datasets and follow the software wizard step-by-step.</li>
	<li>At the region of interest, deposit carbon or platinum (400 nm) on top of the ROI using a 3 nA current to allow even milling and reduce artifacts induced by charging. 
	NOTE: By drawing the region of interest to be milled on the specimen surface imaged with the FIB (width, height, depth), the software calculates and superimposes the sizes of the different sample preparation features, such as the trenches to be milled or area for platinum/carbon deposition. Also, be cautious of imaging the sample surface with too-high FIB currents, as this can damage the sample surface. A current of 50 pA is sufficient for imaging.</li>
	<li>Use the focused ion beam to mill a trench to expose the region of interest (ROI) using a 15/30 nA current.</li>
	<li>Use the focused ion beam to polish cross-section with a 7 nA current.</li>
	<li>After finishing the sample preparation, set up the following imaging parameters: FIB milling parameters in the FIB setup tab (FIB milling current); SEM imaging parameters in the setup tab and the image tuning parameters in the SEM AutoTune tab (perform autofocus and autostigmation every 60 min, 50 nm pixel size, dwell time 3, line average 3). Optimize this for every sample.</li>
	<li>To prevent any thermal drift causing the block-face to drift during the run, leave the room for at least 2 h before starting the acquisition.</li>
	<li>Acquire the dataset in a continuous mill and acquire mode with a 700 pA FIB current. Use 1.5 kV (analytic mode, 900 V) ESB detector with a grid voltage of 400 V to acquire images each with 5 nm pixel size using the advanced software (see Table of Materials) and 50 nm slice thickness. One image acquisition takes about 1.5 min with a dwell time of 6 &#181;s.</li>
	<li>Start the data acquisition. A FIB image at the milling current is acquired to ensure that the sample did not move and the right region of interest is selected. Adjust if necessary.</li>
</ol>
6. Data visualization
<ol>
	<li>Open images in Fiji by dragging the folder containing all the images into Fiji and select Virtual Stack.</li>
	<li>Crop the region of interest using the rectangle tool (Image &#62; Crop).</li>
	<li>Invert the data to show the traditional transmission electron microscopy contrast with membranes showing up in dark (Edit &#62; Invert).</li>
	<li>Use TrackEM214 (Fiji15) for alignment. Load the data into TrackEM2 (File &#62; New &#62; TrackEM2) by clicking on layer. After all images are loaded, align them using multi-layer mosaic function (right-click on Image &#62; Align &#62; Align multi-layer mosaic). After alignment, the images are exported as individual tiff-files (right-click on image &#62; Export &#62; Make flat image). Select all images that should be exported and choose save to file.</li>
	<li>For post-processing, use a Gaussian blur (Process &#62; Filters &#62; Gaussian blur; sigma 2) and local contrast enhancement (Process &#62; Enhance Local Contrast; CLAHE: blocksize 127, histogram bins 256, maximum slope 1.5), which are applied in Fiji to smooth the image and obtain a better signal-to-noise ratio.</li>
	<li>IMOD16 is used to perform a manual segmentation and visualize the structure of interest in 3D. Open the dataset in IMOD and select Drawing tools (Special &#62; Drawing Tools) to perform the manual segmentation. Different manual tools can be used to follow the structure of interest throughout the volume (for example Join, Sculpt). Use Microscopy Image Browser (MIB17) for semi-automated segmentation.</li>
</ol>

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No conflicts of interest declared.

The protocol was developed to illustrate the optimal preservation and contrast to perform serial block-face imaging with a FIB-SEM. Therefore, we chose to apply cryo-immobilization followed by post-staining using freeze substitution and microwave-assisted processing. Therefore, this protocol is limited to samples that are small enough for high-pressure freezing. The size limitations of 3 to 6 mm in width and thickness of ~200 &#181;m are established by the size of the sample carrier, which matches the sample size that can be properly frozen with this technique. This is relevant for the mouse nerve sample, since the sciatic nerve is too large in diameter to fit into the 0.2 mm carriers that are required to ensure proper freezing. Therefore, careful dissection of a smaller nerve such as the tibial nerve or other thin nerve such as the femoral nerve is recommended. Since the myelin sheath is sensitive to stretching, great care must be taken during dissection of the fresh and viable nerve to avoid handling artifacts. In general, only viable samples should be used for electron microscopic studies.
Microwave-assisted processing and minimal resin embedding are designed for speeding up the preparation and targeting process. The microwave-assisted processing applying a modified OTO protocol4 is used for room temperature chemical fixation. A household microwave will not yield the same results, since there is no homogenous distribution of microwaves, its temperature is not controlled, and there is no vacuum that can be applied. The smaller a sample, the better penetration of the chemicals; therefore, the best results are achieved by smaller samples. To avoid damage to the sample by overheating, temperature control and application of the minimally required microwave power are critical. The microwave-assisted processing steps can be performed on the bench if there is no microwave available, which will lead to longer processing times. To directly target structures in the SEM, it is crucial to remove as much resin as possible from the top of the sample. After recording a dataset, post-processing of the raw data is necessary to reduce file size and improve the signal-to-noise ratio. Modern volume imaging techniques produce large amounts of data. Therefore, to perform the data processing in a fast and sufficient manner, sufficient RAM on the workstation is needed. For alignment operations at least two times as much RAM as the dataset size is required.
This protocol has been tested on the nervus tibialis of the mouse as well as in C. elegans. Hall et al.12 used a similar enhancement step after their freeze substitution on the bench for preparation of C. elegans. For any other model organism such as the zebrafish, adjustments of the protocol are likely necessary. One possible modification is to change the composition of the freeze substitution cocktail, such as by the addition of water that is used for contrast enhancement18. Moreover, the duration of the freeze substitution has to be adapted to the sample and can be considerably shortened according to the quick freeze substitution protocol19. One possibility is the application of agitation for accelerating the freeze substitution process20. After the freeze substitution, further modifications are possible, such as repeated application of enhancing chemicals and osmium tetroxide21. During microwave processing, the temperature, incubation times, and power settings can be varied to optimize results for the respective sample.
This protocol shows that such an enhancement can be combined with other freeze substitution protocols and different types of samples as described by Hall12 that are imaged in a FIB-SEM or by serial block-face scanning electron microscopy. These imaging techniques require enhanced contrast, which is less important for transmission electron microscopy.

High-pressure freezing is the sample preparation method of choice for obtaining high-quality ultrastructural preservation, which represents the native state of a sample much better than conventional preparation methods using chemical fixation1. This cryo-preparation method is useful for samples such as myelinated mouse tissue2 and a strict requirement for use of the model organism Caenorhabditis elegans3. After freeze substitution and resin embedding, these samples are usually analyzed by transmission electron microscopy (TEM) or electron tomography (ET). If bigger volumes must be imaged using FIB-SEM or serial block-face imaging for high-resolution large-scale 3D reconstructions, in our experience proper imaging by SEM is often hampered by the lack of contrast. In the FIB-SEM, the image is usually recorded by the detection of backscattered electrons from the primary electron beam. The yield of backscattered electrons is proportional to the content of heavy metals in the sample. Therefore, protocols were especially designed for volume imaging to enhance the contrast by additional heavy metal impregnation. Such methods are based on chemically fixed samples and apply a combination of osmium tetroxide-thiocarbohydrazide-osmium tetroxide4, as described by Knott et al.5, for serial block-face and focused ion beam scanning electron microscopy. Modifications including the use of formamide and pyrogallol6 or lead aspartate7 have been successfully applied for different imaging techniques.
The protocol provided here combines the cryo-preparation of specimens by HPF and freeze substitution with subsequent microwave-assisted processing for enhanced contrast using thiocarbohydrazide/osmium tetroxide in acetone at room temperature. We demonstrate this on the myelinated nervus tibialis of mice and in Caenorhabditis elegans, which represent samples that require high-pressure freezing for high-quality ultrastructural preservation. In addition, it is shown how, after dehydration and infiltration, the samples are embedded with as little resin as possible. This minimal resin embedding8 allows for faster targeting of the structure of interest and reduces the time spent on sample processing, including less time required for exposing the region of interest with the ion beam. After performing further sample preparation steps inside the microscope, imaging and milling of the sample is carried out continuously to acquire a stack of images. For 3D visualization, image processing software (IMOD) is used to reconstruct parts of the dataset.
Our workflow describes how the most suitable contrasting of samples for volume imaging can be combined with the best ultrastructural preservation by HPF and freeze substitution. This is useful for samples that strictly require cryo-preparation. Applications are limited to small samples that can be prepared by HPF. In samples of different nature, such as plant material or microorganisms, this protocol requires adaptation.

<table>
	<tbody>
		<tr>
			<td>Instrumentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica HPM100</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Freeze Substitution</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory microwave with temperature control unit</td>
			<td>Ted Pella</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EM ACE600 with gold target</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crossbeam 540</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Halogen lamp 12 V/ 20 W</td>
			<td>Osram</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>M9</td>
			<td>Homemade</td>
			<td></td>
			<td>According to C. Elegans- A practical approach I.A.Hope</td>
		</tr>
		<tr>
			<td>Hexadecene</td>
			<td>Sigma-Aldrich</td>
			<td>52276</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>Sigma-Aldrich</td>
			<td>P2307</td>
			<td></td>
		</tr>
		<tr>
			<td>A type carrier</td>
			<td>Wohlwend GmbH</td>
			<td>#241</td>
			<td></td>
		</tr>
		<tr>
			<td>B type carrier</td>
			<td>Wohlwend GmbH</td>
			<td>#242</td>
			<td></td>
		</tr>
		<tr>
			<td>Slit carrier</td>
			<td>Wohlwend GmbH</td>
			<td>#446</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Pasteur pipettes</td>
			<td>VWR</td>
			<td>612-1684</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>11200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze substitution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>science services</td>
			<td>10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>EMS</td>
			<td>19100</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>SPI-Chem</td>
			<td>02624-AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>EMS</td>
			<td>10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide</td>
			<td>Sigma-Aldrich</td>
			<td>223220</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc CryoTubes</td>
			<td>Sigma-Aldrich</td>
			<td>V7884-450EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass dishes, 150 mm</td>
			<td>VWR</td>
			<td>216-2189H</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>0030 120.094</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan resin</td>
			<td>Sigma-Aldrich</td>
			<td>44610</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SEM stubs</td>
			<td>Science Services</td>
			<td>E75200</td>
			<td></td>
		</tr>
		<tr>
			<td>Aclar</td>
			<td>Science Services</td>
			<td>50425-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>filter paper</td>
			<td>VWR</td>
			<td>512-3618</td>
			<td></td>
		</tr>
		<tr>
			<td>conductive silver resin</td>
			<td>EMS</td>
			<td>12670-EE</td>
			<td>EPO-TEK EE 129-4</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition</td>
			<td>Zeiss</td>
			<td>SmartSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition</td>
			<td>Zeiss</td>
			<td>Atlas5 A3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing</td>
			<td>Open source</td>
			<td>Fiji</td>
			<td>http://fiji.sc/#download</td>
		</tr>
		<tr>
			<td>Image visualization</td>
			<td>Open source</td>
			<td>IMOD</td>
			<td>http://bio3d.colorado.edu/imod/</td>
		</tr>
	</tbody>
</table>

biological sample preparation by high-pressure freezing, microwave-assisted contrast enhancement, and minimal resin embedding for volume imaging

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

The workflow starts with a sample (here, a freshly dissected mouse nervus tibialis) being placed in metal carriers for high-pressure freezing (Figure 1a). The carriers are recovered from liquid nitrogen (Figure 1b) and placed in a freeze substitution unit on top of the frozen first chemical cocktail (Figure 1c). After a long freeze substitution protocol including 2% osmium tetroxide and 0.1% uranyl acetate, the samples are removed from the carriers at room temperature (Figure 1d). To further enhance the contrast, the samples are transferred to plastic tubes to be processed in the microwave (Figure 1e). The vacuum chamber and temperature control unit are used to optimize the process (Figure 1f).
To be able to perform the minimal resin embedding, toothpicks and sheets of plastic film are needed (Figure 2a). After infiltrating the samples with resin using the microwave, they are placed on pieces of plastic film and moved around until no resin is left on the sample surface. A halogen lamp is used to help drain the remaining resin and leave the sample minimally embedded (Figure 2b) on the plastic film. It should be noted that more resin removed from the top of the sample is good. There should still be a small amount left underneath the sample to keep it attached to the substrate. The sample being polymerized on the plastic film is cut and mounted on top of SEM stub with silver conductive resin (Figure 2c). The stub is polymerized for at least 4 h at 60 &#176;C (Figure 2d). The components should be mixed thoroughly or the mixture may not polymerize correctly. To avoid charging inside the scanning electron microscope, the stub is sputter coated with gold or platinum/palladium (Figure 2e).
The samples are placed in the FIB-SEM and imaged with a secondary electron detector to target the region of interest (Figure 3a,e). An ion beam is used to remove material directly in front of the region of interest to expose a cross section (Figure 3b-d, 3f-h). Standard protocols often suffer from a lack of membrane contrast (Figure 3b,f), whereas the enhanced protocol provides a strong membrane contrast (Figure 3c-d, 3g-h).
The EM data (after post-processing) are visualized using IMOD, an image processing and modelling program. To achieve a better understanding of the 3D information, virtual reslicing of the data is used (Figure 4a). Different structures of the dataset are segmented manually (Figure 4b-d).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59156/59156fig1.jpg" /> 
Figure 1: High-pressure freezing, freeze-substitution, and microwave-assisted processing. (a) Sample carrier containing the mouse nervus tibialis, scale bar 3 mm. (b) Sample carrier containing the mouse nervus tibialis after high-pressure freezing, scale bar 3 mm. (c) Automatic freeze substitution (AFS) unit with samples. Inset: custom-made metal container for up to 23 sample vials and two large vials containing chemicals in the AFS. (d) Samples being removed from carriers in a glass dish in acetone, scale bar 3 mm. (e) Samples in reaction tubes to be put in the microwave for processing. (f) Vacuum chamber and temperature control unit of the microwave. <a href="https://www.jove.com/files/ftp_upload/59156/59156fig1large.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/59156/59156fig2.jpg" /> 
Figure 2: Minimal resin embedding and preparation for the FIB-SEM. (a) Plastic film and toothpicks that are used for the minimal resin embedding, scale bar 4 cm. (b) Nervus tibialis drained of resin on top of the plastic film, scale bar 250 &#181;m. (c) Nervus tibialis polymerized on plastic film, then cut and mounted on top of the SEM stub with silver conductive resin, scale bar 250 &#181;m. (d) Samples polymerized on top of the SEM stub, scale bar 3 mm. (e) Samples coated with gold on the SEM stub, scale bar 3 mm. <a href="https://www.jove.com/files/ftp_upload/59156/59156fig2large.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/59156/59156fig3.jpg" /> 
Figure 3: Preparation of the sample inside the FIB-SEM. (a and e) Secondary electron image inside the FIB-SEM of the sample surface (a)&#160;Nervus tibialis, scale bar 100 &#181;m. (e) C. elegans, scale bar 2 &#181;m. (b-d) and (f-h) Cross-section through sample using ESB detector for imaging. (b and c) Nervus tibialis, scale bar 2 &#181;m. (f and g) C. elegans, scale bar 1 &#181;m and 200 nm. (b and f) Shown are the results of high-pressure freezing and freeze substitution without enhancement, whereas all other images show results of enhanced freeze substitution. (d and h) Detailed image of the sample using the ESB detector. (d) Nervus tibialis, scale bar 200 nm. (h) C. elegans, scale bar 200 nm. <a href="https://www.jove.com/files/ftp_upload/59156/59156fig3large.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/59156/59156fig4.jpg" /> 
Figure 4: Image acquisition and visualization. (a) EM data shown in IMOD with virtually resliced x/z- and y/z-planes, scale bar 2 &#181;m. (b) Segmented axons on EM data (blue), Remak bundles (red), myelin sheaths (yellow and orange), and mitochondria (turquoise), scale bar 2 &#181;m. (c and d) 3D model, scale bar 2 &#181;m and 500 nm. <a href="https://www.jove.com/files/ftp_upload/59156/59156fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging at JoVE.com

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

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

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

biological-sample-preparation-high-pressure-freezing-microwave

Research

JoVE Journal

Biology

10.3K Views.  Max Planck Institute of Experimental Medicine. Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

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

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.15-18 Therefore, significant effort has devoted to the synthesis and exploration of 18F-labeled biomolecules. Although there are elegant examples of the direct 18F-labeling of peptides,19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.23,24
N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB),25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the 18F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [18F]SFB, the resulting [18F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.1 Most [18F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [18F]SFB syntheses have been reported,29-32, 41-42 they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [18F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.
Herein, we report a concise preparation of [18F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.38 Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.28,43 The novelty of our improved [18F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [18F]SFB.

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques.

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...