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Serial block face-scanning electron microscopy for volume electron microscopy
CHAPTER
Serial block face-scanning electron microscopy for volume electron microscopy
4
Saskia Lippensa,b,c,*, Anna Kremera,b,c, Peter Borghgraefa,b,c, Christopher J. Guerina,b,c a
VIB BioImaging Core, VIB, Ghent, Belgium VIB Inflammation Research Center, VIB, Ghent, Belgium c Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium *Corresponding author: e-mail address: [email protected] b
Chapter outline 1 Introduction........................................................................................................70 2 Methods.............................................................................................................71 2.1 From tissue to block.............................................................................71 2.2 From block to sample mounting.............................................................75 2.3 Image acquisition.................................................................................76 2.4 Image visualization...............................................................................76 3 Materials and instrumentation..............................................................................77 3.1 From tissue to block.............................................................................77 3.2 From block to sample mounting.............................................................78 3.3 Image acquisition.................................................................................78 3.4 Image visualization...............................................................................78 4 Results...............................................................................................................78 5 Discussion..........................................................................................................82 Acknowledgments....................................................................................................83 References..............................................................................................................84 Further reading........................................................................................................85
Abstract There are different technologies that can be used to obtain a 3D image at nanometer resolution. Over the past decade, there has been a growing interest in applying Serial Block Face Scanning Electron Microscopy (SBF-SEM) in different fields of life science research. This technology has the advantage that it can cover a range of volumes, going from monolayers to multiple
Methods in Cell Biology, Volume 152, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.04.002 © 2019 Elsevier Inc. All rights reserved.
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tissue layers in all three dimensions. SBF-SEM was originally used in neuroscience and then expanded to other research domains. The whole process of sample preparation for SBF-SEM is very long and consists of many steps, which makes adjustment of a given workflow very challenging. Here we describe the SBF-SEM workflow and those steps in the process that can be tweaked for any sample.
1 Introduction Currently, there are different volume EM techniques available, from TEM tomography, serial sectioning followed by TEM/SEM imaging to the serial block-face imaging techniques using an ultramicrotome (SBF-SEM) or focused ion beam (FIB-SEM) (Heymann et al., 2006). All of these allow us to obtain a 3D view from a biological specimen at the nanoscale and have their specific advantages, but come with a compromise in the volume that can be handled, image resolution, requirements for sample preparation or labor intensity of the techniques. From this range of technologies, SBF-SEM has seen a great increase in implementation across different research labs and facilities (Smith & Starborg, 2018). This slice-and-view technique is based on removing the top layer of a given sample with a diamond-knife ultramicrotome inside the SEM vacuum chamber, then consecutive imaging of the block-face with an SEM. The very first use of an SEM with a miniature microtome inside the chamber was published in the 1980s (Leighton, 1981) and was further developed by Denk and Horstmann, who introduced a microtome in a SEM with variable pressure, which solved the problem of charging of the uncoated sample block during imaging (Denk & Horstmann, 2004). At this point, true automation of the acquisition process was possible due to improvements in computing and digital imaging and soon the technology became available as a commercial product from where it found its way into research laboratories and microscopy facilities. Initially, this technology was used in aid of neuroscience, where it was desired for neurons to be visualized in the context of an intact specimen of brain tissue. Neuronal processes can extend throughout a large volume of the tissue, so visualization of an entire cell requires a substantial tissue block to be imaged, preferentially with a fast and automated technique. In the mean time, the use of SBF-SEM has expanded outside the field of neuroscience and has successfully contributed to many domains in life science research (Kremer et al., 2015; Peddie & Collinson, 2014). It took time to broaden the applicability, because the general workflow is very long and consists of many steps, making it challenging to adapt to non-neuronal tissues. While SBF-SEM made the slice-and-view imaging technique faster and automated, the bottlenecks in the workflow shifted to the preceding sample preparation steps that require en bloc staining and to the data analysis of the huge and complex image stacks. For optimal image quality, it is essential to have conductive samples, thus providing contrast and avoiding charging artifacts. Both pitfalls are tackled by getting a large quantity of heavy metal staining into the sample block. For preparation of brain tissue,
2 Methods
an en bloc staining was established quite early on by Deerinck and colleagues (Deerinck, Bushong, Thor, & Ellisman, 2010), and most protocols that are in use for other tissues are modified versions of this original staining method. It is not possible to obtain a universal recipe, but certain details and an understanding of the imaging requirements can generally contribute to the establishment of optimized protocols. Here, we describe the different general steps of an SBF-SEM workflow for diverse biological samples and highlight the commonalities between established methods.
2 Methods 2.1 From tissue to block SBF-SEM was originally developed in the context of brain research, but it has subsequently been applied to a broader range of biological samples. This entailed numerous steps of optimization in the volume EM workflow. Although there will never be a universal recipe or a SBF-SEM working method that can be applied to all samples, there are certain general steps that apply to any sample type (Fig. 1). Basically, an entire workflow from sample to final 3D visualization consists of four major steps: (A) sample preparation from fixation to contrast staining and embedding in a resin block, (B) trimming and mounting of the block to prepare it for imaging, (C) image acquisition and (D) image processing and visualization. Sample preparation is the step that is most prone to adjustment, as it needs to be tailored for each tissue type and structure that has to be visualized. Most protocols have been derived from the Deerinck protocol that was customized for brain tissue (see Table 1). Frequently, when applying this protocol to another tissue, contrast is insufficient and sample conductivity is not optimal so steps that introduce more heavy metals into the sample need to be introduced. For any EM application, the initial fixation step is essential for good preservation of ultrastructure. A variation on classical Karnovsky fixative (Karnovsky & Deane, 1955) is most often used for chemical fixation of EM samples. When animal model systems are used, it is best practice to fix the animal by cardiac perfusion before dissection of the organ of interest. Before fixation, the animals are first perfused with physiological saline containing heparin (20 units/mL) for 2 min to remove blood from the vascular system and subsequently with fixation buffer for 12 min at a pressure that does not exceed normal mouse blood pressure (e.g., 100 mm Hg for mice). Buffer and fixative are used at the same body temperature as the animal (e.g., 37 °C for isothermic vertebrates). For more details and a diagram of the perfusion apparatus please see chapter “Combining serial block face and focused ion beam scanning electron microscopy for 3D studies of rare events” by Guerin et al. To increase sample contrast needed for efficient signal generation in SEM imaging standard SEM sample preparation protocols must be modified to introduce more heavy metals into the sample. The success of penetration of stains decreases with the size of the sample. Therefore, the samples are best dissected into smaller pieces with a scalpel or vibratome after initial fixation and left at 4 °C in the fixation solution overnight.
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A SAMPLE BLOCK PREPARATION GENERAL ADJUSTMENTS
BASIS PROTOCOL
SAMPLE SPECIFIC ADJUSTMENTS agarose embedding
Karnovsky fix
BioWave for incubation
En bloc contrast staining:
additional contrast steps UA
LS
propylene oxide
dehydration - aceton
different resin
embedding
e.g. viscosity
for lower
flat embedding
B SAMPLE BLOCK MOUNTING trim away empty resin conductive epoxy!
max. contact sample-pin
C IMAGE ACQUISITION ultramicrotome - FCC pin with sample
FCC nozzle
D IMAGE PROCESSING
volume compilation
image restoration
Visualisation
stack registration 3D stack compilation
denoising cropping image adjustment
segmentation rendering
FIG. 1 See legend on opposite page.
+ FCC
2 Methods
Table 1 En bloc staining and embedding protocol. Time
Incubation buffer
5 30 1h 5 30 200 5 30 300 5 30 ON 5 30 300 5 30 50 2 100 3 h to ON ON 2h Embed 48 h
0.1 M cacodylate pH 7.4 2% Reduced Osmium in 0.1 M cacodylate pH 7.4 Ultrapure water (UPW) 1% Thiocarbohydrazide (TCH) in UPW 0.1 M cacodylate 2% OsO4 in UPW UPW 2% uranyl acetate in UPW at 4 °C UPW Walton’s lead at 65 °C UPW Each 70%, 90%, 100%, 100% EtOH Propylene oxide 50% Spurr’s resin in propylene oxide 100% Spurr’s resin 100% Spurr’s resin Fresh Spurr’s resin At 65 °C
(1) 1 part 4% OsO4 + 1 part 3% ferricyanide in 0.2 M cacodylate pH 7.4; (2): 0.1 g TCH in UPW. 1 h in an oven at 60 °C. Shake ervery 100 . (3) or ON 1:3 uranyl acetate replacement (EMS) and UPW (4) add 0.998 g L-Aspartic acid to 250 mL UPW Adjust to pH 3.8 with KOH. Store at 4 °C. Add 0.066 g lead nitrate to 10 mL l-Aspartic Acid pH 3.8 and adjust to pH 5.5 with 0.1 M KOH. (5) Aceton in the original Deerinck protocol. (6) Durcupan in the original Deerinck protocol. Based on Deerinck, T.J., Bushong, E., Thor, A., and Ellisman, M.H. (2010). NCMIR methods for 3D EM: A new protocol for preparation of biological specimens for serial block-face SEM. Microscopy, 6–8; Deerinck, T.J., Shone, T.M., Bushong, E.A., Ramachandra, R., Peltier, S.T., and Ellisman, M.H. (2018). High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injectionbased charge compensation. Journal of Microscopy 270, 142–149.
FIG. 1 The SBF-SEM workflow. Four major steps can be distinguished in the SBF-SEM workflow: sample block preparation (A), sample block mounting (B), image acquisition (C) and image processing (D). (A) Sample preparation is the most prone to adjustments. Most protocols are variations on a basis protocol, developed by Deerinck et al. (2010). We have introduced general adjustments that we apply for most of our experiments. In addition to that different steps, such as fixation, contrast staining and resin infiltration can be tailored for specific samples. (B) Sample block mounting has requirements that are general for any sample type. (C) Although image acquisition always has to be adapted for each image stack, a general improvement for image quality is the use of an FCC. (D) Image processing can entail many different manipulations in order to compile a 3D stack, improve image quality by image restoration techniques and segmentation and volume rendering for final 3D visualization.
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The vast majority of SBF-SEM en bloc heavy metal stainings are based on the protocol developed by Deerinck and colleagues (Deerinck et al., 2010) for mouse brain tissue. In this OTO protocol, a post-fixation step with reduced Osmium is followed by a thiocarbohydrazide (TCH) incubation and a second Osmium step (Willingham & Rutherford, 1984). The TCH allows for amplification of the contrast that comes from the first Osmium, by binding to it and in turn allowing for additional binding of Osmium during the second step (Seligman, Wasserkrug, & Hanker, 1966). All of this is followed by additional incubations in uranyl acetate and lead aspartate (Fig. 1A). Upon dehydration, the samples are embedded in Durcupan. A more detailed description of the protocol that we use can be found in Table 1. A general change that was introduced in our staining protocols was the replacement of UA with Lanthanide Salts (LS), commercialized as Uranyl Acetate Replacement (EMS). After dehydration, the sample is infiltrated with Epoxy resin and again, based on the Deerinck OTO protocol, the use of Durcupan is quite widespread for SBF-SEM preparations. However, this formulation is rather viscous and for better penetration of certain tissues like heart, skin or plant we use the less viscous Spurr’s resin (Larsimont et al., 2015; Spurr, 1969; Vanslembrouck et al., 2018). Depending on the size of the tissue and importance of a specific orientation, we make use of different embedding molds. As little resin as possible around the overall sample is an advantage for the mounting in the later steps and to reduce charging artifacts. To achieve this, we often use a flat embedding method, as described above (Fig. 1A). In order to image the root tip of Arabidopsis thaliana with SBF-SEM, we have made adjustments to the protocol described in Tables 1 and 2. A first change that we introduced was the fixation buffer, which we adjusted to the lower pH of plant tissue. To accomplish this a Phosphate buffer at pH 6.8 containing 2% PFA and 3% glutaraldehyde was used. The fixative was poured on seedlings that were grown on agar plates (20 mL of fixative for a square 12 cm diameter Petri dish) and the plates were placed on a orbital shaking platform to maximize the penetration of the fixative. Plates were left on the orbital shaker for 2 h at RT (100 rpm). After fixation, we took precautions to avoid damage of the fragile sample during the numerous incubation and washing steps. Therefore, root tips were “enveloped” individually in low-melt agarose (Wu, Baskin, & Gallagher, 2012), as follows: 0.6% low-melt point agarose in H2O was pipetted (120 μL) onto a microscope slide and covered with a square 22 mm cover glass. After the agarose was solidified (at least 5 min), the cover glass was removed and 2–4 seedlings were placed on the agarose surface. A droplet of melted agarose (at just above gelling temperature) was added close to the root tip, on the roots and finally onto the root tip itself. After the agarose was solidified, a rectangle of agarose with the root tip was cut out with a sharp razor blade. From then on, the samples were further processed as root tip-agarose blocks during the remaining sample preparation procedure. After leaving the samples in fixative ON at 4 °C, the en bloc staining was performed. The different steps are listed in Table 2. Embedding was done with Spurr’s resin. For embedding we used a flat-embedding method, where we punch a hole in a 60mm 25 mm 1 mm thick silicon sheet, and use this as the mold that
2 Methods
Table 2 Sample preparation for Arabidopsis thaliana root tips. Time
Incubation buffer
5 30 1h 5 30 20’ 5 30 200 53 300 5 30 ON 5 30 300 5 30 50 2 300 3 h to ON ON 8h ON 8h Embed 48 h
0.1 M PB pH 6.8 2% Osmium, 0.2% ruthenium red in 0.1 M PB pH 6.8 Ultrapure water (UPW) 1% Thiocarbohydrazide (TCH) in UPW UPW 1% Thiocarbohydrazide (TCH) in UPW UPW 2% OsO4 in UPW UPW % uranyl acetate in UPW at 4 °C UPW Walton’s lead at 65 °C UPW Each 70%, 90%, 100%, 100% EtOH Propylene oxide 33% Spurr’s resin in propylene oxide 66% Spurr’s resin in propylene oxide 100% Spurr’s resin 100% Spurr’s resin 100% Spurr’s resin Fresh Spurr’s resin At 65 °C
is filled with Epoxy resin and the sample. This is then put between two sheets of Aclar with microscope glass slides on the outside. This “sandwich” is then fastened with spring clips and put in the oven for 48h until the resin is fully polymerized.
2.2 From block to sample mounting In the next stage, the sample is prepared for imaging by mounting the tissue containing resin block trimmed to a pyramid shape on an aluminum pin that fits the holder of the in-chamber ultramicrotome (Fig. 1B). This preparation is general for any type of sample but there are several procedures to follow. It is crucial to have as little resin as possible around the sample. For conductivity reasons it is important to assure that the bottom side of the block contains tissue that is in direct contact with the conductive epoxy and aluminum pin. If necessary, the block has to be back-trimmed to remove bare resin. First, the larger resin block is roughly trimmed with a fine jeweler’s saw or razor blades to a smaller cube of maximum 0.5 mm in any direction. This block is then
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glued onto the aluminum pin with conductive Epoxy resin, and positioned in the middle of the upper platform of the pin. The sample and pin are put in a 65 °C oven overnight to allow the conductive glue to harden. Second, the block is precision trimmed into a pyramid shape with side angles between 45° and 60°, using razor blades, an ultramicrotome or an automated trimming device. As mentioned before, it is essential to remove as much empty resin as possible to increase conductivity. The block-face is smoothed with a glass or diamond knife until the sample tissue is exposed at the surface. An additional coating of the sample block with a layer of 2–5 nm gold or platinum is helpful to improve conductivity on the outside of the block.
2.3 Image acquisition SBF-SEM makes use of imaging with a back-scattered electron (BSE) detector. As the electron beam scans over the block-face, electrons that are scattered back from beneath the sample surface will reach the detector. From each point, BSEs are collected for a certain amount of time (the dwell time) before the beam moves to the next position. As such, a block-face image is built up “point by point” and “line by line.” Once a 2D image of the surface is collected, the ultramicrotome will remove the top slice from the block, revealing a new surface face to be imaged. The image quality in terms of signal-to-noise ratio depends on the primary beam power and is positively correlated to the dwell time. However, with this technique it is an advantage to have the beam current and voltage as low as possible and use a short dwell time in order to decrease the effective Z depth of the information, thus improving the Z resolution. Secondary electrons produced by the primary beam will affect the image quality, because when they are detected by the BSE detector this results in charge artifacts in the image, or when they accumulate in the sample they can locally generate magnetic fields that distort the image. Even if all precautions are taken to maximize the conductivity of the sample, a compromise has to be made between increased resolution resulting from higher beam current/longer dwell times and image quality, in order to avoid the image degradation due to charging artifacts. Recently, a focal gas injection-based charge compensator (FCC) became available. This add-on, developed by Deerinck and colleagues, permits imaging of nonconductive biological samples at high column voltage and longer dwell times without charge artifacts (Deerinck et al., 2018). The technique is very straightforward, as nitrogen gas is locally applied through a nozzle in the chamber precisely over the block face during acquisition (Fig. 1C).
2.4 Image visualization Once a stack of images is recorded, multiple image processing manipulations can be performed (Fig. 1D). In order to obtain a volume dataset, it is necessary to compile the stack of 2D images into a 3D volume image. When doing this, it is also
3 Materials and instrumentation
important to correct for translational movement between two consecutive images in the stack by a process called image registration. It is then possible to apply image restoration techniques to improve the overall quality of the recorded images by either adjusting contrast or removing noise (Roels et al., 2018). To interpret the data set in 3D, it is necessary to perform segmentation, i.e., delineating specific structures of interest in the 3D volume. Segmentation allows for the visualization of individual cellular and subcellular objects and the rendering of their 3D structure (Belevich, Joensuu, Kumar, Vihinen, & Jokitalo, 2016; Cocks, Taggart, Rind, & White, 2018; Kreshuk et al., 2015).
3 Materials and instrumentation 3.1 From tissue to block Instrumentation Stereo microscope (Stemi 2000C, Zeiss) Vibratome (VT1200S, Leica) Vacuum oven (LabLine, ThermoFisher) Materials 0.2 M Cacodylate (sodium) buffer pH 7.4 (11652, EMS) 10% paraformaldehyde (15712, EMS) 25% glutaraldehyde (G-5882, Sigma) Karnovsky fixative: 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4, Reduced Osmium: 1 part 4% OsO4 (19150, EMS) + 1 part 3% ferricyanide in 0.2 M cacodylate pH 7.4 1% Thiocarbohydrazide: dissolve 0.1 g TCH (21900, EMS) in UPWIncubate 1 h in an oven at 60 °C. Shake every 100 Osmium (19150, EMS) Uranyl acetate (EMS) Uranyl Acetate Replacement solution: 1:3 uranyl acetate replacement (22405, EMS) and UPW Walton’s Lead Aspartate staining solution: Prepare L-Aspartic acid solution by dissolving 0.998 g L-Aspartic acid (A8949, Sigma) in 250 mL UPW. Adjust to pH 3.8 with KOH. Dissolve 0.066 g Lead Nitrate (07905CJ, Aldrich) in 10 mL L-Aspartic acid pH 3.8. Adjust to pH 5.5 with 1 N KOH. Check pH with pH indicator strip (109584.1111, Millipore) Ethanol (1.00983, Merck) Propylene oxide (82320, Sigma) Durcupan (Sigma) Spurr’s resin (EMS) Low-melt agarose (A4018, Sigma)
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3.2 From block to sample mounting Instrumentation Ultramicrotome (Ultracut, Riechert-Jung) Sputter Coater (Q150T ES, Quorum) Materials Aluminum pin (Gatan) Conductive epoxy (Chemtronics)
3.3 Image acquisition Instrumentation Merlin SEM (Zeiss) with 3View (Gatan) and Focal Charge Compensator (Zeiss)
3.4 Image visualization Fiji, https://fiji.sc MIB, http://mib.helsinki.fi Imaris, https://www.bitplane.com
4 Results We optimized an en bloc staining for Arabidopsis thaliana root tips, initially based on the protocol described in Table 1 (Fendrych et al., 2014). However, many adjustments were required to tailor the sample preparation for this type of plant specimen. Our detailed protocol is explained step-by-step in Table 2. A first change that was introduced is the pH of the fixation, from classical Karnovsky pH 7.4 to 6.8 to more closely match the pH value of the buffer to that of the specimen. Because the sample is fragile, manipulation of the root tip became easier by enveloping it in low-melt agarose. It is an additional advantage that the agarose also binds osmium and therefore the empty resin around the root tip is less prone to electron charging. To increase contrast in the sample itself, we added Ruthenium Red at the beginning of the en bloc staining, and an additional round of TCH and osmium staining. Mounting of the sample and trimming of the block was done with an orientation of the root tip upward so that the slice-and-view procedure was perpendicular to the growth axis. About 2000 images were acquired with a slice thickness set to 70 nm (Fig. 2A). Images were registered using Fiji and orthogonal viewing was done by making use of Imaris (Fig. 2B). For a selected region, we applied intensity thresholding in order to segment the electron dense structures, in this case the plasmodesmata (Fig. 2C; Supplementary Movie 1 in the online version at https://doi.org/10.1016/ bs.mcb.2019.04.002).
A
B
C
FIG. 2 SBF-SEM imaging of Arabidopsis thaliana root tips. An Arabidopsis thaliana root tip was prepared for imaging with SBF-SEM. (A) A single 2D image from the recorded stack is shown. (B) After registration of the different 2D images and compilation in one volume dataset, representation with orthogonal views is shown. (C) A region of interest was cropped from the original dataset and intensity thresholding was applied, in Fiji. We used the Fiji 3DViewer for rendering, showing two cells with plasmodesmata in the cell wall between them.
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The protocol for Arabidopsis root tips (Table 2) differs substantially from the protocol described in Table 1 and these adjustments are specific for this sample type. For any other tissues optimizing the sample preparation may be required. However, we also introduced modifications that we now generally use in the workflows for SBFSEM in our facility. Specifically, we have tested replacement of UA with Lanthanide salts (Uranyl Acetate Replacement) for reasons of lab safety. In Fig. 3A and B we show an SBF-SEM image of mouse brain tissue that was prepared according to the protocol given in Table 1 using either LS or UA, respectively. We have observed that the contrast with LS is similar to that with UA and have generally switched to using LS instead of UA for our working protocols.
A
C
B
D
E
FIG. 3 General modifications for SBF-SEM sample preparation. Mouse brain tissue was prepared for SBF-SEM, making use of the protocol described in Table 1 with either Lantanide Salts (A) or Uranyl Acetate (B). Single 2D image from an SBF-SEM dataset with LS or UA, respectively. Mouse liver tissue was prepared according to the protocols in Table 3. (C) Single 2D image from an SBF-SEM dataset, making use of sample that was prepared with incubations on the bench. (D) Sample prepared with BioWave incubation. (E) Sample with BioWave incubation and with FCC for imaging.
4 Results
The en bloc stainings described in Tables 1 and 2 can easily take up multiple days. The use of a microwave to assist sample preparation may substantially speed up the process because of the increased speed of reagent penetration and thus shorter incubation times. Therefore, we applied the same staining protocol to prepare mouse liver tissue by either incubation on the bench or using the microwave (see Table 3). An image of the sample preparation without microwave use is shown in Fig. 3C and with microwave use in Fig. 3D. We noticed that due to electron charging of bare resin in a block, SBF-SEM has been difficult to apply to tissues that contain non-cytoplasmic areas, such as plant vacuoles, the alveoli of lungs, or cultured cell monolayers that are surrounded by bare resin. An improvement was achieved when the focal charge compensator, developed by Deerinck and colleagues became available (Deerinck et al., 2018). We have also applied the use of the FCC on microwave processed liver tissue and noticed substantial improvements in the image quality. We no longer observed charging Table 3 En bloc contrast staining protocol for mouse liver tissue with either bench or microwave incubation steps. Bench incubation
Incubation buffer
BioWave step
5 30
0.1 M cacodylate pH 7.4 Reduced Osmium in 0.1 M cacodylate pH 7.4 Ultrapure water (UPW)
200
1% Thiocarbohydrazide (TCH) in UPW
5 30
UPW
300
2% OsO4 in UPW (bench) 1% OsO4 in UPW (BioWave) UPW
2 4000 250 W, Vacuum Off 5 20 alternating 100 W and power Off, Vacuum On 2 50 at bench + 2 4000 250 W, Vacuum Off 20 at bench + 3 1’ alternating 150 W and power Off, Vacuum On 2 50 at bench + 2 4000 250 W, Vacuum Off 5 20 alternating 100 W and power Off, Vacuum On 2 50 at bench + 2 4000 250 W, Vacuum Off 7 10 alternating 150 W and power Off, Vacuum On 2 50 at bench + 2 4000 250 W, Vacuum Off 300 2 50 at bench + 2 4000 250 W, Vacuum Off Each 4000 150 W, Vacuum Off
5 30 1h
5 30
5 30
2% uranyl acetate (UA) in UPW at 4 °C (bench) 1% UA in UPW (BioWave) UPW
300 5 30
Walton’s lead at 65 °C UPW
50
Each 70%, 90%, 100%, 100% EtOH (bench) 50%, 70%, 90%, 100%, 100% EtOH (BioWave)
ON
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artifacts even at relatively high accelerating voltages (Fig. 3E). In addition, the general image quality was also improved in terms of increased signal to noise ratio and throughout an image stack we noticed less translational movement between two images, which can make the image registration step unnecessary.
5 Discussion SBF-SEM has become an established technique to gather volume data on various biological samples. Initially, the neuroscience community was the main beneficiary of this technology, but in the recent years SBF-SEM has proven its value in many different areas of life science research; e.g., inflammation, cardiology, botany, etc. The great advantage of the technology is the volume range that can be imaged. However, the workflow for SBF-SEM is a long and multistep process. While the acquisition has become more efficient because of the automation of the slice-and-view process, the initial focus for optimization was on obtaining high quality images, determined by contrast and good cutting of the sample block. Clearly, sample preparation is the determining factor here and by the turn of the century reliable en bloc heavy metal staining protocols for brain tissue were distributed within the volume EM community. The “Deerinck protocol” was used by several labs and facilities after the implementation of the technology and tailored for diverse sample types (Deerinck et al., 2010). Further improvements of the SBF-SEM method arose from adjustments of sample preparation procedures. The Deerinck protocol works very well for lipid-rich tissues, such as brain, and based on this, variations have been worked out in order to apply the en bloc contrast staining to other samples. For instance, the use of tannic acid and ruthenium red was useful for adding contrast to specific structures like the plant cell wall, collagen or desmosomes (Fendrych et al., 2014; Starborg et al., 2013; Vanslembrouck et al., 2018). Others have explored more selective stainings in order to localize specific structures. For instance, the 3D visualization of ER in animal cells and plant samples has been achieved by staining with Horse Radish Peroxidase or Zink Iodide (Kittelmann, Hawes, & Hughes, 2016; Puhka, Joensuu, Vihinen, Belevich, & Jokitalo, 2012). Additional variations came about by necessity, e.g., certain labs had to avoid the use of uranyl acetate and therefore permanently switched to Lanthanide salts. All of the many improvements, usually worked out empirically, have contributed to the growing use of SBF-SEM in life science research. In addition to these changes in staining of the samples improvements in sample handling have been made. The use of agarose or gelatin embedding for protecting fragile samples or encasing pellets of cells (immune cells, yeast, bacteria) allows for the processing of these samples as blocks to prevent damage during sample manipulation. As far as fixation of the samples is concerned, the use of highpressure freezing, which may preserve samples in a more natural morphological state due to rapid fixation, has been explored (Webb & Schieber, 2018).
Acknowledgments
Webb and Webb also introduced microwave-assisted processing in their working protocols for shortening lengthy sample preparation times (Webb & Webb, 2015). To gather more functional information, certain labs and facilities have also explored the use of SBF-SEM in correlated light and electron microscopy (CLEM). Armer and colleagues performed CLEM with live confocal imaging and SBF-SEM to study blood vessel fusion in Zebrafish (Armer et al., 2009). Others have worked out experiments with Near Infra Red Branding as a method to introduce landmarks to mark a region interest in a sample that was first imaged with high resolution confocal microscopy, then with SBF-SEM (Bishop et al., 2011; Lees, Peddie, Collinson, Ashby, & Verkade, 2017). A very elegant solution to combine fluorescent imaging with SBF-SEM, is the introduction of a miniature fluorescence microscope inside the chamber of the SBF-SEM (Brama et al., 2016). Further improvements in preparation and imaging will make SBF-SEM more efficient and widespread. Since its beginnings the issues of electron charging of bare resin in samples had been a drawback to the use of SBF-SEM at high accelerating voltages or for imaging of tissues with cavities such as vacuoles or blood vessels, samples that are low in lipids, or tissues that are difficult to stain with heavy metal dyes. This was tackled by the development of the Focal Charge Compensator (Deerinck et al., 2018). Both improvements now allow laboratories to get the best out of the technology, because all artifacts due to electron charging are minimized and the image quality is significantly improved. Even samples with low contrast, like archived blocks prepared for standard TEM imaging, can now be used for SBF-SEM. The next issue that is now being addressed is how to handle the data that are gathered with this technique. Size and complexity of data sets are preventing ease of analysis. Several solutions from other fields, such as computer assisted image recognition, are currently being explored. Also solutions from within the volume EM community or through collaborations with image analysis specialists have produced optimized tools for segmentation and visualization of volume EM data (Belevich et al., 2016; Luengo et al., 2017). Alongside that, repositories for data and annotations are being set up and the community is discussing quality standards for SBF-SEM images. It is clear that more and more technical solutions and expertise in SBFSEM will help to turn this technique into a well-established and accessible tool for life science research. The ability to image large samples in 3D at the nanoscale will deliver a rich source of morphological information to combine with functional and molecular scale information.
Acknowledgments The authors would like to thank the VIB Core Facilities program, VIB Inflammation Research Center, VIB Technology Fund and a generous Grant from minister Ingrid Lieten of the government of Flanders for support in implementing the SBF-SEM technology. We are grateful to Matyas Fendrych and Moritz Nowack for the data on Arabidopsis thaliana and their input for data visualization.
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References Armer, H. E., Mariggi, G., Png, K. M., Genoud, C., Monteith, A. G., Bushby, A. J., et al. (2009). Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy. PLoS One, 4, e7716. Belevich, I., Joensuu, M., Kumar, D., Vihinen, H., & Jokitalo, E. (2016). Microscopy image browser: A platform for segmentation and analysis of multidimensional datasets. PLoS Biology, 14, e1002340. Bishop, D., Nikic, I., Brinkoetter, M., Knecht, S., Potz, S., Kerschensteiner, M., et al. (2011). Near-infrared branding efficiently correlates light and electron microscopy. Nature Methods, 8, 568–570. Brama, E., Peddie, C. J., Wilkes, G., Gu, Y., Collinson, L. M., & Jones, M. L. (2016). ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy. Wellcome Open Research, 1, 26. Cocks, E., Taggart, M., Rind, F. C., & White, K. (2018). A guide to analysis and reconstruction of serial block face scanning electron microscopy data. Journal of Microscopy, 270, 217–234. Deerinck, T. J., Bushong, E., Thor, A., & Ellisman, M. H. (2010). NCMIR methods for 3D EM: A new protocol for preparation of biological specimens for serial block-face SEM. Microscopy, 6–8. Deerinck, T. J., Shone, T. M., Bushong, E. A., Ramachandra, R., Peltier, S. T., & Ellisman, M. H. (2018). High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation. Journal of Microscopy, 270, 142–149. Denk, W., & Horstmann, H. (2004). Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biology, 2, e329. Fendrych, M., Van Hautegem, T., Van Durme, M., Olvera-Carrillo, Y., Huysmans, M., Karimi, M., et al. (2014). Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis. Current Biology, 24, 931–940. Heymann, J. A., Hayles, M., Gestmann, I., Giannuzzi, L. A., Lich, B., & Subramaniam, S. (2006). Site-specific 3D imaging of cells and tissues with a dual beam microscope. Journal of Structural Biology, 155, 63–73. Karnovsky, M. L., & Deane, H. W. (1955). Aldehyde formation in the lipide droplets of the adrenal cortex during fixation, as demonstrated chemically and histochemically. The Journal of Histochemistry and Cytochemistry, 3, 85–102. Kittelmann, M., Hawes, C., & Hughes, L. (2016). Serial block face scanning electron microscopy and the reconstruction of plant cell membrane systems. Journal of Microscopy, 263, 200–211. Kremer, A., Lippens, S., Bartunkova, S., Asselbergh, B., Blanpain, C., Fendrych, M., et al. (2015). Developing 3D SEM in a broad biological context. Journal of Microscopy, 259, 80–96. Kreshuk, A., Walecki, R., Koethe, U., Gierthmuehlen, M., Plachta, D., Genoud, C., et al. (2015). Automated tracing of myelinated axons and detection of the nodes of Ranvier in serial images of peripheral nerves. Journal of Microscopy, 259, 143–154. Larsimont, J. C., Youssef, K. K., Sanchez-Danes, A., Sukumaran, V., Defrance, M., Delatte, B., et al. (2015). Sox9 controls self-renewal of oncogene targeted cells and links tumor initiation and invasion. Cell Stem Cell, 17, 60–73. Lees, R. M., Peddie, C. J., Collinson, L. M., Ashby, M. C., & Verkade, P. (2017). Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps. In Vol. 140. Correlative light and electron microscopy III (pp. 245–276). Academic Press.
Further reading
Leighton, S. B. (1981). SEM images of block faces, cut by a miniature microtome within the SEM—A technical note. Scanning Electron Microscopy, 73–76. Luengo, I., Darrow, M. C., Spink, M. C., Sun, Y., Dai, W., He, C. Y., et al. (2017). SuRVoS: Super-region volume segmentation workbench. Journal of Structural Biology, 198, 43–53. Peddie, C. J., & Collinson, L. M. (2014). Exploring the third dimension: Volume electron microscopy comes of age. Micron, 61, 9–19. Puhka, M., Joensuu, M., Vihinen, H., Belevich, I., & Jokitalo, E. (2012). Progressive sheet-totubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells. Molecular Biology of the Cell, 23, 2424–2432. Roels, J., Aelterman, J., Luong, H. Q., Lippens, S., Pizurica, A., Saeys, Y., et al. (2018). An overview of state-of-the-art image restoration in electron microscopy. Journal of Microscopy, 271, 239–254. Seligman, A. M., Wasserkrug, H. L., & Hanker, J. S. (1966). A new staining method (OTO) for enhancing contrast of lipid-containing membranes and droplets in osmium tetroxide-fixed tissue with osmiophilic thiocarbohydrazide(TCH). The Journal of Cell Biology, 30, 424–432. Smith, D., & Starborg, T. (2018). Serial block face scanning electron microscopy in cell biology: Applications and technology. Tissue and Cell, 57, 111–122. Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research, 26, 31–43. Starborg, T., Kalson, N. S., Lu, Y., Mironov, A., Cootes, T. F., Holmes, D. F., et al. (2013). Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization. Nature Protocols, 8, 1433–1448. Vanslembrouck, B., Kremer, A., Pavie, B., van Roy, F., Lippens, S., & van Hengel, J. (2018). Three-dimensional reconstruction of the intercalated disc including the intercellular junctions by applying volume scanning electron microscopy. Histochemistry and Cell Biology, 149, 479–490. Webb, R. I., & Schieber, N. L. (2018). Volume scanning electron microscopy: Serial block-face scanning electron microscopy focussed ion beam scanning electron microscopy. In Cellular imaging (pp. 117–148). Springer. Webb, R., & Webb, R. (2015). Quick freeze substitution processing of biological samples for serial block-face scanning electron microscopy. Microscopy and Microanalysis, 21, 1115–1116. Willingham, M. C., & Rutherford, A. V. (1984). The use of osmium-thiocarbohydrazideosmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells. The Journal of Histochemistry and Cytochemistry, 32, 455–460. Wu, S., Baskin, T. I., & Gallagher, K. L. (2012). Mechanical fixation techniques for processing and orienting delicate samples, such as the root of Arabidopsis thaliana, for light or electron microscopy. Nature Protocols, 7, 1113–1124.
Further reading Urwyler, O., Izadifar, A., Dascenco, D., Petrovic, M., He, H., Ayaz, D., et al. (2015). Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy. Development, 142, 394–405.
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4
Saskia Lippensa,b,c,*, Anna Kremera,b,c, Peter Borghgraefa,b,c, Christopher J. Guerina,b,c a
VIB BioImaging Core, VIB, Ghent, Belgium VIB Inflammation Research Center, VIB, Ghent, Belgium c Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium *Corresponding author: e-mail address: [email protected] b
Chapter outline 1 Introduction........................................................................................................70 2 Methods.............................................................................................................71 2.1 From tissue to block.............................................................................71 2.2 From block to sample mounting.............................................................75 2.3 Image acquisition.................................................................................76 2.4 Image visualization...............................................................................76 3 Materials and instrumentation..............................................................................77 3.1 From tissue to block.............................................................................77 3.2 From block to sample mounting.............................................................78 3.3 Image acquisition.................................................................................78 3.4 Image visualization...............................................................................78 4 Results...............................................................................................................78 5 Discussion..........................................................................................................82 Acknowledgments....................................................................................................83 References..............................................................................................................84 Further reading........................................................................................................85
Abstract There are different technologies that can be used to obtain a 3D image at nanometer resolution. Over the past decade, there has been a growing interest in applying Serial Block Face Scanning Electron Microscopy (SBF-SEM) in different fields of life science research. This technology has the advantage that it can cover a range of volumes, going from monolayers to multiple
Methods in Cell Biology, Volume 152, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.04.002 © 2019 Elsevier Inc. All rights reserved.
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tissue layers in all three dimensions. SBF-SEM was originally used in neuroscience and then expanded to other research domains. The whole process of sample preparation for SBF-SEM is very long and consists of many steps, which makes adjustment of a given workflow very challenging. Here we describe the SBF-SEM workflow and those steps in the process that can be tweaked for any sample.
1 Introduction Currently, there are different volume EM techniques available, from TEM tomography, serial sectioning followed by TEM/SEM imaging to the serial block-face imaging techniques using an ultramicrotome (SBF-SEM) or focused ion beam (FIB-SEM) (Heymann et al., 2006). All of these allow us to obtain a 3D view from a biological specimen at the nanoscale and have their specific advantages, but come with a compromise in the volume that can be handled, image resolution, requirements for sample preparation or labor intensity of the techniques. From this range of technologies, SBF-SEM has seen a great increase in implementation across different research labs and facilities (Smith & Starborg, 2018). This slice-and-view technique is based on removing the top layer of a given sample with a diamond-knife ultramicrotome inside the SEM vacuum chamber, then consecutive imaging of the block-face with an SEM. The very first use of an SEM with a miniature microtome inside the chamber was published in the 1980s (Leighton, 1981) and was further developed by Denk and Horstmann, who introduced a microtome in a SEM with variable pressure, which solved the problem of charging of the uncoated sample block during imaging (Denk & Horstmann, 2004). At this point, true automation of the acquisition process was possible due to improvements in computing and digital imaging and soon the technology became available as a commercial product from where it found its way into research laboratories and microscopy facilities. Initially, this technology was used in aid of neuroscience, where it was desired for neurons to be visualized in the context of an intact specimen of brain tissue. Neuronal processes can extend throughout a large volume of the tissue, so visualization of an entire cell requires a substantial tissue block to be imaged, preferentially with a fast and automated technique. In the mean time, the use of SBF-SEM has expanded outside the field of neuroscience and has successfully contributed to many domains in life science research (Kremer et al., 2015; Peddie & Collinson, 2014). It took time to broaden the applicability, because the general workflow is very long and consists of many steps, making it challenging to adapt to non-neuronal tissues. While SBF-SEM made the slice-and-view imaging technique faster and automated, the bottlenecks in the workflow shifted to the preceding sample preparation steps that require en bloc staining and to the data analysis of the huge and complex image stacks. For optimal image quality, it is essential to have conductive samples, thus providing contrast and avoiding charging artifacts. Both pitfalls are tackled by getting a large quantity of heavy metal staining into the sample block. For preparation of brain tissue,
2 Methods
an en bloc staining was established quite early on by Deerinck and colleagues (Deerinck, Bushong, Thor, & Ellisman, 2010), and most protocols that are in use for other tissues are modified versions of this original staining method. It is not possible to obtain a universal recipe, but certain details and an understanding of the imaging requirements can generally contribute to the establishment of optimized protocols. Here, we describe the different general steps of an SBF-SEM workflow for diverse biological samples and highlight the commonalities between established methods.
2 Methods 2.1 From tissue to block SBF-SEM was originally developed in the context of brain research, but it has subsequently been applied to a broader range of biological samples. This entailed numerous steps of optimization in the volume EM workflow. Although there will never be a universal recipe or a SBF-SEM working method that can be applied to all samples, there are certain general steps that apply to any sample type (Fig. 1). Basically, an entire workflow from sample to final 3D visualization consists of four major steps: (A) sample preparation from fixation to contrast staining and embedding in a resin block, (B) trimming and mounting of the block to prepare it for imaging, (C) image acquisition and (D) image processing and visualization. Sample preparation is the step that is most prone to adjustment, as it needs to be tailored for each tissue type and structure that has to be visualized. Most protocols have been derived from the Deerinck protocol that was customized for brain tissue (see Table 1). Frequently, when applying this protocol to another tissue, contrast is insufficient and sample conductivity is not optimal so steps that introduce more heavy metals into the sample need to be introduced. For any EM application, the initial fixation step is essential for good preservation of ultrastructure. A variation on classical Karnovsky fixative (Karnovsky & Deane, 1955) is most often used for chemical fixation of EM samples. When animal model systems are used, it is best practice to fix the animal by cardiac perfusion before dissection of the organ of interest. Before fixation, the animals are first perfused with physiological saline containing heparin (20 units/mL) for 2 min to remove blood from the vascular system and subsequently with fixation buffer for 12 min at a pressure that does not exceed normal mouse blood pressure (e.g., 100 mm Hg for mice). Buffer and fixative are used at the same body temperature as the animal (e.g., 37 °C for isothermic vertebrates). For more details and a diagram of the perfusion apparatus please see chapter “Combining serial block face and focused ion beam scanning electron microscopy for 3D studies of rare events” by Guerin et al. To increase sample contrast needed for efficient signal generation in SEM imaging standard SEM sample preparation protocols must be modified to introduce more heavy metals into the sample. The success of penetration of stains decreases with the size of the sample. Therefore, the samples are best dissected into smaller pieces with a scalpel or vibratome after initial fixation and left at 4 °C in the fixation solution overnight.
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A SAMPLE BLOCK PREPARATION GENERAL ADJUSTMENTS
BASIS PROTOCOL
SAMPLE SPECIFIC ADJUSTMENTS agarose embedding
Karnovsky fix
BioWave for incubation
En bloc contrast staining:
additional contrast steps UA
LS
propylene oxide
dehydration - aceton
different resin
embedding
e.g. viscosity
for lower
flat embedding
B SAMPLE BLOCK MOUNTING trim away empty resin conductive epoxy!
max. contact sample-pin
C IMAGE ACQUISITION ultramicrotome - FCC pin with sample
FCC nozzle
D IMAGE PROCESSING
volume compilation
image restoration
Visualisation
stack registration 3D stack compilation
denoising cropping image adjustment
segmentation rendering
FIG. 1 See legend on opposite page.
+ FCC
2 Methods
Table 1 En bloc staining and embedding protocol. Time
Incubation buffer
5 30 1h 5 30 200 5 30 300 5 30 ON 5 30 300 5 30 50 2 100 3 h to ON ON 2h Embed 48 h
0.1 M cacodylate pH 7.4 2% Reduced Osmium in 0.1 M cacodylate pH 7.4 Ultrapure water (UPW) 1% Thiocarbohydrazide (TCH) in UPW 0.1 M cacodylate 2% OsO4 in UPW UPW 2% uranyl acetate in UPW at 4 °C UPW Walton’s lead at 65 °C UPW Each 70%, 90%, 100%, 100% EtOH Propylene oxide 50% Spurr’s resin in propylene oxide 100% Spurr’s resin 100% Spurr’s resin Fresh Spurr’s resin At 65 °C
(1) 1 part 4% OsO4 + 1 part 3% ferricyanide in 0.2 M cacodylate pH 7.4; (2): 0.1 g TCH in UPW. 1 h in an oven at 60 °C. Shake ervery 100 . (3) or ON 1:3 uranyl acetate replacement (EMS) and UPW (4) add 0.998 g L-Aspartic acid to 250 mL UPW Adjust to pH 3.8 with KOH. Store at 4 °C. Add 0.066 g lead nitrate to 10 mL l-Aspartic Acid pH 3.8 and adjust to pH 5.5 with 0.1 M KOH. (5) Aceton in the original Deerinck protocol. (6) Durcupan in the original Deerinck protocol. Based on Deerinck, T.J., Bushong, E., Thor, A., and Ellisman, M.H. (2010). NCMIR methods for 3D EM: A new protocol for preparation of biological specimens for serial block-face SEM. Microscopy, 6–8; Deerinck, T.J., Shone, T.M., Bushong, E.A., Ramachandra, R., Peltier, S.T., and Ellisman, M.H. (2018). High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injectionbased charge compensation. Journal of Microscopy 270, 142–149.
FIG. 1 The SBF-SEM workflow. Four major steps can be distinguished in the SBF-SEM workflow: sample block preparation (A), sample block mounting (B), image acquisition (C) and image processing (D). (A) Sample preparation is the most prone to adjustments. Most protocols are variations on a basis protocol, developed by Deerinck et al. (2010). We have introduced general adjustments that we apply for most of our experiments. In addition to that different steps, such as fixation, contrast staining and resin infiltration can be tailored for specific samples. (B) Sample block mounting has requirements that are general for any sample type. (C) Although image acquisition always has to be adapted for each image stack, a general improvement for image quality is the use of an FCC. (D) Image processing can entail many different manipulations in order to compile a 3D stack, improve image quality by image restoration techniques and segmentation and volume rendering for final 3D visualization.
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The vast majority of SBF-SEM en bloc heavy metal stainings are based on the protocol developed by Deerinck and colleagues (Deerinck et al., 2010) for mouse brain tissue. In this OTO protocol, a post-fixation step with reduced Osmium is followed by a thiocarbohydrazide (TCH) incubation and a second Osmium step (Willingham & Rutherford, 1984). The TCH allows for amplification of the contrast that comes from the first Osmium, by binding to it and in turn allowing for additional binding of Osmium during the second step (Seligman, Wasserkrug, & Hanker, 1966). All of this is followed by additional incubations in uranyl acetate and lead aspartate (Fig. 1A). Upon dehydration, the samples are embedded in Durcupan. A more detailed description of the protocol that we use can be found in Table 1. A general change that was introduced in our staining protocols was the replacement of UA with Lanthanide Salts (LS), commercialized as Uranyl Acetate Replacement (EMS). After dehydration, the sample is infiltrated with Epoxy resin and again, based on the Deerinck OTO protocol, the use of Durcupan is quite widespread for SBF-SEM preparations. However, this formulation is rather viscous and for better penetration of certain tissues like heart, skin or plant we use the less viscous Spurr’s resin (Larsimont et al., 2015; Spurr, 1969; Vanslembrouck et al., 2018). Depending on the size of the tissue and importance of a specific orientation, we make use of different embedding molds. As little resin as possible around the overall sample is an advantage for the mounting in the later steps and to reduce charging artifacts. To achieve this, we often use a flat embedding method, as described above (Fig. 1A). In order to image the root tip of Arabidopsis thaliana with SBF-SEM, we have made adjustments to the protocol described in Tables 1 and 2. A first change that we introduced was the fixation buffer, which we adjusted to the lower pH of plant tissue. To accomplish this a Phosphate buffer at pH 6.8 containing 2% PFA and 3% glutaraldehyde was used. The fixative was poured on seedlings that were grown on agar plates (20 mL of fixative for a square 12 cm diameter Petri dish) and the plates were placed on a orbital shaking platform to maximize the penetration of the fixative. Plates were left on the orbital shaker for 2 h at RT (100 rpm). After fixation, we took precautions to avoid damage of the fragile sample during the numerous incubation and washing steps. Therefore, root tips were “enveloped” individually in low-melt agarose (Wu, Baskin, & Gallagher, 2012), as follows: 0.6% low-melt point agarose in H2O was pipetted (120 μL) onto a microscope slide and covered with a square 22 mm cover glass. After the agarose was solidified (at least 5 min), the cover glass was removed and 2–4 seedlings were placed on the agarose surface. A droplet of melted agarose (at just above gelling temperature) was added close to the root tip, on the roots and finally onto the root tip itself. After the agarose was solidified, a rectangle of agarose with the root tip was cut out with a sharp razor blade. From then on, the samples were further processed as root tip-agarose blocks during the remaining sample preparation procedure. After leaving the samples in fixative ON at 4 °C, the en bloc staining was performed. The different steps are listed in Table 2. Embedding was done with Spurr’s resin. For embedding we used a flat-embedding method, where we punch a hole in a 60mm 25 mm 1 mm thick silicon sheet, and use this as the mold that
2 Methods
Table 2 Sample preparation for Arabidopsis thaliana root tips. Time
Incubation buffer
5 30 1h 5 30 20’ 5 30 200 53 300 5 30 ON 5 30 300 5 30 50 2 300 3 h to ON ON 8h ON 8h Embed 48 h
0.1 M PB pH 6.8 2% Osmium, 0.2% ruthenium red in 0.1 M PB pH 6.8 Ultrapure water (UPW) 1% Thiocarbohydrazide (TCH) in UPW UPW 1% Thiocarbohydrazide (TCH) in UPW UPW 2% OsO4 in UPW UPW % uranyl acetate in UPW at 4 °C UPW Walton’s lead at 65 °C UPW Each 70%, 90%, 100%, 100% EtOH Propylene oxide 33% Spurr’s resin in propylene oxide 66% Spurr’s resin in propylene oxide 100% Spurr’s resin 100% Spurr’s resin 100% Spurr’s resin Fresh Spurr’s resin At 65 °C
is filled with Epoxy resin and the sample. This is then put between two sheets of Aclar with microscope glass slides on the outside. This “sandwich” is then fastened with spring clips and put in the oven for 48h until the resin is fully polymerized.
2.2 From block to sample mounting In the next stage, the sample is prepared for imaging by mounting the tissue containing resin block trimmed to a pyramid shape on an aluminum pin that fits the holder of the in-chamber ultramicrotome (Fig. 1B). This preparation is general for any type of sample but there are several procedures to follow. It is crucial to have as little resin as possible around the sample. For conductivity reasons it is important to assure that the bottom side of the block contains tissue that is in direct contact with the conductive epoxy and aluminum pin. If necessary, the block has to be back-trimmed to remove bare resin. First, the larger resin block is roughly trimmed with a fine jeweler’s saw or razor blades to a smaller cube of maximum 0.5 mm in any direction. This block is then
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glued onto the aluminum pin with conductive Epoxy resin, and positioned in the middle of the upper platform of the pin. The sample and pin are put in a 65 °C oven overnight to allow the conductive glue to harden. Second, the block is precision trimmed into a pyramid shape with side angles between 45° and 60°, using razor blades, an ultramicrotome or an automated trimming device. As mentioned before, it is essential to remove as much empty resin as possible to increase conductivity. The block-face is smoothed with a glass or diamond knife until the sample tissue is exposed at the surface. An additional coating of the sample block with a layer of 2–5 nm gold or platinum is helpful to improve conductivity on the outside of the block.
2.3 Image acquisition SBF-SEM makes use of imaging with a back-scattered electron (BSE) detector. As the electron beam scans over the block-face, electrons that are scattered back from beneath the sample surface will reach the detector. From each point, BSEs are collected for a certain amount of time (the dwell time) before the beam moves to the next position. As such, a block-face image is built up “point by point” and “line by line.” Once a 2D image of the surface is collected, the ultramicrotome will remove the top slice from the block, revealing a new surface face to be imaged. The image quality in terms of signal-to-noise ratio depends on the primary beam power and is positively correlated to the dwell time. However, with this technique it is an advantage to have the beam current and voltage as low as possible and use a short dwell time in order to decrease the effective Z depth of the information, thus improving the Z resolution. Secondary electrons produced by the primary beam will affect the image quality, because when they are detected by the BSE detector this results in charge artifacts in the image, or when they accumulate in the sample they can locally generate magnetic fields that distort the image. Even if all precautions are taken to maximize the conductivity of the sample, a compromise has to be made between increased resolution resulting from higher beam current/longer dwell times and image quality, in order to avoid the image degradation due to charging artifacts. Recently, a focal gas injection-based charge compensator (FCC) became available. This add-on, developed by Deerinck and colleagues, permits imaging of nonconductive biological samples at high column voltage and longer dwell times without charge artifacts (Deerinck et al., 2018). The technique is very straightforward, as nitrogen gas is locally applied through a nozzle in the chamber precisely over the block face during acquisition (Fig. 1C).
2.4 Image visualization Once a stack of images is recorded, multiple image processing manipulations can be performed (Fig. 1D). In order to obtain a volume dataset, it is necessary to compile the stack of 2D images into a 3D volume image. When doing this, it is also
3 Materials and instrumentation
important to correct for translational movement between two consecutive images in the stack by a process called image registration. It is then possible to apply image restoration techniques to improve the overall quality of the recorded images by either adjusting contrast or removing noise (Roels et al., 2018). To interpret the data set in 3D, it is necessary to perform segmentation, i.e., delineating specific structures of interest in the 3D volume. Segmentation allows for the visualization of individual cellular and subcellular objects and the rendering of their 3D structure (Belevich, Joensuu, Kumar, Vihinen, & Jokitalo, 2016; Cocks, Taggart, Rind, & White, 2018; Kreshuk et al., 2015).
3 Materials and instrumentation 3.1 From tissue to block Instrumentation Stereo microscope (Stemi 2000C, Zeiss) Vibratome (VT1200S, Leica) Vacuum oven (LabLine, ThermoFisher) Materials 0.2 M Cacodylate (sodium) buffer pH 7.4 (11652, EMS) 10% paraformaldehyde (15712, EMS) 25% glutaraldehyde (G-5882, Sigma) Karnovsky fixative: 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4, Reduced Osmium: 1 part 4% OsO4 (19150, EMS) + 1 part 3% ferricyanide in 0.2 M cacodylate pH 7.4 1% Thiocarbohydrazide: dissolve 0.1 g TCH (21900, EMS) in UPWIncubate 1 h in an oven at 60 °C. Shake every 100 Osmium (19150, EMS) Uranyl acetate (EMS) Uranyl Acetate Replacement solution: 1:3 uranyl acetate replacement (22405, EMS) and UPW Walton’s Lead Aspartate staining solution: Prepare L-Aspartic acid solution by dissolving 0.998 g L-Aspartic acid (A8949, Sigma) in 250 mL UPW. Adjust to pH 3.8 with KOH. Dissolve 0.066 g Lead Nitrate (07905CJ, Aldrich) in 10 mL L-Aspartic acid pH 3.8. Adjust to pH 5.5 with 1 N KOH. Check pH with pH indicator strip (109584.1111, Millipore) Ethanol (1.00983, Merck) Propylene oxide (82320, Sigma) Durcupan (Sigma) Spurr’s resin (EMS) Low-melt agarose (A4018, Sigma)
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3.2 From block to sample mounting Instrumentation Ultramicrotome (Ultracut, Riechert-Jung) Sputter Coater (Q150T ES, Quorum) Materials Aluminum pin (Gatan) Conductive epoxy (Chemtronics)
3.3 Image acquisition Instrumentation Merlin SEM (Zeiss) with 3View (Gatan) and Focal Charge Compensator (Zeiss)
3.4 Image visualization Fiji, https://fiji.sc MIB, http://mib.helsinki.fi Imaris, https://www.bitplane.com
4 Results We optimized an en bloc staining for Arabidopsis thaliana root tips, initially based on the protocol described in Table 1 (Fendrych et al., 2014). However, many adjustments were required to tailor the sample preparation for this type of plant specimen. Our detailed protocol is explained step-by-step in Table 2. A first change that was introduced is the pH of the fixation, from classical Karnovsky pH 7.4 to 6.8 to more closely match the pH value of the buffer to that of the specimen. Because the sample is fragile, manipulation of the root tip became easier by enveloping it in low-melt agarose. It is an additional advantage that the agarose also binds osmium and therefore the empty resin around the root tip is less prone to electron charging. To increase contrast in the sample itself, we added Ruthenium Red at the beginning of the en bloc staining, and an additional round of TCH and osmium staining. Mounting of the sample and trimming of the block was done with an orientation of the root tip upward so that the slice-and-view procedure was perpendicular to the growth axis. About 2000 images were acquired with a slice thickness set to 70 nm (Fig. 2A). Images were registered using Fiji and orthogonal viewing was done by making use of Imaris (Fig. 2B). For a selected region, we applied intensity thresholding in order to segment the electron dense structures, in this case the plasmodesmata (Fig. 2C; Supplementary Movie 1 in the online version at https://doi.org/10.1016/ bs.mcb.2019.04.002).
A
B
C
FIG. 2 SBF-SEM imaging of Arabidopsis thaliana root tips. An Arabidopsis thaliana root tip was prepared for imaging with SBF-SEM. (A) A single 2D image from the recorded stack is shown. (B) After registration of the different 2D images and compilation in one volume dataset, representation with orthogonal views is shown. (C) A region of interest was cropped from the original dataset and intensity thresholding was applied, in Fiji. We used the Fiji 3DViewer for rendering, showing two cells with plasmodesmata in the cell wall between them.
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The protocol for Arabidopsis root tips (Table 2) differs substantially from the protocol described in Table 1 and these adjustments are specific for this sample type. For any other tissues optimizing the sample preparation may be required. However, we also introduced modifications that we now generally use in the workflows for SBFSEM in our facility. Specifically, we have tested replacement of UA with Lanthanide salts (Uranyl Acetate Replacement) for reasons of lab safety. In Fig. 3A and B we show an SBF-SEM image of mouse brain tissue that was prepared according to the protocol given in Table 1 using either LS or UA, respectively. We have observed that the contrast with LS is similar to that with UA and have generally switched to using LS instead of UA for our working protocols.
A
C
B
D
E
FIG. 3 General modifications for SBF-SEM sample preparation. Mouse brain tissue was prepared for SBF-SEM, making use of the protocol described in Table 1 with either Lantanide Salts (A) or Uranyl Acetate (B). Single 2D image from an SBF-SEM dataset with LS or UA, respectively. Mouse liver tissue was prepared according to the protocols in Table 3. (C) Single 2D image from an SBF-SEM dataset, making use of sample that was prepared with incubations on the bench. (D) Sample prepared with BioWave incubation. (E) Sample with BioWave incubation and with FCC for imaging.
4 Results
The en bloc stainings described in Tables 1 and 2 can easily take up multiple days. The use of a microwave to assist sample preparation may substantially speed up the process because of the increased speed of reagent penetration and thus shorter incubation times. Therefore, we applied the same staining protocol to prepare mouse liver tissue by either incubation on the bench or using the microwave (see Table 3). An image of the sample preparation without microwave use is shown in Fig. 3C and with microwave use in Fig. 3D. We noticed that due to electron charging of bare resin in a block, SBF-SEM has been difficult to apply to tissues that contain non-cytoplasmic areas, such as plant vacuoles, the alveoli of lungs, or cultured cell monolayers that are surrounded by bare resin. An improvement was achieved when the focal charge compensator, developed by Deerinck and colleagues became available (Deerinck et al., 2018). We have also applied the use of the FCC on microwave processed liver tissue and noticed substantial improvements in the image quality. We no longer observed charging Table 3 En bloc contrast staining protocol for mouse liver tissue with either bench or microwave incubation steps. Bench incubation
Incubation buffer
BioWave step
5 30
0.1 M cacodylate pH 7.4 Reduced Osmium in 0.1 M cacodylate pH 7.4 Ultrapure water (UPW)
200
1% Thiocarbohydrazide (TCH) in UPW
5 30
UPW
300
2% OsO4 in UPW (bench) 1% OsO4 in UPW (BioWave) UPW
2 4000 250 W, Vacuum Off 5 20 alternating 100 W and power Off, Vacuum On 2 50 at bench + 2 4000 250 W, Vacuum Off 20 at bench + 3 1’ alternating 150 W and power Off, Vacuum On 2 50 at bench + 2 4000 250 W, Vacuum Off 5 20 alternating 100 W and power Off, Vacuum On 2 50 at bench + 2 4000 250 W, Vacuum Off 7 10 alternating 150 W and power Off, Vacuum On 2 50 at bench + 2 4000 250 W, Vacuum Off 300 2 50 at bench + 2 4000 250 W, Vacuum Off Each 4000 150 W, Vacuum Off
5 30 1h
5 30
5 30
2% uranyl acetate (UA) in UPW at 4 °C (bench) 1% UA in UPW (BioWave) UPW
300 5 30
Walton’s lead at 65 °C UPW
50
Each 70%, 90%, 100%, 100% EtOH (bench) 50%, 70%, 90%, 100%, 100% EtOH (BioWave)
ON
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artifacts even at relatively high accelerating voltages (Fig. 3E). In addition, the general image quality was also improved in terms of increased signal to noise ratio and throughout an image stack we noticed less translational movement between two images, which can make the image registration step unnecessary.
5 Discussion SBF-SEM has become an established technique to gather volume data on various biological samples. Initially, the neuroscience community was the main beneficiary of this technology, but in the recent years SBF-SEM has proven its value in many different areas of life science research; e.g., inflammation, cardiology, botany, etc. The great advantage of the technology is the volume range that can be imaged. However, the workflow for SBF-SEM is a long and multistep process. While the acquisition has become more efficient because of the automation of the slice-and-view process, the initial focus for optimization was on obtaining high quality images, determined by contrast and good cutting of the sample block. Clearly, sample preparation is the determining factor here and by the turn of the century reliable en bloc heavy metal staining protocols for brain tissue were distributed within the volume EM community. The “Deerinck protocol” was used by several labs and facilities after the implementation of the technology and tailored for diverse sample types (Deerinck et al., 2010). Further improvements of the SBF-SEM method arose from adjustments of sample preparation procedures. The Deerinck protocol works very well for lipid-rich tissues, such as brain, and based on this, variations have been worked out in order to apply the en bloc contrast staining to other samples. For instance, the use of tannic acid and ruthenium red was useful for adding contrast to specific structures like the plant cell wall, collagen or desmosomes (Fendrych et al., 2014; Starborg et al., 2013; Vanslembrouck et al., 2018). Others have explored more selective stainings in order to localize specific structures. For instance, the 3D visualization of ER in animal cells and plant samples has been achieved by staining with Horse Radish Peroxidase or Zink Iodide (Kittelmann, Hawes, & Hughes, 2016; Puhka, Joensuu, Vihinen, Belevich, & Jokitalo, 2012). Additional variations came about by necessity, e.g., certain labs had to avoid the use of uranyl acetate and therefore permanently switched to Lanthanide salts. All of the many improvements, usually worked out empirically, have contributed to the growing use of SBF-SEM in life science research. In addition to these changes in staining of the samples improvements in sample handling have been made. The use of agarose or gelatin embedding for protecting fragile samples or encasing pellets of cells (immune cells, yeast, bacteria) allows for the processing of these samples as blocks to prevent damage during sample manipulation. As far as fixation of the samples is concerned, the use of highpressure freezing, which may preserve samples in a more natural morphological state due to rapid fixation, has been explored (Webb & Schieber, 2018).
Acknowledgments
Webb and Webb also introduced microwave-assisted processing in their working protocols for shortening lengthy sample preparation times (Webb & Webb, 2015). To gather more functional information, certain labs and facilities have also explored the use of SBF-SEM in correlated light and electron microscopy (CLEM). Armer and colleagues performed CLEM with live confocal imaging and SBF-SEM to study blood vessel fusion in Zebrafish (Armer et al., 2009). Others have worked out experiments with Near Infra Red Branding as a method to introduce landmarks to mark a region interest in a sample that was first imaged with high resolution confocal microscopy, then with SBF-SEM (Bishop et al., 2011; Lees, Peddie, Collinson, Ashby, & Verkade, 2017). A very elegant solution to combine fluorescent imaging with SBF-SEM, is the introduction of a miniature fluorescence microscope inside the chamber of the SBF-SEM (Brama et al., 2016). Further improvements in preparation and imaging will make SBF-SEM more efficient and widespread. Since its beginnings the issues of electron charging of bare resin in samples had been a drawback to the use of SBF-SEM at high accelerating voltages or for imaging of tissues with cavities such as vacuoles or blood vessels, samples that are low in lipids, or tissues that are difficult to stain with heavy metal dyes. This was tackled by the development of the Focal Charge Compensator (Deerinck et al., 2018). Both improvements now allow laboratories to get the best out of the technology, because all artifacts due to electron charging are minimized and the image quality is significantly improved. Even samples with low contrast, like archived blocks prepared for standard TEM imaging, can now be used for SBF-SEM. The next issue that is now being addressed is how to handle the data that are gathered with this technique. Size and complexity of data sets are preventing ease of analysis. Several solutions from other fields, such as computer assisted image recognition, are currently being explored. Also solutions from within the volume EM community or through collaborations with image analysis specialists have produced optimized tools for segmentation and visualization of volume EM data (Belevich et al., 2016; Luengo et al., 2017). Alongside that, repositories for data and annotations are being set up and the community is discussing quality standards for SBF-SEM images. It is clear that more and more technical solutions and expertise in SBFSEM will help to turn this technique into a well-established and accessible tool for life science research. The ability to image large samples in 3D at the nanoscale will deliver a rich source of morphological information to combine with functional and molecular scale information.
Acknowledgments The authors would like to thank the VIB Core Facilities program, VIB Inflammation Research Center, VIB Technology Fund and a generous Grant from minister Ingrid Lieten of the government of Flanders for support in implementing the SBF-SEM technology. We are grateful to Matyas Fendrych and Moritz Nowack for the data on Arabidopsis thaliana and their input for data visualization.
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References Armer, H. E., Mariggi, G., Png, K. M., Genoud, C., Monteith, A. G., Bushby, A. J., et al. (2009). Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy. PLoS One, 4, e7716. Belevich, I., Joensuu, M., Kumar, D., Vihinen, H., & Jokitalo, E. (2016). Microscopy image browser: A platform for segmentation and analysis of multidimensional datasets. PLoS Biology, 14, e1002340. Bishop, D., Nikic, I., Brinkoetter, M., Knecht, S., Potz, S., Kerschensteiner, M., et al. (2011). Near-infrared branding efficiently correlates light and electron microscopy. Nature Methods, 8, 568–570. Brama, E., Peddie, C. J., Wilkes, G., Gu, Y., Collinson, L. M., & Jones, M. L. (2016). ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy. Wellcome Open Research, 1, 26. Cocks, E., Taggart, M., Rind, F. C., & White, K. (2018). A guide to analysis and reconstruction of serial block face scanning electron microscopy data. Journal of Microscopy, 270, 217–234. Deerinck, T. J., Bushong, E., Thor, A., & Ellisman, M. H. (2010). NCMIR methods for 3D EM: A new protocol for preparation of biological specimens for serial block-face SEM. Microscopy, 6–8. Deerinck, T. J., Shone, T. M., Bushong, E. A., Ramachandra, R., Peltier, S. T., & Ellisman, M. H. (2018). High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation. Journal of Microscopy, 270, 142–149. Denk, W., & Horstmann, H. (2004). Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biology, 2, e329. Fendrych, M., Van Hautegem, T., Van Durme, M., Olvera-Carrillo, Y., Huysmans, M., Karimi, M., et al. (2014). Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis. Current Biology, 24, 931–940. Heymann, J. A., Hayles, M., Gestmann, I., Giannuzzi, L. A., Lich, B., & Subramaniam, S. (2006). Site-specific 3D imaging of cells and tissues with a dual beam microscope. Journal of Structural Biology, 155, 63–73. Karnovsky, M. L., & Deane, H. W. (1955). Aldehyde formation in the lipide droplets of the adrenal cortex during fixation, as demonstrated chemically and histochemically. The Journal of Histochemistry and Cytochemistry, 3, 85–102. Kittelmann, M., Hawes, C., & Hughes, L. (2016). Serial block face scanning electron microscopy and the reconstruction of plant cell membrane systems. Journal of Microscopy, 263, 200–211. Kremer, A., Lippens, S., Bartunkova, S., Asselbergh, B., Blanpain, C., Fendrych, M., et al. (2015). Developing 3D SEM in a broad biological context. Journal of Microscopy, 259, 80–96. Kreshuk, A., Walecki, R., Koethe, U., Gierthmuehlen, M., Plachta, D., Genoud, C., et al. (2015). Automated tracing of myelinated axons and detection of the nodes of Ranvier in serial images of peripheral nerves. Journal of Microscopy, 259, 143–154. Larsimont, J. C., Youssef, K. K., Sanchez-Danes, A., Sukumaran, V., Defrance, M., Delatte, B., et al. (2015). Sox9 controls self-renewal of oncogene targeted cells and links tumor initiation and invasion. Cell Stem Cell, 17, 60–73. Lees, R. M., Peddie, C. J., Collinson, L. M., Ashby, M. C., & Verkade, P. (2017). Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps. In Vol. 140. Correlative light and electron microscopy III (pp. 245–276). Academic Press.
Further reading
Leighton, S. B. (1981). SEM images of block faces, cut by a miniature microtome within the SEM—A technical note. Scanning Electron Microscopy, 73–76. Luengo, I., Darrow, M. C., Spink, M. C., Sun, Y., Dai, W., He, C. Y., et al. (2017). SuRVoS: Super-region volume segmentation workbench. Journal of Structural Biology, 198, 43–53. Peddie, C. J., & Collinson, L. M. (2014). Exploring the third dimension: Volume electron microscopy comes of age. Micron, 61, 9–19. Puhka, M., Joensuu, M., Vihinen, H., Belevich, I., & Jokitalo, E. (2012). Progressive sheet-totubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells. Molecular Biology of the Cell, 23, 2424–2432. Roels, J., Aelterman, J., Luong, H. Q., Lippens, S., Pizurica, A., Saeys, Y., et al. (2018). An overview of state-of-the-art image restoration in electron microscopy. Journal of Microscopy, 271, 239–254. Seligman, A. M., Wasserkrug, H. L., & Hanker, J. S. (1966). A new staining method (OTO) for enhancing contrast of lipid-containing membranes and droplets in osmium tetroxide-fixed tissue with osmiophilic thiocarbohydrazide(TCH). The Journal of Cell Biology, 30, 424–432. Smith, D., & Starborg, T. (2018). Serial block face scanning electron microscopy in cell biology: Applications and technology. Tissue and Cell, 57, 111–122. Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research, 26, 31–43. Starborg, T., Kalson, N. S., Lu, Y., Mironov, A., Cootes, T. F., Holmes, D. F., et al. (2013). Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization. Nature Protocols, 8, 1433–1448. Vanslembrouck, B., Kremer, A., Pavie, B., van Roy, F., Lippens, S., & van Hengel, J. (2018). Three-dimensional reconstruction of the intercalated disc including the intercellular junctions by applying volume scanning electron microscopy. Histochemistry and Cell Biology, 149, 479–490. Webb, R. I., & Schieber, N. L. (2018). Volume scanning electron microscopy: Serial block-face scanning electron microscopy focussed ion beam scanning electron microscopy. In Cellular imaging (pp. 117–148). Springer. Webb, R., & Webb, R. (2015). Quick freeze substitution processing of biological samples for serial block-face scanning electron microscopy. Microscopy and Microanalysis, 21, 1115–1116. Willingham, M. C., & Rutherford, A. V. (1984). The use of osmium-thiocarbohydrazideosmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells. The Journal of Histochemistry and Cytochemistry, 32, 455–460. Wu, S., Baskin, T. I., & Gallagher, K. L. (2012). Mechanical fixation techniques for processing and orienting delicate samples, such as the root of Arabidopsis thaliana, for light or electron microscopy. Nature Protocols, 7, 1113–1124.
Further reading Urwyler, O., Izadifar, A., Dascenco, D., Petrovic, M., He, H., Ayaz, D., et al. (2015). Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy. Development, 142, 394–405.
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