We sincerely thank Shigeo Kita for his valuable suggestions and discussion. We also thank John and Sumire Eckstein for their critical reading of the manuscript.

NOTE: The specimens used in this study were microorganisms, rapidly frozen with propane in liquid nitrogen, freeze substituted in acetone containing 2% osmium tetroxide, and embedded in epoxy resin1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18.
1. Preparation of support film (Figure 1-3)
NOTE: Silver-colored Formvar support film is prepared using the cast-on-glass method19.
<ol>
	<li>Add 100 mL of 1.5% Formvar in ethylene dichloride to a Formvar-making apparatus (Figure 1). Dip one-half of a glass slide (76 mm x 26 mm x 1.3 mm) in Formvar solution in the upper column of the apparatus by pressing the solution with air through &#39;a&#39; using a rubber ball19.
	<ol>
		<li>Drain the solution from the column by opening the three-way stopcock and releasing air through &#39;b&#39; to reduce pressure. Take out the glass slide from the apparatus and dry in air to form a film on the surface of the glass slide. Use an incandescent lamp to accelerate drying of the Formvar film.</li>
	</ol>
	</li>
	<li>After scraping off the four edges of the film on the glass slide with a razor blade (Figure 2a), and breathing onto the slide to facilitate the separation of the film from the slide20, float off the film on water by immersing the glass slide into the water slowly at a low horizontal angle (about 10&#176;, Figure 2b).</li>
	<li>Scoop up the Formvar film from the water using an aluminum rack (30 mm x 25 mm x 3 mm) with holes (4 mm in diameter) (Figure 3a). Keep the rack with the Formvar film in a desiccator until use (Figure 3b).</li>
</ol>
2. Trimming the specimen block with an ultrasonic trimming blade and a razor blade 21 under a stereomicroscope (Figure 4)
<ol>
	<li>Make sure that there are cells on the surface of the block by observing the tip of the block with a light microscope (Figure 4a).</li>
	<li>Mount the specimen block in the chuck (block holder), and mount the chuck on a trimming stage21 (Figure 4b). This trimming stage has an illumination mechanism from the back of the specimen.</li>
	<li>Trim the block to a size of 0.7 mm x 1.0 mm (Figures 4c, 5d). Since the epoxy resin is very hard, trim the blocks first using an ultrasonic trimming blade. The ultrasonic trimming blade is a newly introduced machine, and the blocks are easily trimmed. Then trim the block further with a razor blade. Cut one shoulder to mark the direction of cutting (see Figure 5d, Figure 8).</li>
</ol>
3. Trimming the specimen block with diamond knife using the microtome (Figure 5)
<ol>
	<li>Set the specimen block chuck in the specimen holder of the ultramicrotome. Place the specimen 90&#176; counterclockwise against the actual position when serial ultrathin sectioning is performed (See Figure 5d).</li>
	<li>Cut the surface of the block with a diamond trimming knife (See Figure 5d). Set the diamond knife edge parallel to the specimen block face, and cut the minimum amount of specimen surface so as not to lose any specimen (Figure 5d). 
	NOTE: This step is done to smoothen the specimen surface. The part of specimen surface of the slim part is left intact (therefore this part is not shining like a mirror, Figure 5d), so as not to lose any part of the specimen for serial sectioning. This is because the specimen is exposed on the surface of the block.</li>
	<li>Place a mirror (Mesa cut, M) on the knife stage to monitor the cutting of the specimen block (Figure 5c).</li>
	<li>Cut the left edge (this becomes the upper side, Figure 5d, of the sample in serial sectioning) of the block using trimming knife by rotating the knife stage 30&#176; to the left (Figure 5a).</li>
	<li>Cut the block face at about 100 &#181;m from the left edge of the specimen (this becomes the lower side of the specimen in serial sectioning, Figure 5d) by rotating the knife stage 30&#176; to the right (Figure 5b). 
	NOTE: The serial sections will be cut from the slim part of the specimen of about 90 nm x about 1 mm in size. The large part will be used to adjust the specimen surface and the knife edge such that they are parallel. After adjusting the specimen surface to be parallel to the knife edge, the large part will be removed with a razor blade. By cutting the upper and lower sides of the specimen using the microtome, both sides of the specimen become smooth and exactly parallel to each other (Figure 5d), which is necessary to get straight and unbroken ribbon sections.</li>
</ol>
4. Adjusting the specimen surface and knife edge so that they face parallel to each other 21
<ol>
	<li>Remove the trimming knife, and rotate the specimen block 90&#176; clockwise.</li>
	<li>Set an ultrathin sectioning knife to the knife stage.</li>
	<li>Adjust the specimen surface and the knife edge so that they face parallel to each other using the large part of the specimen surface. 
	NOTE: The large part of the specimen is used for adjustment because the cutting face of serial sectioning is so small, making it difficult to adjust specimen surface and knife edge using this part only, especially in vertical direction. Thus, using the large part of the specimen makes adjustment easy.</li>
</ol>
5. Spreading neoprene solution on the specimen block (Figure 6)
<ol>
	<li>To place the specimen chuck at exactly the same position as the original position, apply tape on the chuck and the chuck holder of the microtome and cut at the boundary (Figure 6a).</li>
	<li>Take the specimen chuck out from the microtome, and place it under the stereomicroscope (Figure 6b). Cut off the large part of the specimen with a razor blade, leaving the slim part, from which serial sections will be obtained.</li>
	<li>Using a Pasteur pipette, drop about 1 &#181;L 0.5% neoprene solution (Figure 6b) onto the specimen to be used for serial sectioning, to make the section sides adhesive.Cover the whole specimen with neoprene solution. Absorb excess neoprene solution immediately with a piece of filter paper placed near the specimen. Getting a ribbon of sections is essential for taking pictures of serial cell sections, and neoprene glue is very useful for sticking the sections together.</li>
	<li>Prepare 3-slit grids (Sizes of slits: middle, 0.4 mm x 2.2 mm, both sides 0.2 mm x 2.2 mm) (Figure 6c) for picking up serial sections by bending the handle of the grids to 60&#176;, treating the grids with 0.5% neoprene solution, and making the grids hydrophilic by glow discharge22.</li>
</ol>
6. Making serial sections (Figure 7-9)
<ol>
	<li>Place the specimen block chuck back in the microtome at the same position as 5.1 (Figure 6a) by aligning the taped parts. This keeps the block face and the knife edge perfectly parallel to each other. Then bring the knife close to the specimen.</li>
	<li>Fill the knife boat with water.</li>
	<li>Cover the microtome with a plastic cover to prevent airflow (Figure 7). 
	NOTE: Airflow during ultrathin sectioning and retrieval of sections often cause problems. The plastic cover has three holes: one hole is for the binocular lenses, and the other two holes are for the arms to allow operation while the cover is on. The wooden armrest is used for placing arms while doing delicate work, such as retrieving serial sections with grids. The wooden armrest is placed on different tables from the microtome table (Figure 7, arrows), so as not to transmit the vibration of the operator&#39;s hands to the microtome.</li>
	<li>Start cutting the specimen at 200 nm section thickness (Figure 8). Setting at 200 nm thickness will save time in bringing the knife to the surface of the specimen.</li>
	<li>After the first section is cut, set the section thickness to 70 nm (Figure 8).</li>
	<li>When the number of serial sections reaches 20 (precisely speaking, after the ribbon reaches about 1.8 mm; the number of sections varies depending on the width of sections), set the section thickness to 10 nm (Figure 8) while continuing to cut. 
	NOTE: Since the microtome cannot cut 10 nm-thick sections, no new section appears, and the previously cut sections become separated from the knife edge. It is important to get separated section groups (Figure 9) for picking up serial sections to avoid difficulty in separating sections manually using a pair of hair strands. Since the field of view in the transmission electron microscope is 2.0 mm, sections should be 1.8 mm long at most.</li>
	<li>Set the section thickness to 60 nm (Figure 8). This will produce 70 nm sections (Figure 8) because the machine will add the previous 10 nm thickness.</li>
	<li>Set the section thickness back to 70 nm (Figure 8) and continue cutting until 1.8-mm-long sections are obtained.</li>
	<li>Repeat 6.6-6.8 until five 1.8 mm long sections are obtained (Figure 9). We usually make five section groups since there are five specimen holders in the transmission electron microscope available in our lab, but five is not the limit.</li>
</ol>
7. Picking up serial sections (Figure 10-12)
<ol>
	<li>Place the &#39;Section-holding loop&#39;23 (Inner diameter of the loop: 5.0 mm) (Figure 10a) on the third section group (Figure 10b). 
	NOTE: It is necessary to retrieve the sections in the order of their sectioning to take pictures in the proper order. However, since there are five section groups on the knife boat, it often gets confusing which groups are which. By placing the loop on the third group, it becomes clear that the two groups near to the operator are the first and second, and the distant two from the operator are the fourth and fifth groups (Figure 10b). This will prevent mixing up the order.</li>
	<li>Place &#39;Water-surface-raising loop&#39;23 (WSRL, inner diameter of the loop: 4.0 mm) (Figure 11a) on the knife stage (Figure 11b). The WSRL will stand firmly on the knife stage because it is equipped with a magnet in the bottom.
	<ol>
		<li>Place the loop of the WSRL just above the serial sections by moving its shaft and handle. Lower the loop of the WSRL onto the water surface so that the loop encircles the sections.</li>
		<li>Push the loop down by turning the screw downwards so that the water surface will raise by surface tension (Figures 11c-d). Move the section to the center of the loop, position it on the apex of the water, and adjust the direction of the section by using a hair strand21.</li>
		<li>Pick up section groups by touching it with a bare slit-grid, which is held by tweezers and kept parallel to the water surface (Figure 11d). It is important for sections to touch the grid first, not the water, to place sections precisely on the center of the grid (Figure 11d).</li>
	</ol>
	</li>
	<li>Place the grids with sections along with a tiny drop of water on the Formvar support film16 (Figure 12), and remove excess water using filter paper. 
	NOTE: Wrinkling of the support film is often a problem when sections are picked up using a grid with support film because membrane (sections) touches membrane (support film) directly. The problem of getting wrinkles on the support film is significantly reduced by placing a section-bearing grid along with a tiny drop of water on the Formvar support film16.</li>
</ol>
8. Staining sections16, 24 (Figure 13)
<ol>
	<li>After the sections are completely dried, tear the Formvar film from around the grids, and remove the grids bearing the sections.</li>
	<li>Set the grids in the groove of the staining tube16 in the proper order (Figure 13), and stain sections with uranyl acetate and lead citrate24. 
	NOTE: There is a groove 0.6 mm deep along the long axis of the tube cut with a razor blade. The tube is useful for preventing accidental breakage of support films when using tweezers, since direct handling of grids is not necessary during the staining and washing process16.</li>
</ol>
9. Observation of serial sections (Figure 14)
<ol>
	<li>Set the 5 grids in the multi-specimen holder in the proper order (Figure 14a), orienting the long axis of the grid slit perpendicular to the specimen holder axis. The handles (arrowheads) of the grids do not have to be cut, because the &#39;grid-fixers&#39; will not touch the handles.</li>
	<li>Fix the grids with &#39;grid-fixers&#39; (Figure 14b) and insert the specimen holder into the transmission electron microscope. 
	NOTE: It is often useful to take low magnification images of all cell sections (Figure 15) to find interesting microorganisms or cells in good condition with no contamination and with good fixation, before taking pictures of the target cell.</li>
	<li>Take pictures of the target cells in all the cell sections at high magnification.</li>
</ol>

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</ol>

The authors have nothing to disclose.

The method presented here requires no expensive equipment. It requires only an aluminum rack (Figure 3), three-slit grids (Figure 6c), section-holding loops (Figure 10a), water-surface-raising loop (Figure 11a), and a staining tube (Figure 13). There are many features of the present method. The large part of the specimen is used to adjust the specimen surface and knife edge...

Proper serial ultrathin sectioning technique is indispensable to study cells and cell components three-dimensionally at the electron microscopic level. We have studied the dynamics of spindle pole body in the cell cycle of yeast cells, and revealed morphological changes of their ultrastructure during the cell cycle and the time of duplication1,2,3,4,5. In 2006, we coined a new word &#39;structome&#39; by combining &#39;structure&#39; and &#39;-ome&#39;, and defined it as the &#39;quantitative and three-dimensional structural information of a whole cell at the electron microscopic level&#39; 6, 7.
By structome analysis, which requires serial ultrathin sectioning technique, it was found that a yeast cell of Saccharomyces cerevisiae and Exophiala dermatitidis had about 200,000 ribosomes7, 8, an Escherichia coli cell had 26,000 ribosomes9, a Mycobacterium tuberculosis cell had 1,700 ribosomes10 and Myojin spiral bacteria had only 300 ribosomes11. This information is useful in not only estimating the growth rate in each organism, but also in identification of species9.
Further, structome analysis led to the discovery of a new organism; Parakaryon myojinensis was found in the deep sea off the coast of Japan, whose cell structure were an intermediate between those of prokaryotes and eukaryotes12,13,14,15. At present, serial ultrathin sectioning technique is considered to be so difficult that it would take a long time to master. In this study, we have developed a reliable method in which anybody can perform serial ultrathin sectioning without difficulty.

<table><tbody><tr><td>Formvar making apparatus</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>652</td><td>W 180 x D 180 x H 300 mm</td></tr><tr><td>Glass slide</td><td>Matsunami Co. Ltd., Osaka</td><td>-</td><td>76 x 26 x 1.3 mm</td></tr><tr><td>Aluminum rack with 4-mm holes</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>658</td><td>W 30 x D 25 x H 3 mm, Refer to this paper</td></tr><tr><td>Stereomicroscope</td><td>Nikon Co. Ltd., Tokyo</td><td>-</td><td>SMZ 645</td></tr><tr><td>LED illumination for stereomicroscope</td><td>Nikon Co. Ltd., Tokyo</td><td>-</td><td>SM-LW 61 Ji</td></tr><tr><td>Trimming stage</td><td>Sunmag Co.Ltd., Tokyo</td><td>-</td><td>Tilting mechanism equipped, Refer to this paper</td></tr><tr><td>LED illumination for trimming stage</td><td>Sunmag Co.Ltd., Tokyo</td><td>-</td><td>Refer to this paper</td></tr><tr><td>Ultrasonic trimming blade</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>5240</td><td>EM-240, Refer to this paper</td></tr><tr><td>Diamond knife for trimming</td><td>Diatome Co. Ltd., Switzerland</td><td>-</td><td>45&amp;deg;</td></tr><tr><td>Diamond knife for ultrathin sectioning</td><td>Diatome Co. Ltd., Switzerland</td><td>-</td><td>45&amp;deg;</td></tr><tr><td>Ultramicrotome</td><td>Leica Microsystems, Vienna</td><td>-</td><td>Ultracut S</td></tr><tr><td>Mesa cut</td><td>Leica Microsystems, Vienna</td><td>-</td><td>Mirror</td></tr><tr><td>0.5% Neoprene W solution</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>605</td><td></td></tr><tr><td>Special 3-slit nickel grid</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>2458</td><td>Refer to this paper</td></tr><tr><td>Special 3-slit copper grid</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>2459</td><td>Refer to this paper</td></tr><tr><td>Section-holding loop</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>526</td><td>Refer to this paper</td></tr><tr><td>Water-surface-raising loop</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>527</td><td>Refer to this paper</td></tr><tr><td>Staining tube</td><td>Nisshin EM Co. Ltd., Tokyo</td><td>463</td><td>Refer to this paper</td></tr><tr><td>Multi-specimen holder</td><td>JEOL Co. Ltd., Tokyo</td><td>-</td><td>EM-11170</td></tr><tr><td>JEM-1400</td><td>JEOL Co. Ltd., Tokyo</td><td>-</td><td>Transmission electron microscope</td></tr></tbody></table>

a method for obtaining serial ultrathin sections of microorganisms in transmission electron microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

In this protocol, three-slit grids were used for picking up serial sections. The grids are made of nickel or copper. The serial sections are placed on the middle slit. The slits on both sides are necessary to view the sections when picking them up with the grid. To keep the grids parallel with the serial sections when picking them up with tweezers (Figure 11d), the handle is bent (Figure 6c, right). A small handle is advantageous...

Watch this Scientific Journal Video about A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy at JoVE.com

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

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14.2K Views.  Chiba University. This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Video: A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Immunology and Infection

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Neuroscience

An Unbiased Approach of Sampling TEM Sections in Neuroscience

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling.

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Biology

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

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

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

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

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

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

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

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

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

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is often limited to two dimensions, hampering the ability to fully describe the three-dimensional (3D) ultrastructure and functional relationship between organelles. Volume electron microscopy (vEM) describes a collection of techniques that enable the interrogation of cellular ultrastructure in 3D at mesoscale, microscale, and nanoscale resolutions. 
This protocol provides an accessible and robust method to acquire vEM data using serial section transmission EM (TEM) and covers the technical aspects of sample processing through to digital 3D reconstruction in a single, straightforward workflow. To demonstrate the usefulness of this technique, the 3D ultrastructural relationship between the endoplasmic reticulum and mitochondria and their contact sites in liver hepatocytes is presented. Interorganelle contacts serve vital roles in the transfer of ions, lipids, nutrients, and other small molecules between organelles. However, despite their initial discovery in hepatocytes, there is still much to learn about their physical features, dynamics, and functions. 
Interorganelle contacts can display a range of morphologies, varying in the proximity of the two organelles to one another (typically ~10-30 nm) and the extent of the contact site (from punctate contacts to larger 3D cisternal-like contacts). The examination of close contacts requires high-resolution imaging, and serial section TEM is well suited to visualize the 3D ultrastructural of interorganelle contacts during hepatocyte differentiation, as well as alterations in hepatocyte architecture associated with metabolic diseases.

A simple and comprehensive protocol to acquire three-dimensional details of membrane contact sites between organelles in hepatocytes from the liver or cells in other tissues.

Transmission electron microscopy has been long considered to be the gold standard for the visualization of cellular ultrastructure. However, analysis is ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Summary

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