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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Cell Biol. 82, 531-538 (2003).</li><li>Yamaguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+dynamic+ultrastructure+of+yeast+cells+by+electron+microscopy.">Quantitative and dynamic ultrastructure of yeast cells by electron microscopy.</a> Recent Res. Dev. Microbiology. 8, 219-243 (2004).</li><li>Yamaguchi, M., Biswas, S. K., Ohkusu, M., Takeo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+spindle+pole+body+of+the+pathogenic+yeast+Cryptococcus+neoformans+examined+by+freeze-substitution+electron+microscopy.">Dynamics of the spindle pole body of the pathogenic yeast Cryptococcus neoformans examined by freeze-substitution electron microscopy.</a> FEMS Microbiol. 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Lett. 219, 17-21 (2003).</li><li>Kozubowski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+kinetochore+assembly+in+the+human+pathogenic+basidiomycetous+yeast,+Cryptococcus+neoformans.">Ordered kinetochore assembly in the human pathogenic basidiomycetous yeast, Cryptococcus neoformans.</a> mBio. 4, e00614-e00613 (2013).</li><li>Yamaguchi, M., Adachi, K., The Japanese Society of Microscopy, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specimen+support.">Specimen support.</a> Guide Book for Electron Microscopy. , 52-57 (2011).</li><li>Hayat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Support+films.">Support films.</a> Principles and Techniques of Electron Microscopy: Biological Application. , 216 (2000).</li><li>Yamaguchi, M., Kita, S., The Japanese Society of Microscopy, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+sectioning.">Ultrathin sectioning.</a> Guide Book for Electron Microscopy. , 40-52 (2011).</li><li>Yamaguchi, M., Aoyama, T., Yamada, N., Chibana, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+hydrophilicity/hydrophobicity+of+the+plasma-polymerized+naphthalene+film+(Super+support+film)+and+other+support+films+and+grids+in+electron+microscopy.">Quantitative measurement of hydrophilicity/hydrophobicity of the plasma-polymerized naphthalene film (Super support film) and other support films and grids in electron microscopy.</a> Microscopy. 65, 444-450 (2016).</li><li>Kita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+method+for+obtaining+serial+ultrathin+sections+using+new+small+tools.">Reliable method for obtaining serial ultrathin sections using new small tools.</a> Kenbikyo. 46, 253-257 (2011).</li><li>Yamaguchi, M., Shimizu, M., Yamaguchi, T., Ohkusu, M., Kawamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+use+of+uranyl+acetate+solution+in+section+staining+in+transmission+electron+microscopy.">Repeated use of uranyl acetate solution in section staining in transmission electron microscopy.</a> Plant Morphology. 17, 57-59 (2005).</li><li>Hayworth, K. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+serial-section+electron+microscopy+in+larval+zebrafish.">Whole-brain serial-section electron microscopy in larval zebrafish.</a> Nature. 545, 345-349 (2017).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> PLoS Biol. 2, e329 (2004).</li><li>Heymann, J. A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+3D+imaging+of+cells+and+tissues+with+a+dual+beam+microscope.">Site-specific 3D imaging of cells and tissues with a dual beam microscope.</a> J. Struct. Biol. 155, 63-73 (2006).</li></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&#176;</td>
		</tr>
		<tr>
			<td>Diamond knife for ultrathin sectioning</td>
			<td>Diatome Co. Ltd., Switzerland</td>
			<td>-</td>
			<td>45&#176;</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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14.0K 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

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. This paper demonstrates the procedure of an in situ TDDB experiment in the transmission electron microscope, which opens a possibility to study the failure mechanism in microelectronic products.

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. ...

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

This protocol describes the operation of a liquid flow specimen holder for scanning transmission electron microscopy of AuNPs in water, as used for the observation of nanoscale dynamic processes.

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a ...

Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and nanocrystalline materials. The traditional techniques associated with scanning electron microscopy (SEM), such as electron backscatter diffraction (EBSD), do not possess the required spatial resolution due to the large interaction volume between the electrons from the beam and the atoms of the material. Transmission electron microscopy (TEM) has the required spatial resolution. However, due to a lack of automation in the analysis system, the rate of data acquisition is slow which limits the area of the specimen that can be characterized. This paper presents a new characterization technique, Transmission Kikuchi Diffraction (TKD), which enables the analysis of the microstructure of UFG and nanocrystalline materials using an SEM equipped with a standard EBSD system. The spatial resolution of this technique can reach 2 nm. This technique can be applied to a large range of materials that would be difficult to analyze using traditional EBSD. After presenting the experimental set up and describing the different steps necessary to realize a TKD analysis, examples of its use on metal alloys and minerals are shown to illustrate the resolution of the technique and its flexibility in term of material to be characterized.

This paper provides a detailed method to characterize the microstructure of ultra-fine grained and nanocrystalline materials using a scanning electron microscope equipped with a standard electron backscatter diffraction system. Metal alloys and minerals presenting refined microstructures are analyzed using this technique, showing the diversity of its possible applications.

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and ...

Fabrication of Magnetic Nanostructures on Silicon Nitride Membranes for Magnetic Vortex Studies Using Transmission Microscopy Techniques

Electron and x-ray magnetic microscopies allow for high-resolution magnetic imaging down to tens of nanometers. However, the samples need to be prepared on transparent membranes which are very fragile and difficult to manipulate. We present processes for the fabrication of samples with magnetic micro- and nanostructures with spin configurations forming magnetic vortices suitable for Lorentz transmission electron microscopy and magnetic transmission x-ray microscopy studies. The samples are prepared on silicon nitride membranes and the fabrication consists of a spin coating, UV and electron-beam lithography, the chemical development of the resist, and the evaporation of the magnetic material followed by a lift-off process forming the final magnetic structures. The samples for the Lorentz transmission electron microscopy consist of magnetic nanodiscs prepared in a single lithography step. The samples for the magnetic x-ray transmission microscopy are used for time-resolved magnetization dynamic experiments, and magnetic nanodiscs are placed on a waveguide which is used for the generation of repeatable magnetic field pulses by passing an electric current through the waveguide. The waveguide is created in an extra lithography step.

A protocol for the fabrication of magnetic micro- and nanostructures with spin configurations forming magnetic vortices suitable for transmission electron microscopy (TEM) and magnetic transmission x-ray microscopy (MTXM) studies is presented.

Electron and x-ray magnetic microscopies allow for high-resolution magnetic imaging down to tens of nanometers. However, the samples need to be prepared ...

Single-Digit Nanometer Electron-Beam Lithography with an Aberration-Corrected Scanning Transmission Electron Microscope

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by employing an aberration-corrected scanning transmission electron microscope as the exposure tool. Here, we present results of single-digit nanometer patterning of two widely used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. The method achieves sub-5 nanometer features in poly (methyl methacrylate) and sub-10 nanometer resolution in hydrogen silsesquioxane. High-fidelity transfer of these patterns into target materials of choice can be performed using metal lift-off, plasma etch, and resist infiltration with organometallics.

We use an aberration-corrected scanning transmission electron microscope to define single-digit nanometer patterns in two widely-used electron-beam resists: poly (methyl methacrylate) and hydrogen silsesquioxane. Resist patterns can be replicated in target materials of choice with single-digit nanometer fidelity using liftoff, plasma etching, and resist infiltration by organometallics.

We demonstrate extension of electron-beam lithography using conventional resists and pattern transfer processes to single-digit nanometer dimensions by ...

Graphene-Assisted Quasi-van der Waals Epitaxy of AlN Film on Nano-Patterned Sapphire Substrate for Ultraviolet Light Emitting Diodes

This protocol demonstrates a method for graphene-assisted quick growth and coalescence of AlN on nano-pattened sapphire substrate (NPSS). Graphene layers are directly grown on NPSS using catalyst-free atmospheric-pressure chemical vapor deposition (APCVD). By applying nitrogen reactive ion etching (RIE) plasma treatment, defects are introduced into the graphene film to enhance chemical reactivity. During metal-organic chemical vapor deposition (MOCVD) growth of AlN, this N-plasma treated graphene buffer enables AlN quick growth, and coalescence on NPSS is confirmed by cross-sectional scanning electron microscopy (SEM). The high quality of AlN on graphene-NPSS is then evaluated by X-ray rocking curves (XRCs) with narrow (0002) and (10-12) full width at half-maximum (FWHM) as 267.2 arcsec and 503.4 arcsec, respectively. Compared to bare NPSS, AlN growth on graphene-NPSS shows significant reduction of residual stress from 0.87 GPa to 0.25 Gpa, based on Raman measurements. Followed by AlGaN multiple quantum wells (MQWS) growth on graphene-NPSS, AlGaN-based deep ultraviolet light-emitting-diodes (DUV LEDs) are fabricated. The fabricated DUV-LEDs also demonstrate obvious, enhanced luminescence performance. This work provides a new solution for the growth of high quality AlN and fabrication of high performance DUV-LEDs using a shorter process and less costs.

A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

This protocol demonstrates a method for graphene-assisted quick growth and coalescence of AlN on nano-pattened sapphire substrate (NPSS). Graphene layers ...

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different materials show variability in resistive switching properties due to the intrinsic nature of the material used, leading to discrepancies in the field because of underlying operation mechanisms. This highlights a need for a reliable technique to understand mechanisms using nanostructural observations. This protocol explains a detailed process and methodology of in&#160;situ nanostructural analysis as a result of electrical biasing using transmission electron microscopy (TEM). It provides visual and reliable evidence of underlying nanostructural changes in real time memory operations. Also included is the methodology of fabrication and electrical characterizations for asymmetric crossbar structures incorporating amorphous vanadium oxide. The protocol explained here for vanadium oxide films can be easily extended to any other materials in a metal-dielectric-metal sandwiched structure. Resistive switching crossbars are predicted to serve the programmable logic and neuromorphic circuits for next-generation memory devices, given the understanding of the operation mechanisms. This protocol reveals the switching mechanism in a reliable, timely, and cost-effective way in any type of resistive switching materials, and thereby predicts the device&#39;s applicability.

In Situ Transmission Electron Microscopy with Biasing and Fabrication of Asymmetric Crossbars Based on Mixed-Phased a-VOx

Presented here is a protocol for analyzing nanostructural changes during in&#160;situ biasing with transmission electron microscopy (TEM) for a stacked metal-insulator-metal structure. It has significant applications in resistive switching crossbars for the next generation of programmable logic circuits and neuromimicking hardware, to reveal their underlying operation mechanisms and practical applicability.

Resistive switching crossbar architecture is highly desired in the field of digital memories due to low cost and high-density benefits. Different ...

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at length scales down to the atomic level. In situ closed-cell gas reaction (CCGR) studies performed using (scanning) transmission electron microscopy (STEM) can separate and identify localized dynamic reactions, which are extremely challenging to capture using other characterization techniques. For these experiments, we used a CCGR holder that utilizes microelectromechanical systems (MEMS)-based heating microchips (hereafter referred to as &#34;E-chips&#34;). The experimental protocol described here details the method for performing in situ gas reactions in dry and wet gases in an aberration-corrected STEM. This method finds relevance in many different materials systems, such as catalysis and high-temperature oxidation of structural materials at atmospheric pressure and in the presence of various gases with or without water vapor. Here, several sample preparation methods are described for various material form factors. During the reaction, mass spectra obtained with a residual gas analyzer (RGA) system with and without water vapor further validates gas exposure conditions during reactions. Integrating an RGA with an in situ CCGR-STEM system can, therefore, provide critical insight to correlate gas composition with the dynamic surface evolution of materials during reactions. In situ/operando studies using this approach allow&#160;for detailed investigation of the fundamental reaction mechanisms and kinetics that occur at specific environmental conditions (time, temperature, gas, pressure), in real-time, and at high spatial resolution.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at ...

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (&#181;CT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. &#181;CT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the ...