Name: A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy (Video) | JoVE Journal
Uploaded: 2018-01-17T10:45:00.000+00:00
Duration: 9 min 46 s
Description: Learn a cost-effective method for serial ultrathin sectioning of microorganisms in transmission electron microscopy. This technique enables high-resolution 3D structural analysis of whole cells without expensive equipment.

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5 minutes

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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<li>Yamaguchi, M. <a target="_blank" href="http://cat.inist.fr/?aModele=afficheN&cpsidt=17883841">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/pubmed?term=((Dynamics+of+the+spindle+pole+body+of+the+pathogenic+yeast+Cryptococcus+neoformans+examined+by+freeze-substitution+electron+microscopy%5BTitle%5D)+AND+%22FEMS+Microbiol.+Lett%22%5BJournal%5D)">Dynamics of the spindle pole body of the pathogenic yeast Cryptococcus neoformans examined by freeze-substitution electron microscopy.</a> FEMS Microbiol. Lett. 296, 257-265 (2009).</li>
<li>Yamaguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+spindle+pole+body+of+the+pathogenic+yeast+Cryptococcus+neoformans%3A+variation+in+morphology+and+positional+relationship+with+the+nucleolus+and+the+bud+in+interphase+cells%5BTitle%5D)+AND+%22J.+Electron+Microsc%22%5BJournal%5D)">The spindle pole body of the pathogenic yeast Cryptococcus neoformans: variation in morphology and positional relationship with the nucleolus and the bud in interphase cells.</a> J. Electron Microsc. 59, 165-172 (2010).</li>
<li>Yamaguchi, M. Structome of Exophiala yeast cells determined by freeze-substitution and serial ultrathin sectioning electron microscopy. Curr. Trends Microbiol. 2, 1-12 (2006).</li>
<li>Yamaguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structome+of+Saccharomyces+cerevisiae+determined+by+freeze-substitution+and+serial+ultrathin+sectioning+electron+microscopy%5BTitle%5D)+AND+%22J.+Electron+Microsc%22%5BJournal%5D)">Structome of Saccharomyces cerevisiae determined by freeze-substitution and serial ultrathin sectioning electron microscopy.</a> J. Electron Microsc. 60, 321-335 (2011).</li>
<li>Biswas, S. K., Yamaguchi, M., Naoe, N., Takashima, T., Takeo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+three-dimensional+structural+analysis+of+Exophiala+dermatitidis+yeast+cells+by+freeze-substitution+and+serial+ultrathin+sectioning%5BTitle%5D)+AND+%22J.+Electron+Microsc%22%5BJournal%5D)">Quantitative three-dimensional structural analysis of Exophiala dermatitidis yeast cells by freeze-substitution and serial ultrathin sectioning.</a> J. Electron Microsc. 52, 133-143 (2003).</li>
<li>Yamada, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structome+analysis+of+Escherichia+coli.+cells+by+serial+ultrathin+sectioning+reveals+the+precise+cell+profiles+and+the+ribosome+density%5BTitle%5D)+AND+%22Microscopy%22%5BJournal%5D)">Structome analysis of Escherichia coli. cells by serial ultrathin sectioning reveals the precise cell profiles and the ribosome density.</a> Microscopy. 66, 283-294 (2017).</li>
<li>Yamada, H., Yamaguchi, M., Chikamatsu, K., Aono, A., Mitarai, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4309607/">Structome analysis of virulent Mycobacterium tuberculosis, which survives with only 700 ribosomes at density per 0.1 fl cytoplasm.</a> PLoS ONE. 10, e0117109(2015).</li>
<li>Yamaguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-voltage+electron+microscopy+tomography+and+structome+analysis+of+unique+spiral+bacteria+from+the+deep+sea%5BTitle%5D)+AND+%22Microscopy%22%5BJournal%5D)">High-voltage electron microscopy tomography and structome analysis of unique spiral bacteria from the deep sea.</a> Microscopy. 65, 363-369 (2016).</li>
<li>Yamaguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prokaryote+or+eukaryote%3F+A+unique+microorganism+from+the+deep-sea%5BTitle%5D)+AND+%22J.+Electron+Microsc%22%5BJournal%5D)">Prokaryote or eukaryote? A unique microorganism from the deep-sea.</a> J. Electron Microsc. 61, 423-431 (2012).</li>
<li>Yamaguchi, M. <a target="_blank" href="https://www.jstage.jst.go.jp/article/cytologia/78/4/78_333/_article">Ultrathin sectioning of the Myojin parakaryote discovered in the deep sea.</a> Cytologia. 78, 333-334 (2013).</li>
<li>Yamaguchi, M., Worman, C. O. <a target="_blank" href="http://protistology.jp/journal/jjp47/JJP47YAMAGUCHI.pdf">Deep-sea microorganisms and the origin of the eukaryotic cell.</a> Jpn. J. Protozool. 47, 29-48 (2014).</li>
<li>Yamaguchi, M. <a target="_blank" href="https://www.jstage.jst.go.jp/article/myco/65/2/65_81/_article/-char/ja/">An electron microscopic study of microorganisms: from influenza virus to deep-sea microorganisms.</a> JSM Mycotoxins. 65, 81-99 (2015).</li>
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<li>Yamaguchi, M., Biswas, S. K., Suzuki, Y., Furukawa, H., Takeo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three-dimensional+reconstruction+of+a+pathogenic+yeast+Exophiala+dermatitidis+cell+by+freeze-substitution+and+serial+sectioning+electron+microscopy%5BTitle%5D)+AND+%22FEMS+Microbiol.+Lett%22%5BJournal%5D)">Three-dimensional reconstruction of a pathogenic yeast Exophiala dermatitidis cell by freeze-substitution and serial sectioning electron microscopy.</a> FEMS Microbiol. Lett. 219, 17-21 (2003).</li>
<li>Kozubowski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ordered+kinetochore+assembly+in+the+human+pathogenic+basidiomycetous+yeast%2C+Cryptococcus+neoformans%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Ordered kinetochore assembly in the human pathogenic basidiomycetous yeast, Cryptococcus neoformans.</a> mBio. 4, e00614-e00613 (2013).</li>
<li>Yamaguchi, M., Adachi, K. Specimen support. <a target="_blank" href="http://microscopy.or.jp/english/">Guide Book for Electron Microscopy</a>. The Japanese Society of Microscopy, , 2nd ed, Kokusaibunkeninsatsusya, Tokyo. 52-57 (2011).</li>
<li>Hayat, M. A. Support films. <a target="_blank" href="https://www.cabdirect.org/cabdirect/abstract/19822298860">Principles and Techniques of Electron Microscopy: Biological Application</a>. , 4th ed, Cambridge, Univ. Press. Cambridge, United Kingdom. 216(2000).</li>
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<li>Yamaguchi, M., Aoyama, T., Yamada, N., Chibana, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+measurement+of+hydrophilicity%2Fhydrophobicity+of+the+plasma-polymerized+naphthalene+film+%28Super+support+film%29+and+other+support+films+and+grids+in+electron+microscopy%5BTitle%5D)+AND+%22Microscopy%22%5BJournal%5D)">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>
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<li>Yamaguchi, M., Shimizu, M., Yamaguchi, T., Ohkusu, M., Kawamoto, S. <a target="_blank" href="https://www.jstage.jst.go.jp/article/plmorphol1989/17/1/17_1_57/_article/-char/ja/">Repeated use of uranyl acetate solution in section staining in transmission electron microscopy.</a> Plant Morphology. 17, 57-59 (2005).</li>
<li>Hayworth, K. J., Kasthuri, N., Schalek, R., Lichtman, J. W. <a target="_blank" href="http://homes.mpimf-heidelberg.mpg.de/~mhelmsta/pdf/2006%20Hayworth%20Kasthuri%20Schalek%20Lichtman%20MicroscMicroanal.pdf">Automating the collection of ultrathin serial sections for large volume TEM reconstructions.</a> Microsc. Microanal. 12, Suppl . 2. 86-87 (2006).</li>
<li>Hildebrand, D. G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-brain+serial-section+electron+microscopy+in+larval+zebrafish%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Whole-brain serial-section electron microscopy in larval zebrafish.</a> Nature. 545, 345-349 (2017).</li>
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<li>Heymann, J. A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Site-specific+3D+imaging+of+cells+and+tissues+with+a+dual+beam+microscope%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">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&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

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...

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 ...

The overall goal of this procedure is to make serial ultrathin sections safely and easily. This method can help answer key questions in the cell biology field, providing quantitative and three-dimensional structural information of a whole cell at the electron microscopic level. The main advantage of this technique is that serial ultrathin sectioning can be done without an expensive machine, and high resolution images can be obtained with this method.To begin this procedure, clean the surface of a glass slide. Then dip one half of the glass slide in 1.5%Formvar solution in the upper column of the Formvar support filmmaking apparatus by pressing the solution with air through the three-way stopcock using a rubber ball. Drain the solution from the column by opening the three-way stopcock and releasing air to reduce the pressure.Then take out the glass slide from the apparatus, and dry in air to form a film on the surface of the glass slide. Scrape off the four edges of the film on the glass slide with a razor blade. After breathing onto the slide to facilitate the separation of the film from the slide, float off the film by immersing the glass slide into water slowly at a low horizontal angle.Then scoop up the Formvar film from the water using an aluminum rack with holes. To trim the specimen block with an ultrasonic trimming blade and a razor blade under a stereo microscope, first make sure that there are cells on the surface of the block by observing the tip of the block with a light microscope. Then mount the specimen block in the chuck, and mount the chuck on a trimming stage.Trim the blocks to a size of 0.7 by 1.0 millimeters. If the epoxy resin is very hard, trim the box first using an ultrasonic trimming blade, then trim the block further with a razor blade. Cut one shoulder to mark the direction of cutting.To trim the specimen block with a diamond knife using the microtome, set the specimen block chuck in the specimen holder of the ultra microtome. Cut the surface of the block with a diamond trimming knife. 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.Cut the left edge of the block using the trimming knife by rotating the knife stage 30 degrees to the left. This edge become the upper side of the sample in serial sectioning. Then cut the block face at about 100 microns from the left edge of the specimen by rotating the knife stage 30 degrees to the right.This edge becomes the lower side of the specimen in serial sectioning. Remove the trimming knife, and rotate the specimen block 90 degrees clockwise. Set an ultrathin sectioning knife to the knife stage.Adjust the specimen surface and the knife edge so that they face parallel to each other using the large part of the specimen surface. Next, apply tape on the chuck and the chuck holder of the microtome, and cut the tape at the boundary. Take the specimen chuck out from the microtome and place it under the stereo microscope.Cut off the large part of the specimen with a razor blade leaving the slim part from which serial sections will be obtained Make the section sides adhesive by using a Pasteur pipette to drop about one microliter of 0.5%of Neoprene solution onto the specimen to be used for serial sectioning. Then cover the whole specimen with Neoprene solution. Absorb excess Neoprene solution immediately with a piece of filter paper placed near the specimen.Next, prepare three slit grids for picking up serial sections. To do so, bend the handle of the grids to 60 degrees, and treat the grids with 0.5%Neoprene solution. Then make the grids hydrophylic by glow discharge.Place the specimen block chuck back in the microtome at the same position 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.After filling the knife boat with water, cover the microtome with a plastic cover to prevent air flow. Start cutting the specimen at a section thickness of 200 nanometers. After the first section is cut, set the section thickness to 70 nanometers.When the number of serial sections reaches 20, set the section thickness to 10 nanometers. Since the microtome cannot cut 10 nanometer thick sections, no new section appears, and the previously cut sections become separated from the knife edge. Next, set the section thickness to 60 nanometers.This will produce 70 nanometer sections because the machine will add the previous 10 nanometer thickness. Finally, set the section thickness back to 70 nanometers, and continuing cutting until 1.8 millimeter-long sections are obtained. To pick up the serial sections, first place the section holding loop on the third section group.Then place water surface raising loop on the knife stage. 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.Push the loop down by turning the screw downward so that the water surface is raised by surface tension. 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 strand.Pick up section groups by touching it with a bare slit grid, which is helped by tweezers and kept parallel to the water surface. Place the grids with sections along with a tiny drop of water onto the Formvar support film. Remove excess water using filter paper.After the sections are completely dried, tear the Formvar film around the grids;and remove the grids bearing the sections. Set the grids in the groove of the staining tube in the proper order, and stain sections with uranyl acetate and lead citrate. Set the five grids in the multi-specimen holder in the proper order, orienting the long axis of the grid slit perpendicular to the specimen holder axis.Fix the grid with grid fixers, and insert the specimen holder into the transmission electron microscope. Proceed to take pictures of the target cells and all the cell sections at high magnification. Shown here is a low-magnification view of five grids that carry serial sections.18-25 sections were mounted on one grid. Note that the sections are attached together and have the same thickness. Serial sections of pericarion myogenesis are shown here.The figure shows 12 out of 67 complete sections. This cell was found to have a large nucleoid consisting of naked DNA fibers with a single nucleoid membrane and endosymbionts that resemble bacteria but no mitochondria. Thus, this organism appears to be an intermediate life form evolving from prokaryote to eukaryote.Shown here are serial sections of Escherichia coli. The figure shows 12 complete sections. Interestingly, this cell was found to contain 21, 700 ribosomes.After watching this video, you should have a good understanding of how to make serial ultrathin sections. Once mastered, this technique can be done in three hours if it is performed properly.

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Source: Lutz, D., et.al. Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord. J. Vis. Exp. (2019).This video demonstrates the preparation and transmission electron microscopy (TEM) analysis of a brain section from a mouse pup. The protocol starts with osmium tetroxide-fixed tissue embedded in resin, followed by precise trimming to isolate the region of interest and ultramicrotomy to produce semithin and ultrathin...

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Learn a cost-effective method for serial ultrathin sectioning of microorganisms in transmission electron microscopy. This technique enables high-resolution 3D structural analysis of whole cells without expensive equipment.

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

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