Name: a method for obtaining serial ultrathin sections of microorganisms in transmission electron microscopy
Uploaded: 2018-01-17T10:45:00
Description: Watch this Scientific Journal Video about A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy at JoVE.com

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,</...

<ol>
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K., Suzuki, Y., Furukawa, H., 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=Three-dimensional+reconstruction+of+a+pathogenic+yeast+Exophiala+dermatitidis+cell+by+freeze-substitution+and+serial+sectioning+electron+microscopy.">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/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. 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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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