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

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 resin^{1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18}.

1. Preparation of support film (Figure 1-3)

NOTE: Silver-colored Formvar support film is prepared using the cast-on-glass method¹⁹.

1. 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 'a' using a rubber ball¹⁹.
   1. Drain the solution from the column by opening the three-way stopcock and releasing air through 'b' 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.
2. 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°, Figure 2b).
3. 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).

2. Trimming the specimen block with an ultrasonic trimming blade and a razor blade ²¹ under a stereomicroscope (Figure 4)

1. 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).
2. 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.
3. 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).

3. Trimming the specimen block with diamond knife using the microtome (Figure 5)

1. Set the specimen block chuck in the specimen holder of the ultramicrotome. Place the specimen 90° counterclockwise against the actual position when serial ultrathin sectioning is performed (See Figure 5d).
2. 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.
3. Place a mirror (Mesa cut, M) on the knife stage to monitor the cutting of the specimen block (Figure 5c).
4. 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° to the left (Figure 5a).
5. Cut the block face at about 100 µm 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° 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.

4. Adjusting the specimen surface and knife edge so that they face parallel to each other ²¹

1. Remove the trimming knife, and rotate the specimen block 90° clockwise.
2. Set an ultrathin sectioning knife to the knife stage.
3. 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.

5. Spreading neoprene solution on the specimen block (Figure 6)

1. 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).
2. 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.
3. Using a Pasteur pipette, drop about 1 µL 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.
4. 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°, treating the grids with 0.5% neoprene solution, and making the grids hydrophilic by glow discharge²².

6. Making serial sections (Figure 7-9)

1. 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.
2. Fill the knife boat with water.
3. 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's hands to the microtome.
4. 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.
5. After the first section is cut, set the section thickness to 70 nm (Figure 8).
6. 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.
7. 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.
8. Set the section thickness back to 70 nm (Figure 8) and continue cutting until 1.8-mm-long sections are obtained.
9. 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.

7. Picking up serial sections (Figure 10-12)

1. Place the 'Section-holding loop'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.
2. Place 'Water-surface-raising loop'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.
   1. 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.
   2. 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.
   3. 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).
3. 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.

8. Staining sections^{16, 24} (Figure 13)

1. After the sections are completely dried, tear the Formvar film from around the grids, and remove the grids bearing the sections.
2. 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.

9. Observation of serial sections (Figure 14)

1. 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 'grid-fixers' will not touch the handles.
2. Fix the grids with 'grid-fixers' (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.
3. Take pictures of the target cells in all the cell sections at high magnification.