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

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.