Preparing teeth for viewing with the scanning electron microscope (SEM) © June 1, 1999 Thomas Martin & John H. Wahlert

1. Specimen data

   Collect isolated incisors and pieces sawed from incisors in jaws and skulls. Place each specimen in a separate box with a label that at least includes specimen number, tooth information (right/left, upper/lower, direction toward worn tip). Save the boxes to receive the blocks that will be made in step 3 and worked in subsequent stages. Decide how you will glue these to the SEM stub. Wahlert prefers the flat, mesial side down.

2. Embedding

   Products
   Polyester resin or epoxy are suitable for embedding specimens. For small quantities, materials are available in some hobby shops. Follow the directions on the product. Large quantities may be obtained from plastics suppliers.
   We used Clear Casting Polyester Resin for casting and embedding (thin castings up to 1/2 inch thick). Industrial Plastics Supply Co., 309 Canal St., New York, NY.		Liquid hardener for hardening polyester resins and compounds
   10 grams: 20 grams: 30 grams (~1 ounce):		7 drops 14 drops 21 drops
   Mix well with wooden stick

   In a small flexible plastic mold (obtainable at a hobby shop), pour a thin layer (1 to 2 mm thick) of mixed polyester; you will hold onto this bottom layer during the grinding procedure (4 below). When polyester is hardened but still tacky, place incisors in orientation for sectioning; place tiny paper labels with identifying numbers in pencil below each specimen. Keep in mind that the plastic must be sawed into squares small enough to fit on an SEM stub. Pour in newly mixed polyester to cover specimens.

   

3. Sawing

   When polyester is hard, remove from mold, saw into cubes, each containing one incisor and its label. For this gross cutting, we use a water-cooled coarse diamond saw, a metal disc of about 1.5 mm thickness. The blocks will probably fit into the specimen clamp of the precision saw, and it is a good idea to make them longer on the side where they will be clamped. Put each block into its box.




In order to make both sagittal and transverse sections of the same tooth, the blocks can be sawed through the specimens using a low speed, thin blade, precision diamond saw for mineralogical samples. The orientation of each tooth can be indicated by scratching on a side of the plastic block an arrow that points in the direction of the wear tip of the incisor. You can also scratch an identifying number into the block.

4. Formatting: Grinding

   It is necessary to grind away the plastic and the tooth down to the desired plane of section. We use a rotating wheel (8 inch diameter) covered with an abrasive paper disc that is bathed in water. The paper is Carbimet ® Special Silicon Carbide grinding paper for metallography, wet or dry; KH Grit 320, manufactured by Buehler, Lake Bluff, IL. The plastic blocks are further reduced and formatted to fit on the stubs by further abrasion. Final polishing of the planes of section is done with Buehler Aluminum Oxide powder, 5.0 micron, in water on a glass plate. The surface should look matte when dry, and there should be no remaining scratches from the abrasive paper. Scratch the specimen number on the side of the finished block.

5. SEM stub preparation

   Aluminum stubs may be sanded to increase surface roughness and adhesion of glue (this step is not necessary, but Wahlert likes it). With a carbide steel point, scratch an identifying number into the top of each SEM stub close to the edge; you will want to be able to see the number when viewing with the SEM.

6. Gluing

   Each plastic block containing a prepared specimen is glued to the SEM stub that bears the corresponding number. Try not to cover the number; you will want to see it with the SEM. A conducting paint such as, SPI Conductive Carbon Paint can be used as glue; this is a colloidal graphite in isopropanol - 20% solids, made by SPI, a division of Structure Probe Inc., West Chester, PA. Paint has no shear strength and makes a weak bond. For a strong bond a glue such as Duco Cement may be used.

7. Etching

   2N HCl is the acid of choice for etching enamel. Etching takes from 2 to 4 seconds depending on the induration of the tooth. You may have to re-polish and re-etch the tooth if the original time is not sufficient.



8. Cleaning

   The etched specimens on stubs may be ultrasonically cleaned in water for 30 seconds to remove any remaining abrasive. SEM stub holders are plastic and have holes in which the stubs fit snugly. The entire holder can float face down in the water of the ultrasonic bath. Rinse gently in distilled water.

9. Coating

   Specimens should be thoroughly dry before coating. Keeping them warm for a few hours is sufficient or they may be dried more quickly in a desiccation chamber. Gold or gold palladium are ideal coatings and can be applied for 2 to 6 minutes. The thickness of the coating depends on the scope. Our new Hitachi SEM requires only 2 minutes of coating.



10. SEM viewing

    For best results, view with the specimens untilted. The new scanning electron microscopes give excellent results at 5 KV.
    
    

Acknowledgments: We thank the Department of Earth and Planetary Science and the Department of Vertebrate Paleontology for preparation facilities, equipment and supplies, and we thank the Interdepartmental Laboratories for access to and assistance with the Hitachi S4700 cold field emission scanning electron microscope at the American Museum of Natural History.

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