William F. Chissoe1 and John J. Skvarla 1, 2
1 University of Oklahoma and Samuel Roberts Noble Microscopy Laboratory;
2 Department of Botany-Microbiology and Oklahoma Biological Survey, Norman, OK 73019
Received: 1995 Aug 12; Revised: 1996 Mar 14
We have increased the efficiency for containing and transferring pollen during chemical drying procedures for conventional scanning electron microscope (CSEM) viewing by replacing standard laboratory containers (glass test tubes, pipettes, etc.) in the final drying stage with smoothed and polished metallic conical wells. The wells, formed by drilling into one end of aluminum CSEM specimen holders, are approximately 4 mm deep, 8 mm in top diameter, and 1 mm in bottom diameter. The large surface area allows rapid elimination of dehydrant. Unlike standard containers, to which dry pollen sticks after evaporation or sublimation of dehydrant, the wells allow pollen to be easily collected, concentrated, and transferred to CSEM specimen holders with minimal loss of sample. The conical wells have been used with alcohol, Peldri-II, and hexamethyldisilazane drying procedures and therefore, universal application seems likely.
Pollen must be dry before it can be examined in a conventional scanning electron microscope (CSEM). The simplest way to do this is by air-drying cleaned (1) and/or treated (2) pollen directly from absolute ethanol (EtOH) onto specimen holders, after the pollen has passed through a graded series of (EtOH-H2O) solutions with ascending concentrations of EtOH. This approach is expedient and insures minimal loss of pollen during dehydration and transfer to specimen holders, but it is often unacceptable because physical drying stresses can cause the pollen to wrinkle or collapse. In addition, the pollen grains are often poorly attached to the specimen holders and may be dislodged before CSEM viewing; they may also be so widely dispersed that they are difficult or impossible to locate; cf. Fig. 1, bottom inset (6).
Other drying procedures employed in conventional scanning electron microscopy [critical point drying (CPD; 3), freeze drying (FD; 4), Peldri-II drying (PD; 5), or hexamethyldisilazane drying (HMDS; 6)] provide better preservation of pollen structure. However, all these procedures require a container, usually a glass pipette or test tube, to complete the drying process (for example, Fig. 1H of reference 5). Prior to examination in the CSEM, chemically dried pollen is transferred to CSEM specimen holders by first tapping the containers to loosen the pollen and then sprinkling the free grains onto a holder, usually covered with an adhesive (Fig. 1I of reference 5;7). Frequently, some of the dry pollen adheres to the sides of the containers (Fig. 1, top and middle insets) and an appreciable amount is lost in the transfer process, as discussed in depth elsewhere (5). Additionally, drying in glass test tubes or pipettes requires extended time periods because the long side walls and small openings retard speedy elimination of dehydrant (for example, evaporation of EtOH and HMDS, and sublimation with PD; Fig. 1, top and middle insets).
We have improved the speed of drying and reduced the amount of pollen lost during transfer by performing the final drying step in shallow, cone-shaped wells that have been drilled into one end of JSM-2 (JEOL Inc.) cylindrical aluminum CSEM specimen holders (10 ( 10 mm), using a 3/8-inch drill bit with a 110° tip. The specimen holders are readily available through commercial electron microscopy supply dealers. The wells are approximately 8 mm in diameter at the top, 4 mm in depth, 1 mm in diameter at the base, and highly polished with metal polish after initial smoothing with steel wool (Fig. 1A). The wide top diameter allows rapid elimination of dehydrant, and the conical shape concentrates the pollen at the well bottom (Figs. 1B-C). The highly pol-
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ished finish promotes nearly total pollen transfer to CSEM specimen holders because dried pollen grains adhering to the interior surface of the well can be easily loosened with an "eyelash" stick (an eyelash glued to a thin wood stick) or fine brush (Figs. 1 D-E).
It is less important to emulate the exact dimensions of the wells than it is to insure a well that provides a large surface area, cone-shaped sidewalls, short depth, small bottom diameter, and highly polished surface. Although we used wells made of aluminum, any metal, such as copper, brass, or stainless steel, is suitable. Similarly, CSEM specimen holders can be of other brands and dimensions; they can also be made from any round metallic rod stock. The important point is that the well should not be plastic as dehydration fluids can react with the synthetic organic components and contaminate the sample. Attainment of these conditions will enable dehydrants to escape rapidly as well as allow direct access to the dried pollen. We have used conical wells with EtOH, HMDS and PD drying and feel that they can be used in other drying procedures. Further, the shallow wells may be suitable for manipulating pollen samples that are sparse, including single pollen grains and spores frequently encountered in geological preparations. Our procedure for drying pollen in conical wells is outlined in Figure 1.
We are deeply grateful to Susan Gray for preparing the technically precise illustrations and to Jeanne Skvarla for critical reading of the manuscript.
1. Chissoe, W.F., and Skvarla, J.J., Sucrose density pads for concentration and purification of pollen grains. Stain. Technol. 49, 123-124 (1974).
2. Erdtman, G., The acetolysis method. Sven. Bot. Tidskr. 54, 561-564 (1960).
3. Garner, G.E., and Bryant, V.M., Preparation of modern palynomorphs for scanning electron microscopy by the critical point drying method. Geoscience and Man 7, 83-88 (1973).
4. Nilsson, S., Nybom, R., and Praglowski, J., Experiments regarding collapsing of pollen grains in scanning electron microscopy. Grana 14, 23-25 (1974).
5. Chissoe, W.F., Vezey, E.L., and Skvarla, J.J., Drying of Pollen with Peldri-II (proprietary fluorocarbon) for scanning lectron microscopy. Rev. Palaeobot. Palynol. 63, 29-34 (1990).
6. Chissoe, W.F., Vezey, E.L., and Skvarla, J.J., Hexamethyldisilazane (HMDS) as a drying agent in pollen scanning electron microscopy. Biotechnic Histochem. 69, 192-198 (1994).
7. Chissoe, W.F., Vezey, E.L., and Skvarla, J.J., Mounting pollen on a thermoplastic adhesive for scanning electron microscopy. J. Am. Microsc. Soc. 113, 72-79 (1994).