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Volume 76—1996

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Combining Sputter Coating with OTOTO Treatment to Eliminate Charging Artifacts in Pollen Preparations

William F. Chissoe1 and John J. Skvarla 1,2
1 University of Oklahoma and Samuel Roberts Noble Microscopy Laboratory, Norman, OK 73019
2 Department of Botany-Microbiology and Oklahoma Biological Survey, Norman, OK 73019

Received: 1996 Mar 25; Revised: 1996 Jul 01

Pollen grains made conductive for SEM by coating in a series of osmium and thiocarbohydrazide solutions (OTOTO) show charging artifacts when nonconductive detritus (sand grains, plant fragments, etc.) is present in the immediate sample area of the SEM scanning probe. Standard sputter coating of the sample makes the detritus electrically conductive and eliminates all electrical instabilities.

When acetolysis (1) is used to prepare pollen for scanning electron microscopy (SEM), some residue such as undigested plant parts, sand grains and other inorganic particles, collectively referred to as detritus, may remain, even following procedures designed to remove it (2, 3). Some of the detrital material does not incorporate osmium metal when the osmium - thiocarbohydrazide - osmium - thiocarbohydrazide - osmium (OTOTO) method is used to make the pollen grains electrically conductive (4). When struck by the electron beam this detritus becomes highly charged and can cause artifacts when it resides within the overscan portion of the raster, yet is outside the frame of the electron micrograph. The escape of secondary electrons from the surface of nearby electrically conductive pollen is essentially blocked giving rise to "shaded images" (Fig. 1) and "fuzzy" pollen surfaces (Fig. 3). Other effects of charging can be seen as scan lines (Fig. 3) and "glowing" pollen grains (Fig. 5).

The above artifacts can be easily removed by pulse sputter coating, usually for 3 minutes, using a gold or gold/palladium (60/40) target. This renders the detritus conductive and allows the secondary electrons from the neighboring pollen grains to be emitted and detected unimpeded to form crisp, artifact-free SEM images (Fig. 2, 4, 6).

Sputter coating of OTOTO-prepared pollen does not diminish the value of the OTOTO technique for pollen SEM or obviate it in favor of traditional sputter coating or vacuum evaporation methods. The consistently high quality of SEM images obtained after OTOTO, in contrast to these other problematic metal deposition methods, and the general paucity of detritus in pollen samples, greatly favor OTOTO as the most efficient way of preparing pollen for SEM. Detrital contamination was not considered in our earlier study (4), and it is included here as a supplement to it.


1.   Erdtman, G., The acetolysis method. Sven. Bot. Tidskr. 54, 561-564 (1960).

2.   Faegri, K., and Iversen, J., Textbook of Modern Pollen Analysis, Munksgaard, Copenhagen (1975) p. 103.

3.   Chissoe, W.F., and Skvarla, J.J., Sucrose density pads for concentration and purification of pollen grains. Stain Tech. 49, 123-124 (1974).

4.   Chissoe, W.F., and Skvarla, J.J., The use of osmium-thiocarbohydrazide for structural stabilization and enhancement of secondary electron images in scanning electron microscopy of pollen. Grana 34, 317-324 (1995).

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[Page 84 consists of Figures 1–6.]

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[The text which accompanies the figures occurs on page 85, but is presented with the images for convenience sake.]