Procedure

In order to take these photos, we used a standard compound microscope (as shown) with a microscope camera mounted on the eyepiece. Unlike with the SEM and the Leica light microscope, we prepared normal microscope slide to view our pollen samples rather than the stub we prepared for the SEM. This allowed us to see color in the photos relatively well due to the way slides were back-lit.

We prepared the microscope slide by dissecting the specific plant to be viewed and locating the anthers. Next, we used a paintbrush to transfer pollen from the anther to a microscope slide. Once we had an adequate amount of pollen on the slide, we added a drop of water and a slide cover before making our observations with the microscope. The process of transferring iris pollen onto the slide is shown on the right. For our experiment, we observed the pollen of Iris (Iris Hollandica), Snapdragon (Antirrhinum) and Millet (Pennisetum glaucum).

Before using the SEM, we took a few photos using a Leica light microscope with a built-in camera. These photos were of a very high resolution, however the magnification of this microscope was only 35X, much lower than that of the compound microscope. While this microscope can pick up and display color, our photos were against the black background of the SEM stub and were only being lit from above. As a result, the photos show the basic exterior color of the pollen (and unintended contaminants in certain photos) but they do not give as much color detail as the
compound microscope.

We took our most detailed photos using a scanning electron microscope. This microscope allowed us to capture photos at up to 20,000X magnification, showing the surface and specific shape of individual grains of pollen. These photos should help us to determine and categorize specific characteristics of the different pollen types. Additionally, they helped us get very accurate measurements of the pollen grain size, letting us compare the different varieties of pollen in quantitative terms as well as qualitative ones. Unlike the optical microscopes, the SEM does not show color in its images because it doesn't use a conventional system of lenses and light for magnification.

Operating the SEM was much more complicated than the other microscopes. First of all, we had to prepare the special SEM stub with the pollen to be inspected in the microscope. To do this, we were given a metal stub part which we applied a circular piece of carbon tape to. Next, we made three sets of small lines, called fiduciary marks, that would assist us in determining where each type of pollen is located on the stub. We then extracted and applied the pollen to the sticky surface of the stub, in much the same way as we did for the light microscope slide.

After all three types of pollen had been added to the stub in their designated (and marked) areas, we blew "canned air" onto the stub. Doing so pushed the pollen deeper into the surface and helped prevent the pollen from coming off of the stub in the microscope, which could damage the SEM and our results. We stored the completed stub in a protective cardboard case (shown) to prevent damage.

When the stub's preparation was finally complete, we were ready to insert it into the SEM's "cup" and rotate it until the stub was 4 "clicks" below being flush with the cup's surface. Next, we opened the door of the SEM and inserted the cup. After closing the door again and seeing a "map" picture of the stub, we selected specific areas of interest and adjusted the focus and contrast as needed. When we found areas that would produce a good, clear picture, we saved images and in some cases used the measurement feature to find the size of the pollen grains. The SEM pictures turned out very well and can be seen here.