Measuring Stomatal Density and Length in Sugar Maples

By Sydney Weimer

This semester, I have been conducting independent research on stomatal density and length on leaves collected from trees in the Control and Nitrogen plots within the MELNHE sites as a member of the Yanai Forest Ecosystem Ecology lab. The species that we focused on, and that I focused on for my project, is Acer saccharum. 

Figure 1. Taking the impressions of the leaves with tape, making sure to not get fingerprints on the slides by wearing a glove (Photo: Jenna Zukswert).

During my time as a lab host in April, I taught a few students how to prepare a slide for microscope analysis with leaves from different stands. I think it went well and everyone seemed to pick up how to make a slide easily. Everyone was interested in learning why we were doing this project and supported the project. It was fun to teach others how to make slides and everyone seemed to enjoy the process.

Stomatal density and length can show us how productive a tree is. Stomatas’ main purposes in plants are gas exchange and transpiration. For a more productive tree the stomata could have longer length or greater density than a tree that has a shorter length and smaller density. This is why we are comparing the Control to the Nitrogen stands, since Nitrogen can help the tree grow and not be limited by that particular resource. 

The first step in the procedure is procuring the materials. We use three glass slides for each tree–one per leaf–and each slide holds three nail-polish films. We use a glove to make sure no fingerprints are transferred to the slide. We used clear nail polish and tape to make and pick up the imprint of the leaf. We are not using the actual leaf, but rather an impression of it. Then finally, we use a sharpie to label the correct stand and numbers on each slide. 

Figure 2. Example of what leaf imprints look like on a slide (Photo: Sydney Weimer).

Each leaf in this experiment was collected with a shotgun, which is why some of the leaves have shotgun pellet holes in them. From each tree there were three leaves collected. I personally made 75 slides (75 leaves), which would equate to 25 trees. On the underside of each leaf, we paint three spots with clear nail polish, each about 1×1 cm, on the top, middle, and bottom, making sure to paint it close to the center vein. We paint close to the vein each time so that we can be consistent from leaf to leaf. We use clear nail polish so that they are easier to see underneath the microscope. After waiting 10 minutes, or until the nail polish looks dry, we put the glove onto our dominant hand. I did three leaves at a time so that once they dry I can do three slides. We then put a piece of tape over each spot of nail polish film, press down to get the film onto the tape, and peel the tape off the leaf and tape it onto the slide. Sometimes bits of leaves get stuck to the film; we try to remove any leaf bits. To view the stomata under the microscope we are trying to get only the nail polish film, because any leaf bits will obscure our view.

The next part of the process is imaging the slides with a microscope. Attached to one of the microscope’s viewing lenses is a camera. When a computer is connected via a USB port we can view the stomata and look around, adjusting the fine and coarse focus knobs to get the best view we can of the stomata (they look like coffee beans in the images). We tried to minimize features of the plant that did not have stomata (e.g., veins) in the image, which would bias our stomatal density results. 

Figure 3. An example of what an image taken by AmScope with a microscope looks like (Photo: Sydney Weimer).

Once the photo is taken, we use ImageJ software to analyze the pictures by counting all the stomata in the image. Each image has the same area on the leaf, which is very small, being only 0.066 mm^2. We use a random number generator to pick three of the stomata and measure their lengths. Then we can compare our data to see if there is a difference in stomatal density or length between the Nitrogen plots and the Control plots. If there are more stomata, or longer stomata, in the Nitrogen plots, this would suggest that, by adding Nitrogen, the trees could have a greater capacity for gas exchange or transpiration. This might suggest that the trees are limited by nitrogen, if adding nitrogen changes their anatomy in a way that helps them grow. We could implement this by helping struggling  forests grow by adding Nitrogen to it, so that they are not limited by it anymore. The forests will eventually be limited by another factor, but this could help our forests grow, which is important in our world today with deforestation among other problems.