The first step in leaf sampling is to collect the leaves from the locations that are being sampled. In this case, the three locations being sampled were in rural, suburban, and urban areas. The rural location (leaf 8) in this experiment was just outside of Jamesville, New York, on Henderson Road. The suburban leaves (leaf 7) were collected in DeWitt, New York, on Short Road. The urban leaves (leaf 9) were collected in downtown Syracuse, New York, on Adams Street, next to the I-81 highway (see map). At least two different species of leaves were collected from each location, to make sure that the differences between species were not factors in the results (see chart). These leaves were all collected on October 31, 1998. The leaves were wrapped in aluminum foil, which was placed into a plastic bag and stored in a freezer at about -20 degrees Celsius.

The scanning electron microscope (SEM) was chosen for this experiment because of its usefulness in analyzing small particles on surfaces. Preparation of samples for the SEM requires several steps. These steps are as follows.

From each location, one leaf was selected at random from the aluminum foil. A portion of approximately 1 to 2 cm² was cut from each of the leaves using a razor blade. Each of these portions was cut roughly in half. A strip of double-sided adhesive carbon tape was placed on a 5x5 cm aluminum sampling plate. One half of each leaf portion was placed top surface up (surface “a”) and the other bottom surface up (surface “b”) onto the carbon tape (see fig.1).

Normally, non-conductive materials, or “wet” samples, such as leaves, are dried and coated with a thin layer of conductive carbon or metal before use in the SEM to prevent a buildup of a charge on the surface of the sample. However, the RJ Lee PSEM that was used for this experiment has a “variable pressure” feature that allows control of the air pressure inside the sample chamber. With higher pressure, more air molecules are present to conduct the charge, so it doesn’t build up on the sample surface. Therefore, instead of being dried and coated, the leaf samples were kept in their natural state to minimize the number of variables and to shorten the preparation procedure.

The sampling plate with the leaf samples was placed into the RJ Lee PSEM sample chamber immediately after preparation. Using the variable pressure feature, the chamber was evacuated to an air pressure of 0.2 torr. The electron beam was turned on to a voltage of 20 kV, the maximum voltage for this SEM, and the filament was saturated. The maximum voltage was used to provide the best backscattered electron (BE) imaging and x-ray information.

The method used to count and measure the particles on the leaves was the automated analysis. In this type of analysis, which is another feature of the RJ Lee PSEM, the area of analysis and the parameters are set, and the computer program controls the analysis.

The first step to setting up the automated analysis is to set the basis of the analysis. In this step, the run number is set, the samples are named and the run letters (i.e. run 89a) for each sample are set. After this the parameters for the analysis are set. This tells the program the limits of the analysis, such as the maximum number of particles to count, the minimum and maximum sizes for particle analysis, the maximum amount of time, etc. This also sets the magnification for the analysis. In this experiment, the analyses were set to analyze up to 500 particles (up to 1000 in run #94) from each leaf portion. The particle size limits set for this experiment were 0.12 µm minimum and 10.63 µm maximum. The maximum time set was 5 hours. The magnification for this experiment was set at 1000x.

The next step is to set the stage coordinates for each leaf sample. This sets the corners of the area of each leaf to be analyzed, which, in this experiment, was a quadrilateral. This also sets the focus for each coordinate, so that if the sampling surface is not flat, the computer can calculate the slope of the surface and set the focus for each part of the surface.

The final step in setting up the automated analysis is setting the particle detection threshold (fig.2a). This setting ensures that the background surface (in this case, the leaf surface) is not analyzed, by setting the minimum brightness of particles that are analyzed. This is set so that the maximum number of particles possible and the least possible background “noise” (folds in the leaf surface, etc.) will be analyzed.

The automated analysis measures two aspects of the particles. First, the computer calculates the average diameter (Dave), which is the average of 16 different diameters of the particle. Next, the SEM does an x-ray analysis of the particle, which determines which elements are present in the particle. This spectrum (see fig.2b), along with the average diameter and the coordinates of the particle, is stored in the memory so that the data can be viewed at a later time. For every tenth particle, a digital image of the particle was stored.

The data collected from the automated analysis were stored on two 100-megabyte Zip™ disks. This data was opened on a PC using the Zepview™ program, a program made for viewing data from automated analyses. From there, the data was exported into Microsoft Excel format. Excel was used to determine median Dave, concentrations, and chemical classification statistics of the particles, after which the different leaves were compared. These comparisons were displayed in graphs (averages and standard error are shown).

The preparation, analysis, and data analysis steps were repeated five times. Three of these runs were repeated using the same sample. The dates of the runs were 11/1/98 (#87), 11/2/98 (#88/89), 1/22/99 (#93), 1/23/99 (#94), 2/9/99 (#97/98), 2/18/99 (#99), 3/5/99 (#123) and 3/6/99 (#124) (see chart). Two of the runs (#87 and #123) were discarded because of problems with the automated analysis.