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2008 Research Grant Recipient

Nicole d’Entremont
Mount Allison University

Cancer in the woods?

Photo: Mary McQuaid
Emissions from a pulp mill, a tire-manufacturing facility and a provincially operated coal plant all contribute to the chemical soup in Nova Scotia’s Pictou County, about 160 kilometres northeast of Halifax. The rate of prostate cancer among the county’s 46,500 residents is 24 percent higher than the provincial average.

Nicole d’Entremont, a recent graduate in physics at Mount Allison University, in Sackville, N.B., is studying tree rings to determine whether the region’s trees also show cancerlike symptoms — such as an increased replication of cells — given that they are exposed to the same environmental conditions as humans.

With funding from The Royal Canadian Geographical Society, d’Entremont (above) ventured into the woods of Pictou County last summer to extract core samples from trees at various distances from the factories. She then applied a process called flow cytometry, typically used in oncology, but rarely applied to plants.

In flow cytometry, a stream of liquid containing biological cells flows through two pressurized containers. The stream is so thin that a laser can illuminate a single cell, allowing scientists to examine each one individually and to monitor how many times a cell duplicates. Excessive duplications may indicate cancer.

But flow cytometers are expensive and are designed to accommodate the cells of animals, not plants. “It’s proving very difficult to get tree cells to suspend in a liquid,” says d’Entremont. Undeterred, she has built her own flow cytometer with the help of one of her professors. Results of her study are pending as she develops a method of analysis that works with various species of trees.

— Marielle Picher

Nicole d’Entremont
Mount Allison University

Enhancing dendrochronology through the use of flow cytometry

Taking a sample from a black spruce in Garden of Eden. This was one of the pristine sites visited.
Flow cytometry is a process used in the field of Physics, where individual cells (typically human) move in suspension past the path of a laser beam. There were two main goals of this project; to build a simple flow cytometer (commercial units are complicated and also very costly), and then to analyze tree cells to see if it is possible to detect various properties such as age, difference between early wood and late wood of a tree and species. This would be particularly important in dendro-archaeology, where often samples from houses and other historic architectures have little indication of species, since they are so old. We also hoped to see if flow cytometry could detect cancerous or other illnesses in trees.

Building a working flow cytometer was the first task. Despite many setbacks, the unit was built and has been pressure tested (necessary for working conditions). This flow cytometer uses basic ideas from commercial units while also implementing new ideas to make analysis simpler, and decrease the cost substantially. It is my hope that this new unit will make flow cytometry more accessible for the study of trees and other plants, as it is currently not widely used due to its costliness. In January, the original design was given to a mechanic, who used sturdier materials and more accurate measurements to make the flow cytometer even better. The new model has passed the pressure test, and cells can flow through with greater ease.

Though it was a cloudy day, the smog rising from one of the factories is still clearly visible.
In order to analyze data, it is necessary to build detectors to transfer laser light signals to an oscilloscope or computer. The detectors were also an original design by the lab. The first detector was designed to detect signals on an oscilloscope; an operational amplifier allows the human eye to easily view the signal from the cells on the oscilloscope screen, which would otherwise be too small to see. The light from the flow cytometer’s laser is picked up by a photodiode, which is what sends the signal to the oscilloscope. Our next detectors will capture the peak voltage (height) of the signal for a short time (100 microseconds) so the data can be read by an analog to digital converter on a computer. This will allow for data to be collected and stored on a PC. This detector is still in progress, and will hopefully be finished in April.

Fieldwork to obtain tree samples was conducted in mid to late August. I visited two pristine sites near Seafoam and Garden of Eden, Nova Scotia, and three sites with more environmental degradation near factories in Granton, Abercrombie and Trenton, NS. These sites had four tree species in common; white birch, red maple, trembling aspen and black spruce. This is an ideal mix of species as we would like to analyze both hard and softwoods using flow cytometry. Samples were taken using a coring tool, which allows for small pencil-sized samples to be extracted from the tree. Two samples were taken from each tree, and two trees per species were sampled at each location (for a total of 16 cores at the sites). By taking samples from different pollution levels, we hope to find a difference in the cells. If this method is successful, it could potentially be used as an indicator of tree health in an area.

We were assisted by two high school students as part of the “Go Global” program run by Mount Allison. Here, one of the students demonstrates the maceration process.
Back in the lab, it was necessary to design a method to separate cells, and put them in suspension (in a liquid solution). Since plant flow cytometry is relatively unheard of, there is currently no set method for separating tree cells, but we were fortunate enough to have Dr. Robert Thompson (a retired biology professor at Mount Allison University) guide us through a method which may work on woody plants (it had previously been used on other plant materials). This method has been adapted for our purposes, and is still being worked on to obtain the optimal results from all species. The current procedure is as follows:

The samples are cut into thin slivers and placed in a bath of glacial acetic acid and hydrogen peroxide. The solution is boiled gently for 3 hours, which somewhat separates the cells and fibers. The solution is filtered out, and a pecinase (dissolves pectin, the material that makes woody material stick together) solution is added. This is allowed to sit overnight (approximately 16 hours), after which the pectinase solution is removed and replaced by a saline solution with which we can use to look at cells under a microscope.

It was suggested that our first attempt be on a dense hardwood, so while perfecting the method local red oak samples were used. The cells separated quite nicely using this method, and two distinct types of cells could be identified under a microscope: xylem vessels and tracheids. These samples were run through the flow cytometer with success, and could be easily viewed using an oscilloscope. However, the cell separation method did not produce satisfactory results when using the white birch species. Thus, the ideal method for separating cells for all species is still being determined. The next step will be to analyze these cells and determine what properties, if any, can be gathered from flow cytometry.

Testing out the new flow cytometer model. Red goggles are necessary as the green laser used can quickly cause permanent damage to the eyes.
Recently, the flow cytometry lab has been working in collaboration with Dr. Zoe Finkel from Mount Allison’s Biology department. Dr. Finkel has a Flow Camera, or Flow Cam for short, which is similar to a flow cytometer, but instead of taking data through laser signals, takes pictures of individual cells as they flow past the lens. Dr. Finkel was interested to see how results from the flow cam compare to results from the flow cytometer. Currently, the flow cam cannot take many pictures in focus, as cells can be various distances from the camera. The next step in this study will be to use a nozzle (similar to the one used in the flow cytometer) to arrange cells so they will consistently be in focus. Then, analysis of the cells can take place.

I gave an oral presentation on this project in September at Mount Allison’s Science Undergraduate Research Fair. Of the many presentations done, this one took second place, and I received many comments on how interesting the project was. I am pleased to see that others are interested in this research and I plan on continuing cell analysis this summer. Many thanks for helping this project happen.

— Nicole d’Entremont

A red oak cell, seen using Dr. Zoe Finkel’s flow cam.


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