2008 Research Grant Recipient
Mount Allison University
Cancer in the woods?
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.
|Photo: Mary McQuaid
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
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
Mount Allison University
Enhancing dendrochronology through the use of flow cytometry
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.
Taking a sample from a black spruce in Garden of Eden. This was one of the pristine sites visited.
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
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.
Though it was a cloudy day, the smog rising from one of the factories is still clearly visible.
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.
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:
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.
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.
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.
Testing out the new flow cytometer model. Red goggles are necessary as the green laser used can quickly cause permanent damage to the eyes.
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.