Walter Dawydiak
At Bigelow Laboratory’s JJ MacIsaac Facility for Aquatic Cytometry, we use a technology called
flow cytometry to analyze samples containing microscopic marine organisms including algae, bacteria,
and zooplankton. We count and characterize these organisms because they’re an extremely important
component of the marine food web and global carbon cycle. Although they’re tiny – we can’t see them
with the naked eye – there are many thousands of them in every drop of seawater and they have a
profound influence on the global environment.
So, how does it work?
Flow cytometers utilize laser light to gather data on several properties of cells being studied,
including size, internal complexity, and fluorescent properties. Cells in a sample begin their journey
through the flow cytometer by passing through the sample injection line and into the flow cell, which is
the heart of the instrument. Here, the cells are organized neatly in a single file stream of cells, as seen in
the figure below.
This organization of the cells is absolutely vital to the proper performance of a flow cytometer and is
achieved through hydrodynamic focusing, whereby a secondary sheath fluid is streamed around the
outside of the sample fluid, similar to how rubber insulation covers the outside of a wire.
Once the cells are in line with the stream, they then pass through the interrogation point where
the laser intersects the stream. The cells pass through the laser, one at a time. When a cell passes
through the laser, the path of the laser is interrupted, and the laser light is scattered. The amount and
direction of the scattered light can be measured using photodetectors placed at strategic locations in
relation to the interrogation point. The nature of the light scatter depends on a cell’s size and internal
complexity.
Beyond simple light scatter, flow cytometers also rely on fluorescence to better understand cells
in a sample. Natural pigments in the organisms we study will fluoresce when exposed to certain types of
energy. Laser light can excite electrons in these pigments and cause this fluorescence to occur. In this
manner, cells not only scatter and redirect the light from the laser, but they also absorb it and re-emit
light of a different, unique wavelength. As cells pass through the laser beam, their scatter characteristics
and fluorescence are measured almost instantaneously using many light filters, mirrors, and
photodetectors. The cells then flow into a waste container for disposal.
This entire process happens extremely fast. Some machines can accurately collect data on
100,000 cells or more every second. As optical, mechanical, electrical, and processing technologies
improve and are further refined, the rate at which we can analyze cells grows faster. This means that we
can process samples faster and more accurately, which opens the door to more extensive
experimentation and analyses.
Why is it useful?
There are several advantages of using flow cytometry over other analytical techniques such as
microscopy. It allows for the quantitative analysis of large populations of cells in a relatively short
amount of time. Although flow cytometers cannot produce images of cells or provide a direct visual view
of a cell, they can provide a multitude of information on the physical characteristics of each cell based
on how it interacts with the laser light. This information can be used to analyze characteristics of
individual cells such as life cycle stage or overall health, as well as facets of the greater microbe
communities present in a sample.
Walter Dawydiak is a University of Pennsylvania student in Bigelow Laboratory for Ocean Science’s Research Experience for Undergraduates program. This intensive experience provides an immersion in ocean research with an emphasis on hands-on, state-of-the-art methods and technologies.