One in a Million
By Jason Socrates
Bardi
"Our main tool of choice is flow cytometry," says Associate
Professor Michael McHeyzer-Williams. This tool allows him
to analyze components of the immune system and immunological
processes one cell at a time.
Flow cytometry was invented early 1970s by a group at Stanford
led by Len Herzenberg, who patented his "fluorescence activated
cell sorter" in 1972. The technology was similar to a device
used by geologists to interrogate samples of stone to determine
mineral composition. The technology arose after scientists
had found a way to generate antibody-containing reagents that
are specific for particular proteins or other individual markers
on the surface of cells. What Herzenberg did was to attach
fluorophores—colors—to these antibodies.
Analytically, the actual detection is based on the fluorescence
of the fluorophores to which antibodies have been attached.
Different antibodies have different fluorophores, which give
different colors. In a typical flow experiment, some tissue
or organ is dissociated into single-cell suspensions and labeled
with the colored antibodies. The more colors you use, the
more separating power you have.
When McHeyzer-Williams was in graduate school, for instance,
he worked with four different antibodies and four different
colors to sort his cells based on four different surface molecules.
Through the years, the technique has developed incrementally
into a powerful tool for everything from the high speed sorting
of cells by cell type to the isolation of a single cell from
within a large population of cells.
Flow cytometry is also now much faster than it used to be.
Instruments typically can sort through 6,000 cells per second
and the fastest ones can sort through 25,000 per second. But
the number of colors that can be used has also improved, and
with it, resolution.
"Every time you add a color, you increase the resolution
of the sub-population of cells you can find by 10-fold," says
McHeyzer-Williams. He himself uses six colors routinely, which
allows him to find one cell in 100,000 or one in a million.
The power of flow is not derived solely from the technology,
the instrument, but also from the biology—the nuanced
strategy that a scientist like McHeyzer-Williams employs to
identify the right population of cells. There is a lot of
strategy involving variables such as how to run the experiment
and which colors and antibodies to use.
Flow cytometry is a powerful way of looking for the proverbial
needle in a haystack. More descriptively, it is a way of taking
a haystack, running it down a conveyor belt one straw at a
time, and finding the needles because they are shinier than
everything else.
In the apparatus, the cells in a buffered solution stream
through a small tube in the instrument, and they flow past
a detector built with one or more lasers (McHeyzer-Williams's
brand new instrument has three). The lasers blast the flowing
cells with light at particular wavelengths, and various wavelengths
excite the various fluorophores if they are present on the
surface of the cells. A lens and an optical bench full of
charged coupled devices like those you might find in a digital
camera resolve the different colors of emission, and a computer
combines these into a picture.
Flow cytometry can be used in tandem with other techniques
and technologies like quantitative PCR or gene microarrays,
which then allow you to sort the cell and then ask what genes
are being expressed therein or how much of one particular
gene is being expressed.
So to further the analogy, flow cytometry is like a way
of finding a needle in a haystack where once you find it,
you can also ask where that needle came from, when it was
made, and who made it.
"Flow is not just the instrument," says McHeyzer-Williams.
"It's the way you design [the experiment]."
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