SIGHTINGS
When Less Might Be More
Felice Frankel
When I first saw the color image on the facing page, I couldn't
figure out what microscopic technique was used to get those
colors (which translate to information). I was pretty sure some
sort of light polarization was used, but it didn't completely
resemble the techniques with which I am familiar as an untrained
user of optical microscopy. So I thought I'd invite myself to
Danielle Cook France's lab. I was ultimately drawn more to a
grayscale rendition from an aesthetic point of view, as I
mention below. Perhaps I am dating myself. But no matter: I put
myself in Danielle's capable hands, knowing there's no better
way to spend time than learning from the next generation.
Danielle Cook France is a graduate student in biological
engineering at the Massachusetts Institute of Technology. She
holds a B.S. in biomedical engineering from Washington
University in St. Louis. At MIT, she works with Paul Matsudaira,
director of the Whitehead-MIT Bioimaging Center (http://bioimaging.wi.mit.edu). Her thesis work,
which focuses on novel physical mechanisms of powering cellular
motility and their applications to devices engineered from
biological components, is supported by a Poitras Fellowship and
the U.S. Army.
F. F. Danielle, can you describe to us how you
produced the color-encoded image and what the colors mean? Why did
you decide to use this particular technique rather than standard
immuno-fluorescence imaging?
D. C. F. We know little about the proteins that
make up the Vorticella stalk—the main calcium-binding
proteins (centrin and spasmin) are known, but no other major
structural proteins have been identified yet. This means that we
exhausted our options for immunofluorescence imaging pretty quickly.
Since TEM (transmission electron microscope) imaging from the 1970s
showed the presence of small fibers, and I believe that centrin
cannot form those fibers on its own, it made sense to go back to
polarization microscopy to have a look at the fibrous structure.
I had seen a lecture by [microscopist] Shinya
Inoué while I was taking the physiology summer course at the
Marine Biological Laboratory at Woods Hole in 2004. I also got to
see some of the microscopes in the lab, including the LCPolScope,
which Rudolf Oldenbourg and collaborators at the MBL developed.
Shinya used polarized light to see microtubules in dividing cells
long before we knew what microtubules were, or that they were even
there. Polarization microscopy has the ability to pick up a
birefringence signal from biological fibers that are only nanometers
in diameter. A lot of modern imaging has moved past that stage of
initial discovery—the major components (like actin and
microtubules) are already identified and easily tagged. But because
I'm working on a relatively forgotten organism, polarization
microscopy is very powerful.
The thing that Rudolf brought to Shinya's lab was the
ability to generate orientation-independent retardance images. The
LCPolScope uses liquid crystal polarizers to rapidly collect
multiple images of the same specimen at different polarizations, or
orientations. From those images, the maximum retardance at each
pixel is calculated and stored in a new image (the black-and-white
image shown was made from a two-frame technique, with the two frames
captured at different orientations). The polarization direction in
which that maximum occurred is also calculated, and can be overlaid
on the black and white image as colorization. That's how we get the
first color image shown.
F. F. I remember sitting with you at the computer
and asking, just out of curiosity, what the image would look like
without the coloring. When you showed the grayscale image to me, I
was fascinated by how much more detail we saw. Do you agree? If so,
why do you think that's the case?
D. C. F. Yes, I think that the black and white
image is a little more striking. However, remember we are looking at
two different images but with the same information in each. It's
just that the black-and-white image shows the fibers within the
stalk more clearly than the color image does; the color image shows
that those fibers line up along the main axis of the stalk while it
is extended. I could get all the alignment information from the
color picture, but it's just a little easier to see distinct
structures in black and white.
I don't have a rigorous optical/physiological
explanation for it, but I think that we are better able to discern
detail in the black and white picture because we better discern the
contrast of white on black. The color picture adds another level of
information which is helpful in a lot of ways, but does actually
obscure some of the detail. I think you can see the same effect in
photographs, and I think it's part of why Ansel Adams is so
popular—he uses the full range of black and white to show all
the immaculate detail that comes through in a landscape or a
textured surface.
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