Confocal Microscopy and higher organism taxonomy

[Mark Holmgren]

This is in response to Lawrence Kirkendall's queries regarding the
applications of the Confocal Microscope to taxonomists, particularly
entomologists.  I am serving as a conduit for the comment of Brian
Matsumoto, whose address follows.
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First, it is my opinion that any field that is dependent on anatomical
characterization of a structure will benefit from confocal microscopy.
Briefly, it provides the capability of taking relatively thick preparations,
say 50 to 100 microns thick and optical sectioning that material.  The
sectioned material can then be used for three-dimensional reconstruction.
This three-dimensional imaging characteristic may be sufficient
justification for the instrument's usefulness for taxonomy.  However, like
any instrument, it does possess potential pitfalls and disadvantages.  The
primary disadvantage for most investigators is that confocal microscopy is
primarily a fluorescence technique.  It can be adapted for reflectance-type
work, but my experience with the instrument's used in this configuration is
that the results are disappointing with biological material.  This may
change, but for the present let us assume that we are limited to
fluroescence microscopy.

The limitation to fluorescent staining is not, in my opinion, a major
problem. Indeed, I view fluorescence imaging to be advantageous for many
biological studies.  First, consider the fact that by emitting light, a
structure that is much smaller than the limit of resolution of the light
microscope can be detected.  This can be very advantageous.  In my studies
on photoreceptors, it becomes possible to follow the path of single
microtubules within a cell.  A task that is technically very difficult to do
by electron microscopy.  Second, the limitations by using fluorescence
imaging can be circumvented by an imaginative use of techniques.  For
example, suppose we are interested in imaging the surface structure of
insect chitin.  First, I would try a pilot experiment with a fluorescent dye
that would bind covalently to chitin.  For example, FITC or RITC can be
added to chitin and they will bind to its surface.  After rinsing, the
fluroescent structure can be placed under the confocal microscope and a
three-dimensional constructure of the chitin's surface obtained.  Advantages
over SEM:  Sample preparation will be extremely quick.  Chemical interaction
of the chitin to dye can be done in an hour and rinsing equally fast.
Second, no need to dehydrate.  Sample's (although this may not be applicable
to chitin it may be to nematodes) won't shrink and delicate structures will
not be lost through critical point evaporation.  Imaging can be accomplished
with the microscope with any objective lens on that microscope. Although I
am experienced with high magnification organelle labeling, the applicability
of the confocal microscope for lower mag work has been underestimated.

            Brian Matsumoto
            Neuroscience Research Institute/Dept. Biological Sciences
            University of California
            Santa Barbara, CA  93106

            Voice phone 805-893-8702
            FAX         805-893-2005
            email       matsumot at lifesci.ucsb.edu
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* Mark Holmgren, Associate Director        805 893-4098    *
* Museum of Systematics & Ecology          fax 893-4724    *
* Ecology, Evolution, and Marine Biology                   *
* University of California                                 *
* Santa Barbara, CA 93106                                  *
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