"Objects Worthy of Notice"
Microscopical Anatomy of Selected Plants Collected by
The Lewis & Clark Expedition
Harry A. Alden1, Roland H. Cunningham1, Kevin Ryan2, Paul T. Jantzen2 and David R. Dobbins3
Smithsonian Center for Materials Research and Education, 2Media Cybernetics, Inc., Silver Spring, MD
3Department of Biological Sciences, Millersville University
aldenh@scmre.si.edu)
Introduction
Plants that are used for taxonomic research are generally preserved by drying them out while they are being pressed flat for easy
storage. This pressed plant material, is placed in a herbarium, in phylogenetic order, based on the species, genus, family and order
designations.
Herbaria are essential for the study and verification of plant classification, the study of geographic distributions, and the standard-
izing of taxonomic nomenclature. Lately, herbarium materials have been utilized in biochemical and genetic studies. The key to these
studies is the state of preservation of the plant material. The preservation of the reproductive and vegetative structures (hairs, stomata,
cuticle, etc.), internal anatomy (vascular tissue size and arrangement), biochemistry and genetic material is essential to make accurate
observations, to collect representative data and answer pertinent research questions. To investigate the potential of herbarium specimens
to be used in non-taxonomic studies, we examined herbarium material collected by the Lewis & Clark Expedition (1803-1806) and
evaluated their state of preservation through microscopic examination of the external and internal features.
Plant material from ten specimens was selected from the plants collected by the Lewis and Clark Expedition and housed at The
Academy of Natural Sciences in Philadelphia.* For the sake of space, only six of these samples will be described. Preliminary microscopic
examination of the specimen surfaces revealed exquisite preservation of trichomes, stomata, and cuticle in most samples. Detailed ex-
amination of the surfaces and the internal anatomy of the plants, using digital image analysis (gradient pseudocoloring), have revealed
fungal hyphae on, and in, all of the samples examined.
Materials and Methods
Light Microscopy: Samples were photographed on a Wild M420 Makroskop prior to embedding or carbon coating. Samples were
embedded in Spurr’s resin (Spurr, 1969), sectioned at 10-15 μm with glass knives, mounted on glass microscope slides and stained with
toluidine blue-O. Slides were viewed in a Leica DMRX research microscope. Images were captured on film (macro photography) with
a Nikon DXM-1200 camera.
Electron Microscopy: 16 mm diameter disks of carbon conductive double-sided adhesive material were applied to the center of
carbon stubs (26 mm X 8 mm). Sample fragments were arranged on the adhesive material. Occasionally, curved or irregular fragments
were mounted on minimal amounts of carbon paste to insure good contact with the adhesive surface and facilitate conductivity. The
mounted samples were then placed in an Edwards E306A vacuum coating unit and given a carbon coating approx. 30 nm thick. Samples
were viewed on a JEOL JXA-840A Scanning Electron Microscope with a Thermo-NORAN TN-5502 energy dispersive analytical attach-
ment and, NORAN Vantage spectrum processing software. With a few exceptions (see specs. on photos), samples were photographed
between 8-15 kV at a working distances ranging from 7-12 mm.
Colorization of SEM Images: Gradient pseudocoloring is a method for bringing out edges and their orientation in the image.
The gradient image is created in Hue-Saturation-Intensity (HSI) space. The original image is used to modulate the intensity, and local
variance is used to calculate saturation, or the range of color to grayscale. The hue of the image is calculated from a derived edge, such
as a Sobel, variance, Roberts, or other edge detecting filter. A gradient phase is calculated from the edge image, indicating the direction
of changes in intensity. This phase is then converted to color hues, so that edges sloping in one direction will have a color distinct from
edges sloping in other directions. The effect is similar to illuminating a three dimensional object with a ring of colored lamps, so that
edges oriented towards different lamps will
reflect the different colors. The three images are
combined in HIS space, and converted to RGB
to produce the gradient pseudocolor image.
Silver Buffalo-Berry
(Shepherdia argentea [Pursh] Nutt.)
Oleaster Family (Eleagnaceae)
This specimen was collected on September
4th, 1804 at the mouth of the Niobrara River,
south of the current border of Nebraska and
South Dakota (Fig. 1). The collection site is
near the current southern range of the plant
(Fig. 2) inferring that it was most likely col-
lected shortly after being seen.
Fig. 1. Collection site for silver
buffalo-berry.
Fig. 2. Current range map for
silver buffalo-berry.
8 MICROSCOPY TODAY May 2005
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
SeeMoreGssLess_MicToday_4_8_05 4/11/05 5:04 PM Page 1
More. Guess Less.
NORAN System SIX for x-ray microanalysis
You no longer need to optimize yourx-ray microanalysis parameters for just a
few elements. That's because Thermo's NORAN System SIX x-ray microanalysis
system gives you a complete data set with every run.
Built around our spectral imaging technology, NORAN System SIX:
• Eliminates guesswork by automatically optimizing data collection
• Gives you a full spectrum for every pixel of your electron microscope image
• Allows analysis and re-analysis of the full data set any time, anywhere.
Open your eyes to the world of NORAN System SIX at www.thermo.com/microanalysis
or contact us for more information.
Telephone: 1-800-532-4752 • Email: analyze@thermo.com
Analyze • Detect • Measure • Control™ Thermo
ELECTRON CORPORATION
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
Fig. 3. Macrophotography of silver buffalo-berry, showing midvein. Upper left.
Fig. 4. Macrophotography of silver buffalo-berry, showing numerous peltate trichomes. Upper center.
Fig. 5. Peltate trichomes in normal light (l.) and polarized light (r.). Upper right.
Fig. 6. SEMs of Peltate trichomes. Lower left and center.
Fig. 7. SEM of Peltate trichome, showing its close association to the leaf surface. Lower right.
Microscopy shows a well preserved leaf (Fig. 3) covered in peltate (shield-like) trichomes (Figs. 4 -7 ) . They are tightly packed and
closely appressed to the surface of the leaf (Fig. 7). The trichomes are an adaptation to a xeric (dry) environment, where they impede
the loss of water by creating a boundary layer of humid air at the leaf surface.
Interior Wild Rice
(Zizania palustris L. var. interior [Fassett] Dore)
Grass Family (Poaceae)
This specimen was collected on September 8th, 1804 along the Missouri River, near the current border
of Nebraska and South Dakota (Fig. 8).
Microscopy shows another well preserved leaf (Fig. 9), with intact cuticle, knobby protuberances
called papillae and bow-tie shaped silica cells (Fig. 10). Also apparent are numerous fungal hyphae on
the surface of the leaf (Figs. 10 & 11). Fungal hyphae extend into the stomata (Fig. 11). Fig. 8. Collection site for wild rice.
.Pier
pid City
Winner,
1
—
Ainsworth [
bluff
Sioux Falls.
1i
Sioux City
Norfolk*
e
 , North Platte
Minnea
MankE
ear t
, O n ^
Fig. 9. Macrophotograph of wild rice leaf. Upper left.
Fig. 10. SEM of wild rice, showing fungal hyphae (arrowheads) and silica cells (S). Upper right.
Fig. 11. SEM's of wild rice, showing fungal hyphae (arrowheads) at a stomatal complex (arrows).
Indian Tobacco
(Nicotiana quadrivalvis Pursh)
Potato Family (Solanaceae)
This specimen was collected on October 12th, 1804 at the Arikara Camp near the mouth of the Grande River (Fig. 12.). The
herbarium sheet shows that the plants were dried while being hung, and not pressed in a book (see the sheet at: http://clade.acnatsci.
org/mccourt/Lewis%20and%20Clark/flattened%20jpgs%2012_10/146.jpg).
The samples of tobacco were not well preserved, with few discernable trichomes and stomata (Figs. 13 - 15). The leaf surface ap-
peared altered and contained considerable extraneous material (Figs. 15 - 17).
10 MICROSCOPY TODAY May 2005
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
June 2(
McCrone Atlas of Microscopic Particles
brought to you by ASSOCIATES, INC
Westmont, Illinois
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
Fig. 12. Collection site for Indian tobacco. Fig. 13. Macrophotograph of Indian tobacco leaf.
Fig. 14. Macrophotograph of Indian tobacco leaf, showing several trichomes.
m*
g. 15. SEM of Indian tobacco leaf showing remnants oftrichomes (left, arrowheads) and cluttered leaf surface (center).
Fig. 16. SEM of Indian tobacco, showing fungal hyphae (right, arrowheads).
Fig. 17. Polarized light micrographs (with λ plate), showing extraneous, granular (arrows) and spherical (arrowheads) birefringent materials.
Oregon Boxleaf
Paxistima myrsinites (Pursh) Raf.
Bittersweet Family (Celastraceae)
This specimen was collected on November 16th, 1805 at Cape Disappointment (Fig. 18).
Microscopy shows leaves that are well preserved (Fig. 19-21). The stomatal apertures are overarched by rodlets of cuticle and the
leaf surface is covered with fungal hyphae (Fig. 22 - 23).
W, W;
Fig. 18. Collection site for Oregon boxleaf. Fig. 19. Macrophotograph of Oregon boxleaf leaf. Fig. 20. Macrophotograph of Oregon boxleaf leaf.
12 • ffllCROSCOPYTODHY May 2005
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
,0.5 |Jtn
J-JJIT)
•»•?£•
( V Peripheral Nerve. TEM Magnification = 30,000X; 80kV.
Sfl Image courtesy of Kenneth L. Tiekotter, University of Portland.
Renal Biopsy. TEM Magnification = 1,100X; 80kV.
I Image courtesy of Kenneth L. Tiekotter, University of Portland. i
^
0 W
i.en
in Booth 1007. www.gatan.com
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
Fig. 21. SEM's of Oregon boxleaf leaf, showing stomata (white flecks). Left.
Fig. 22. SEM's of Oregon boxleaf, showing fungal hyphae (arrowheads). Normal SEM (center) and gradient pseudocoloring (right).
Fig. 23. SEM's of Oregon boxleaf stomatal complex, showing cuticular rods (arrows) overarching the guard cells and fungal hyphae (arrowheads). Normal
SEM (left) and gradient pseudocoloring (center).
Fig. 24. Collection site for American Dune Grass.
American Dune Grass
Leymus arenarius (L.) Hochst.
Grass Family (Poaceae)
This specimen was collected on December, 1805 at Fort Clatsop, Oregon (Fig. 24, above).
Microscopy shows yet another well preserved leaf (Figs. 25 - 28), with an intact cuticle and tough, pointed trichomes (Figs. 27 &
28). Apparent are numerous fungal hyphae on the surface (Figs. 27 & 28) and in the cavity formed by the rolled leaf (Fig. 29). The septate
hyphae (Fig. 29) (hyphae with cross walls) indicate it is a basidiomycete (club fungus). There are also numerous spherical structures,
likely conidiospores on the inner surface (Figs. 27 & 28).
Fig. 25. Macrophotograph of American Dune Grass (left).
Fig. 26. SEM of American Dune Grass, showing vascular bundles (arrows) and abaxial surface (AB) Second image..
Fig 27. SEM's of American Dune Grass, showing numerous, pointed trichomes and fungal hyphae (arrowheads) on the inner (adaxial) surface. 3rd & 4th
Fig. 28. SEM's of American Dune Grass, showing pointed trichomes, a single fungal hypha (arrowhead) on the adaxial surface and thin cuticle covering the
surface. Normal SEM (left) and gradient pseudocoloring (second).
Fig. 29. Light microscopy showing septate fungal hyphae (arrowheads) from an airspace formed by the curled leaf of American Dune Grass (3rd). Image on the
right is a digital compilation of 9 focal planes using the Extended Depth of Focus feature in ImagePro Plus© software.
14 • ffllCROSCOPYTODHY May 2005
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
MicroscopyToday_2 0 05_May.qxp 5/4/2005 2:46 PM Page 1
thinkforward
/ w
o
a
I
Si
a
• Compact design
• Sure_Cal® automated
calibration
• Automatic fluorescence
removal by SERDSTM
• High wavelength precision
of <0.1 cm-1
• Multi laser excitation
• Confocal depth profiling
• Chemical mapping
• Real time color video
Tired of spending all your time calibrating
your expensive Raman System ?
Introducing the new SENTERRATM
affordable Dispersive Raman Microscope
with patented Sure_Cal® technology
The new SENTERRA combines the sensitivity of dispersive
technology and the wavelength accuracy of Fourier transform Raman
spectroscopy. It provides unsurpassed performance, flexibility and
ease of use by utilizing patented Sure_Cal® automatic calibration
technology. The SENTERRA is a full-featured confocal system with the
highest possible spatial resolution, that can accommodate multiple
excitation wavelengths. It incorporates a revolutionary and patented
automatic fluorescence rejection method for rejecting fluorescence
from many samples.
of
IRUKER
www.brukeroptics.com/microscopy
1-888-4BRUKER | microscopy@brukeroptics.com
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
Big-leaf Maple
Acer macrophyllum Pursh
Maple Family (Aceraceae)
This specimen was collected on April 10th, 1806 along the Columbia River near The Great Rapids (Fig. 30). The collection site is
near the current eastern range of the plant (Fig. 31), showing that it was most likely collected shortly after being seen.
Microscopy reveals a well preserved leaf with large hairs (Figs. 32 - 34) on the veins of the lower (abaxial) surface, cuticle (Fig.
35) and stomata (Figs. 36 & 37). The leaf shows macroscopic signs of being infected with Tar Spot fungus (Fig. 32). The leaf surface is
covered with fungal hyphae (Fig. 36). Hyphae can be seen inserted into the stomatal pore (Fig. 37).
Fig. 30. Collection site for big-leaf maple.
Fig. 31. Current range map for big-leaf maple.
Fig. 32. Macrophotograph of big-leaf maple, showing trichomes (arrowheads) and tar spot fungus (arrows).
Fig. 33. SEM's of big-leaf maple.
Fig. 34. SEM's of big-leaf maple, showing trichomes (arrowheads) and leaf veins (V). Adaxial surface (left) and abaxial surface (center)
Fig. 35. Adaxial surface of big-leaf maple, showing intact cuticular ridges. This image is the same as Fig. 2E in Teece, et al (2002). Right.
Fig. 36. SEM's of big-leaf maple, showing fungal hyphae (arrowheads). Original image (left) and digitally enhanced image (2nd). Normal SEM (left) and
gradient pseudocoloring (2nd). The normal SEM image is the identical one used in Figure 2B in Teece, et al (2002).
Fig. 37. SEM's of big-leaf maple, showing fungal hyphae (arrowheads) at two different stomatal apertures (outlined in red). 3rd & 4th.
Discussion
It is clear from the above images that the plants collected by Meriwether Lewis, while structurally sound, have been contaminated
by fungi on the surface and internally. This finding is in disagreement with previously published research (Teece, et al, 2002). •
Acknowledgements
A gracious acknowledgement to Dr. Richard M. McCourt, Department of Botany, The Academy of Natural Sciences of Philadel-
phia* for providing samples for this study. Range maps of woody species were scanned from Little, Jr. (1976), digitally cleaned up and
colorized.
*The Academy of Natural Sciences, an international museum of natural history operating since 1812, undertakes research and public
education that focuses on the environment and its diverse species. The mission of the Academy is to create the basis for a healthy and sus-
tainable planet through exploration, research and education. The Academy of Natural Sciences' Lewis & Clark Herbarium is a collection
of 226 sheets of dried, pressed plants collected by Meriwether Lewis during the Lewis & Clark Expedition of 1804-06. These plants are the
most well documented and historically important collection of American plants in existence. Recognizing their value, the federal government
declared the herbarium an "American Treasure" through its Save America's Treasures program.
16 • ffllCROSCOPYTODHY May 2005
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579
Literature Cited
Little, Jr., E.L. 1976. Atlas of United States tree. Volume 3. Minor western hardwoods.
USD A, Forest Service, Misc. Publ. No. 1314.
Spurr, A.R. 1969. A low-viscosity epoxy resin embedding medium for electron
microscopy. Clin. Microbiol. Res., 3:197-218.
Teece, M. A., M. L. Fogel, N. Tuross, E., R. M. McCourt and E. Spamer, (2002) The
Lewis and Clark Herbarium of the Academy of Natural Sciences, Part 3. Modern
Environmental Applications of a Historic Nineteenth Century Botanical Col-
lection, Notulae Naturae, No. 477.
Related Web-Sites:
http://www.lewis-clark.org/
http://www.acnatsci.org/library/scipubs/collections.html
http://www.acnatsci.org/~mccourt/lc_today.html
http://www.edgate.com/lewisandclark/
http://www.inform.umd.edu/PBIO/L&C/L&Cpublic1.html
http://www.lewisclark.net/maps/index.html
http://www.acnatsci.org/lewisclark/herbarium.html
http://www.acnatsci.org/lewisclark/preservation.html
http://clade.acnatsci.org/mccourt/Lewis%20and%20Clark/flattened%20jpgs%2012_
10/herbsheetsrev.html
... Continued from page 28.
diatom a helpful test sample for accessing the quality of the microSEMOperatorPositin-
scope images [6]. Figure 2 shows fine structure of three fragments of
Pleurosigma angulatum taken from different sources.
A variety of living cells have been observed and photographed
with this technique, including cancer cells (Figures 3,4), pollen (FigUniverstyofAlabamatBirmingham-
ure 5), and bacteria (Figure 6). Spirochete (Figure 6), a microscopic
bacterial organism, has a worm-like, spiral-shaped form. It wiggles
vigorously when viewed under a microscope. The photograph shows a
fine structure of bacterium, represented by 12 small additional "coils"
between major periods of a spiral body.
In summary, CytoViva 150 is a highly effective tool for direct-
view, light (optical) microscopy to enable the resolution of cellular
features down to 100-250 nm. The system provides a unique view of
live cells and cell processes while they are occurring. It achieves high
light efficiency and utilizes a low power light source. Performance
advantages include enhanced spatial resolution of 150 n m or less, high
contrast, detection less than 60 nm, magnifying power greater than
6,000, reduction of stray light, and the capability of three-dimensional
sectioning. Contrary to phase contrast microscopic imaging it has
better resolution and no image distortions. It is similar to DIC but
does not require a prerequisite orientation, has a better resolution
and contrast, and can visualize very small particles. It is superior to
conventional darkfield due to higher contrast, better resolution, and
light economy. It produces low heating and provides a reduced photo^^-
toxicity. Sample preparation techniques such as freezing, dehydration,
staining, labeling, and metal deposition can be avoided. All these
features are combined in a unit that is easy to install and use. •
Acknowledgements:
This work is supported by grants from DAADOJ-02-C-0016,
Fetzer Institute, #2143, Aetos Technologies, Inc. Images were pro-
duced by Oleg Pustovyy with CytoViva150.
References
1. Foster B., Focus on Microscopy: A Technique for Imaging Live Cell Interactions
and Mechanisms, Amer ican Laboratory, November 2004, 21-27.
2. Born, M. and Wolf, E. Principles of Optics, Pergamon Press, New York, 1980.
3. Cragg, GE, So, PTC. ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^Lateral Resolution Enhancement with Standing Evanescent
Waves. Optics Letters, 25 ,46-48, 2000.
4. Mar t in LC, Johnson BK, Practical Microscopy, Blackie & Son, London, 1949.
5. Richardson, T.M. Test slides: Diatoms To Divisions. - What Are You Looking At?
Proc Roy Microsc Soc 33, 3 -9 , 1998.
6. Sterrenburg, FAS, Crystal Palaces—Diatoms for Engineers, J. Nanoscience and
Nanotechnology, 5, 100-107, 2005.
Get it wit
ElnJ
Electronics
SEM Products
Chamber Surveillance Systems
iroductory Special
25% OIF
Infrared Chamberscope
laencesi
800-992-9037 or 413-786-9322
email: ebs@ebsciences.com
M I C R O S C O P Y TODAYMarch2005 May 2005 17
D
ow
nloaded from
 https://w
w
w
.cam
bridge.org/core . Sm
ithsonian Institution Libraries , on 20 Sep 2018 at 18:17:27 , subject to the C
am
bridge C
ore term
s of use, available at https://w
w
w
.cam
bridge.org/core/term
s . https://doi.org/10.1017/S1551929500051579