Figure 8. Electron micrograph of nuclear pole region found in
rat ventricular wall. (N) nucleus; (M) mitochondri
(G) Golgi apparatus; (NM) nuclear membrane (A.
Tonti).

which have been implicated in outbreaks of gastro-
enteritis. EM studies at this facility have detected a
number of these virus particles in fecal material
obtained from afflicted humans or animals (Figures 9
and 10).

Algal Identification

Much of the structural detail of microalgae is so small
it can be resolved only by TEM or SEM analyses. Both
procedures have been used to examine algae from
surface waters and to prepare identification manuals
for field biologists to determine the effects of pollu-
tants on the species composition of algal communi-
ties in receiving waters (Figures 11 and 12).

Intra-Cellular Deposition of Particulates

When animals are exposed to diesel exhaust fumes,
the emitted product in whole particulate form affects
certain target organs of the body. The lung is a
primary receptor for absorption of this particulate,
which is phagocytized by selective cells. Electron
microscopy magnifies the ingested particulate and
the surrounding area of deposition (Figure 13). In
contrast to that seen with the light microscope, this

enables the laboratory to obtain a more detailed per-
spective of intra-cellular relationships.

Summary

There is no doubt that the TEM, SEM, and EDS
systems are powerful analytical tools for structural
determination and chemical identification. These
systems support important objectives of the research
groups at ERC-Cincinnati through their various
engineering, biological and mineralogical applica-
tions.

Figure 9. Rotavirus: “‘complete’’ or “smooth” double-shelled
virions can be seen in the upper portion of the elec-
tron micrograph, while a group of “rough” single-
shelled virions appear below. Two stain-penetrated
virions of each type can be seen (F. Williams).

Figure 10. Electron micrograph of astrovirus particles, some
possessing a distinctive star-shaped surface mor-
phology and smaller parvovirus particles showing

no such distinctive morphology (F.



Figure 6. Characteristic: spectra of chrysotile asbestos dis-
played on the ORTEC EEDS I! console.

The SEM has been extremely useful in the examina-
tion for surface deterioration of asbestos-cement
(A/C) products (water distribution pipes and roofing
tiles). Continued surface deterioration commonly
results in the release of asbestos fibers into drinking
water supplies. Health and economic factors result-
ing from the deterioration of A/C materials have
stimulated research on corrosion-preventive com-
pounds, such as zinc orthophosphate. The SEM-EDS
can be used to determine the coating effectiveness of
these corrosion inhibitors (Figure 7a, b, and c).

Size characterization of asbestos fibers according to
length and. width involves EM analysis. It is believed
that the size of the fiber, especially the length, may

have an effect on its ability to cause cancer. It is there-
fore important to characterize the asbestos fibers
in prepared standards used for cancer research. Once
it is confirmed that a standard contains fibers of a
certain size range, the effects of fiber size in the
biological testing can be better ascertained. Similar
characterization has also been applied to fibrous or
fibrous-like clay particulates.

Monitoring drinking water for asbestos utilizes TEM
analysis. Therefore, quality assurance pertaining to
sample preparation and analytical technique is
needed. To fulfill such a need, asbestos standards
similar to known indigenous fiber concentrations and
size distributions are being prepared to be sent to
laboratories analyzing water samples for asbestos.

Cardiovascular Tissue Changes

EM studies are used in the evaluation of environ-
mental factors related to ultrastructural cardiovas-
cular tissue changes. The ultra-microtome, modified
with a CryoKit, can obtain ultra-thin frozen tissue
sections of the ventricular wall of the heart, which are
then analyzed by TEM-EDS. Observations are made
for any soluble toxic chemicals that may have accu-
mulated in the tissue, with special attention given to
binding in the mitochondria. Morphometric analyses
of electron micrographs are also being used to
observe changes in the nuclear pole areas for early
detection of cardiovascular disease (Figure 8).

Virus Detection
Electron microscopy plays a crucial role in the
detection of noncultivable viral agents, many of

Figure 7.

(a) SEM photograph of surface of unused A/C distribution pipe, (b) SEM photograph of surface of corroded A/C pipe,

(c) SEM photograph of surface of A/C pipe coated with zinc orthophosphate.


Figure 11, Scanning electron micrograph of the diatom,
Stephanodiscus hantzschii, showing the micro-
structure of the cell wall (B. McFarland).

Figure 12. Transmission electron micrograph of the small dia-
tom, Thalassiosira pseudonana, common in eutro-
phic waters (C. Weber).

Figure 13. Transmission electron micrograph of rat lung tis-
sue. Arrow points to a macrophage that has phago-
cytized deposited diese! fuel exhaust particulates
inhaled into the lungs (H. Ball).

4 US GOVERNMENT PRICTING OFFICE: 1985 -757-012/7098


United States
Environmental Protection
Agency

Andrew W Bretdenbach
Environmental Research Center
Cincinnati OH 45268

Research and Development

EPA-600/D-81-113 April 1981

EPA

ENVIRONMENTAL

RESEARCH BRIEF

Electron Microscopy in Environmental Research

Richard L. Boone’, Patrick J Clark'?, James

Background

Research conducted at the U.S. Environmental Pro-
tection Agency's Cincinnati laboratories ts aided by a
shared electron microscope (EM) facility. This facility
includes two transmission electron microscopes
(TEM), a scanning electron microsope (SEM), a con-
necting darkroom, and a specimen preparation
laboratory containing light microscopes, vacuum
evaporators, and two ultramicrotomes.

Since inception, the new facility has been regulated
by a committee of principal scientists representing
the major program users. This shared operation, now
in its eighth year, has been a complete success. In
addition to enacting operating policies insuring equi-
table use of the facility, the committee's responsibil-
ities also include periodic evaluation of the facility’s
performance; EM operator certification; acquisition of
new or additional accessory equipment; and super-
vision of facility maintenance. The uniqueness is also
reflected in the fact that each program desiring use Is

"RL Boone, P J Clark, and J R Millette are with the Health Effects
Research Laboratory, Cincinna, OH 45268

?P J Clark was formerly with the Municipal Environmental Research
Laboratory, Cincinnati, OH 45268

5R § Safferman 1s with the Environmental Monitoring and Support Labora-
tory, Cincinnati, OH 45268

R Millette’, and Robert S. Safferman®

responsible for providing its own personnel with
expertise in their specific area of interest.

The multi-faceted needs were first met with the
acquisition of a JEOL* 100B TEM, which was later
upgraded by the addition of a high-resolution
scanning attachment. This made specimen examina-
tion possible in scanning, scanning transmission, and
conventional transmission modes. To add micro-
analytical capability to the 100B, an energy dispersive
X-ray spectrometer (EDS) was obtained, allowing
elemental analysis directly from observed specimens.
The recent development of even more sophisticated
microanalytical instrumentation resulted in replacing
the obsolete console of the ORTEC Delphi with that of
the ORTEC EEDS II multichannel analyzer and data
processor.

The increasing work load at the EM facility led to the
acquisition of a second transmission electron micro-
scope (the JEOL 100CX) in 1977 (Figure 1). Like the
100B TEM, the 100CX is equipped with a side-entry
goniometer and an ORTEC EDS detector The EEDS ll

“The use of a specific manufactuer’s name 1s for identification purposes only
and does not constitute endorsement by the US Environmental Protection
Agency

» D>


Figure 1. JEOL 100CX transmission
electron microscope and
the ORTEC EEDS I. The
EM is housed in a room
free from EM-interfering
mechanical vibrations and
magnetic fields,

Figure 2, ETEC scanning electron micro-
scope with electron column
specimen chamber (right),
instrument console and cath-
ode ray tube viewing screens
(left).

Figure 3. LKB ultramicrotome,



display console, obtained originally for the 100B, can
be detached and moved to the 100CX with minor ad-
justments.

The facility has also added an ETEC SEM toits group
of analytical tools (Figure 2). The SEM provides a
three-dimensional view and an added depth of field,
as well as allowing the use of relatively large
samples. The SEM has recently been fitted with two

viewing screens allowing simultaneous specimen.

observation at both low and high magnification and
an EDS detector for use with the EEDS II console. The
EEDS II console is now equipped to obtain data on two
independent channels from separate electron micro-
scopes. This results in faster and more efficient use of
the microscopes without loss of precise analysis.

Instruments are available for conventional- and cryo-
ultramicrotomy. Cryo-ultramicrotomy is used when
frozen thin sectioning of tissue is needed for the
microanalysis of certain elements which would be
leached by usual sectioning techniques. For this
purpose the LKB Ultrotome IV (Figure 3) has been
fitted with an LKB CryoKit. An LKB* Ultrotome V is
available for preparing conventional sections.

Research Applications

Researchers are continually increasing their use of
the EM facility for studies in the areas of methods
development, air and water toxicology, and water
supply. The TEM, SEM, and EDS systems have been
very useful in the developmental work on asbestos
standards, and also the analyses of surface waters,
drinking water, minerals, volcanic ash, and micro-
particulates from various sources. High resolution
microscopy has had a large impact on the detection of
noncultivable viral agents associated with outbreaks
of gastroenteritis. It is also important in inhalation
toxicology studies to detect changes in tissue
morphology after animal exposure to smoke and dust
Particulates. Moreover, research concerning ultra-
structural tissue changes related to the ingestion of
trace metals has been heavily dependent on EM
analysis.

Asbestos

inhalation of asbestos fibers is a known cause of lung
cancer. Ingestion of asbestos is suspect as a cause of
gastrointestinal cancer among workers occupation-
ally exposed to asbestos, and possibly may be a
hazard to those who have ingested asbestos in drink-

ing water. The TEM-EDS system is used as a
complete analytical tool in the identification, charac-
terization, and deturmination of the concentration of
asbestos fibers in drinking water supplies throughout
the United States. TEM is required because asbestos
fibers which are as small as , cannot be resolved

using the conventional light microscope. The exact
mineral variety, elemental composition, and crystal
structure are determined using TEM by direct
morphology, selected area electron diffraction, and
EDS (Figures 4, 5, and 6).

Figure 4. Electron micrograph of chrysotile asbestos fibers in
Prepared standard showing distinct morphology and
selected area electron diffraction pattern (upper left).

Figure 5, Electron micrograph of amphibole ssbestoe fiber in
distribution system with selected area
caetion aetivection patton, tarpon right