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Faster than a speeding virus
Digital image analysis in electron-microscopical viral diagnostics
Application Report: Digital image analysis in electron-microscopical viral diagnostics
Faster than a speeding virus
Fighting animal epidemics is the responsibility of government agencies. In Brandenburg, a state in the eastern part of Germany, the Landeslaborbetrieb is responsible for
the diagnosis of animal epidemics and infectious animal diseases. The scientists employed at this lab can detect the viruses directly via electron microscope. Taking a comprehensively digital approach enables them to obtain diagnoses quickly and reliably.
Acquisition, evaluation and management of the images and the documentation of
findings is done using a special digital camera and the image analysis software iTEM.
Cases that raise particularly thorny questions can be discussed with experts located
elsewhere via the internet.
Dr. Peter Stallknecht, Olympus Soft Imaging Solutions
GmBH, Germany
From animal to animal
In the first six months of 2003, avian influenza (fowl plague)
had much of Europe holding its collective breath. The
Netherlands, Belgium and Germany were directly affected. About 30 million chickens, geese and ducks had to
be exterminated. There were several countries around the
world that imposed import bans as self-protective measures. Following the death of a Dutch veterinarian due to the
avian influenza virus, there was speculation as to whether
or not the danger of people becoming infected had been
underestimated up until then. The fact that livestock is kept
in large numbers in cramped quarters and often transported
great distances does not help to restrict the transmission of
such infectious diseases from animal to animal. Some of
the diseases, such as foot and mouth disease, put animals
into much pain, not to mention the economic damage to
agriculture and trade which easily runs into millions of Euros.
narians, state veterinarians and state agencies have to work
well together, often on an international level. As a team,
they all have to move faster than the viruses spread. This
is one of the reasons why electron microscopy is used for
diagnosing numerous viral diseases. The speed with which
this detection method can be conducted along with the
clear view of the pathogens are decisive advantages.
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Pox viruses - 200 to 300 nm in length - are the largest sized viruses.
The lepori pox virus shown was prepared from the eyelid of a rabbit.
This virus is not harmful to humans. Pox viruses harmful to humans are
considered to have been eradicated. The last case recorded worldwide was back in 1977. The often discussed danger of a bio-terrorist
attack using pox viruses underlines the significance of fast and reliable
diagnostics.
From the pasture to the store counter
Fighting diseases
When such an infectious disease breaks out, immediate
countermeasures are absolutely necessary. With fast and
reliable diagnosis, the origin of the epidemic can be isolated
early on and the distribution of dangerous pathogens contained. When fighting epidemics, animal breeders, veteri-
To protect the health of consumers in Germany, the state
requires a series of thorough lab tests - from the pasture to
the store counter. These ensure the health of the animals
as well as guaranteeing the quality of the food products.
In the state of Brandenburg, this task is the responsibility
of the State Laboratory Agency [Ger. Landeslaborbetrieb].
This agency is based in Frankfurt an der Oder, Brandenburg, Germany and is directly responsible to the State
Ministry for Environment, Protection of Nature and Regional
Planning.
Application Report: Digital image analysis in electron-microscopical viral diagnostics
Investigations for detecting infectious animal diseases are
a primary focus of the State Laboratory Agency. Dr. Dirk
Soike and his team of lab specialists are responsible for the
electron microscopical virus diagnostic work at the lab in
Potsdam (also in Brandenburg). They conduct these investigations using a JEOL transmission electron microscope
– a JEM 1010, with the MegaView II, side mounted digital
camera by Olympus Soft Imaging Solutions.
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specific, previously known pathogen. This is why it plays
such a key role in the detection of new viral pathogens as
well.
Searching for virus particles in samples of organs, excretions or cultured media usually takes place at a magnification
of 40 000x. In order to identify the characteristic structural features of the virus reliably, the scientists use higher
magnifications - ie, ranging from 150 000 to 300 000x. This
would not be feasible using a light microscope as it can
only magnify up to 1000x.
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Adenoviruses - acquisition at 200 000x magnification. Due to the high
magnification electron microscopes are capable of, images of viruses
can be acquired directly. Locating the viruses is usually done at 40 000x
magnification. Investigating characteristic structural features is done at
magnifications ranging from 100 000x to 300 000x.
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Some viruses can only be distinguished from one another according to
size via electron microscope. This is why it is so important that digital
acquisitions are correctly calibrated and easy to measure. Two viruses
that can be distinguished are the circovirus (a) and the parvovirus (b).
Why use electron microscopy?
The electron microscopical negative-contrast method
makes it possible to detect virus particles directly. This
means scientists can display the viruses greatly magnified, thus being able to classify them using morphological
criteria. Considering the course of the disease and other
investigations, results often lead to a definite diagnosis very
quickly. In contrast with many other diagnostical methods,
electron microscopy is not limited to the detection of one
Reovirus - acquired at 100 000x magnification. Scientists drag and
drop all digitally acquired images right into the database along with all
measurement data, results and any other related documents. Documenting results is done using the integrated report generator. Images,
data and finished reports can be e-mailed directly within the software.
This saves much time and expense.
Application Report: Digital image analysis in electron-microscopical viral diagnostics
Why ‚go digital‘?
The MegaView II digital camera is side mounted on the
wide-angle port of the electron microscope. The camera
has a prism that is moved into the beam path of the microscope via pressurized air and intercepts the electrons in the
intermediate image plane. In the intermediate image plane,
the image segment is larger than in the final image plane.
The camera then transmits the acquired image directly to
the PC. The software for reading out the image data and
for operating the MegaView II is fully integrated with iTEM.
iTEM is the TEM software environment installed on the PC
developed by Olympus Soft Imaging Solutions for digital
TEM image analysis and digital TEM image management.
The scientists in Potsdam use this software environment for
acquisition, evaluation, management and archiving of their
images and for the documentation of their findings. This
comprehensive digital approach has one primary advantage
for them: they can diagnose viral diseases more quickly and
more reliably. Other advantages include:
• Once the investigatory work at the microscope has been
completed, all the image documentation is already completed as well. An extra step is no longer required.
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• Elimination of all the steps involved with actual film means
time and money are saved with the benefit of environmentally friendly digital imaging.
• The digital camera makes it possible to focus and adjust
the electron microscope using test samples at significantly
higher magnifications (up to 600 000x). This optimizes the
performance capacity the microscope can offer. In any
case, at these magnification ranges, structural details cannot be clearly made out by the naked eye on the electron
microscope‘s fluorescence screen.
• Images can also be acquired at significantly higher magnifications.
• The live image on the PC screen makes it possible to
monitor image quality during the investigation via microscope. This makes it possible to interactively optimize imaging
conditions.
• Brightness and contrast of the digital images can be adjusted post-acquisition as necessary. Special digital filters
can be used to emphasize important structural details.
• Size is decisive for distinguishing between viruses of similar appearance. This is where iTEM increases diagnostical
certainty because the images are automatically calibrated
and can be immediately measured on the PC screen – including a statistical evaluation of the measurement results.
• All images and findings are stored in the integrated database simply by dragging and dropping them. Retrieving
them is quick and convenient using search terms selected
by users themselves. This is a real time saver.
• The images collected in the database can also serve as
reference images – for use at demonstrations, training courses or for discussing findings where issues need clarifying.
• The internet makes it much easier for experts to share
images and any other data.
Viral diagnostics in the lab in Potsdam, Germany exploits the tremendous advantages of electron microscopy and doing things ‚the digital
way‘. Getting a clear view of the viruses and the digital image acquisition and analysis mean that a reliable diagnosis can be obtained fast.
Unknown viruses are identified quickly as well. The column of the JEOL
JEM 1010 has a MegaView II camera mounted on it (on the left, covered
up). This camera transfers the microscope image directly to the iTEM
image analysis software.
Exchange via internet
„Not a second to waste“ is the first thought in scientists‘
minds when working to diagnose potentially infectious
diseases. Taking a comprehensively digital approach
makes it possible to achieve results in the least time
possible. Fighting animal epidemics and animal diseases
means the faster results are obtained, the better. This is
especially true with regard to minimizing and/or preventing
endangerment to the health of people and animals, not to
mention limiting negative economic effects. Furthermore,
the digital approach lowers the cost of diagnosis as well as
increasing diagnostical certainty. The internet‘s role is one
of increasing significance in this regard. Respected for their
extensive experience in the field of electron-microscopical
virus diagnostics, the scientists at the state lab in Potsdam
use the internet to discuss digital images and other data
with experts located elsewhere. The internet makes it
much easier to clarify difficult issues in diagnostical cases
involving questions difficult to resolve.
Application Report: Digital image analysis in electron-microscopical viral diagnostics
Acknowledgements
Text written in close collaboration with:
Dr. Dirk Soike, Landeslaborbetrieb, Brandenburg, Laborbereich Potsdam, Germany
Image source: Dr. Dirk Soike
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Application Report Carbon #1, 2007
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