Faster than a speeding virus Digital image analysis in electron-microscopical viral diagnostics

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1 Digital Imaging Solutions In Practice TEM Imaging Faster than a speeding virus Digital image analysis in electron-microscopical viral diagnostics

2 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. 2 1 Pox viruses 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 The often discussed danger of a bio-terrorist attack using pox viruses underlines the significance of fast and reliable diagnostics. 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- From the pasture to the store counter 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.

3 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. 3 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 x. In order to identify the characteristic structural features of the virus reliably, the scientists use higher magnifications - ie, ranging from to x. This would not be feasible using a light microscope as it can only magnify up to 1000x. 4 Adenoviruses - acquisition at x 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 x magnification. Investigating characteristic structural features is done at magnifications ranging from x to x. 5 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 x 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 ed directly within the software. This saves much time and expense.

4 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. 6 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 x). 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. 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. The internet makes it much easier for experts to share images and any other data.

5 Acknowledgements Text written in close collaboration with: Dr. Dirk Soike, Landeslaborbetrieb, Brandenburg, Laborbereich Potsdam, Germany Image source: Dr. Dirk Soike All rights including translation are reserved. Any and all forms of reproduction must be authorized by Olympus Soft Imaging Solutions. We are unable to assume any liability for unrequested consignments received. Application Report Carbon #1, 2007 Johann-Krane-Weg Münster Germany Phone: +49 (251) Fax: +49 (251) West Bayaud Ave., Suite 300 Lakewood, CO USA Phone: +1 (303) Fax: +1 (303) , Ayer Rajah Crescent #04-02, Tempco Technominium, Singapore Singapore Phone: Fax: