University of Ulm. Nadja El-Mecharrafie Grade 10. Division: Electron Microscopy

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1 University of Ulm Nadja El-Mecharrafie Grade 10 February 20-24th 2006 University of Ulm Division: Electron Microscopy Universität Ulm Zentrale Einrichtung Elektronenmikroskopie Materialwissenschaftliche Elektronenmikroskopie Albert Einstein Allee 11 D ULM Contact person: Ms Dr. Kleinschmidt Further participant: Alice Conrad Translation from German: Nadja El-Mecharrafie

2 Content Page 1: Page 2: Page 3: Page 4: Page 5: Page 6: Page 7: Page 8: Introduction About the University procedure of preparation for TEM: - Fixation - Embedding - Sectioning - Staining Images of a TEM TEM: - Transmission electron microscope - Viruses - HFF-cells SEM: - Scanning electron microscope - Preparation - Examination under the SEM - Postgraduates Miscellaneous Conclusion Images of an SEM ANNEX: - Pictures taken using SEM - Pictures taken using TEM

3 Account Introduction In grade ten all students at German grammar schools are asked to apply to companies for a career orientation. In German this is called BOGY and involves an unpaid one-week internship. Since I am mostly interested in foreign languages and like airports a lot, I mainly applied to language schools and to Stuttgart Airport but received letters of refusal only. So in the end, although I had started my search for a career orientation very early, my efforts stayed without success and I eventually ran out of time. Therefore I was all the happier to be given a chance to join my friend Alice. She was going to spend a week at the University of Ulm where her grandmother works as a scientist. In the end it was good we came as a pair, because as we had been warned we were not occupied all the time and by oneself one would have been bored easily. University of Ulm As already mentioned, Alice and I spent our BOGY-week at the University of Ulm in the electron microscopy division ( EM ). The University itself is a large complex of buildings featuring the scientific fields of Maths, Physics, Chemistry, Biology and Medicine. The electron microscopy division does not form a part of any of these because of its interdisciplinary use. Its location is in the sector of Medicine between Pathology and Microbiology. Companies and scientists from the university use it for experiments and experimental research. Unfortunately, our career orientation took place a week before semester vacations, which is the time when the last exams are being written and because of this we were not able to attend any lectures. The University of Ulm was founded in 1967 as the ninth University in Baden-Württemberg. Students can choose from a wide range of subjects such as: - Medicine - Computer Science - Maths - Economics - Engineering technologies - Natural Sciences (Biology, Physics, Chemistry) All of these are divided again into different branches. Apart from regular studies scientific research is of significant importance.

4 Preparations for the transmission electron microscope (TEM) Fixation, Embedding, Sectioning, Staining On our first day we were presented to the staff working in and around the electron microscopy division. The team consists of scientists and technical assistants, all of them very friendly people, who showed us a lot during the following days. Both companies and the university use this division for experimental research. Each day of a week is designed for a specific step or method. On Thursdays for example, probes that are to be examined under the microscope, mostly cells or viruses, are embedded into synthetic resin. This longsome procedure takes up the time of a whole day and is therefore performed once a week only. Fixation One or two days before embedding, probes are fixated in a dilution. We were allowed to help putting the probes into a dilution of Glutaraldehyde and a buffer which adjusts variations of the dilution s ph. The phosphate buffer we used consists of Na 2 HPO 4 + NaH 2 PO 4 + NaCl. The fixation process is important to keep the cell it in its momentary state, i.e. to protect it from either growth or decay. As a positive side effect undesired organisms such as bacteria are killed. After the first fixation preparations are washed in a buffer and fixated again in Osmium Tetroxide. The cell s membranes will color black when in Osmium which makes them easily observable. For dehydration probes are now put into 30% ethanol first, then 50% and at last 70 % ethanol around 2-3 minutes each. They can be left in the latter a while without harm; in all cases they have to be dehydrated in 90% ethanol before they can be put into a heating furnace at 37 C. As a next step, the synthetic resin is prepared. A special gadget turns the resin-containing bottle around and upside down continually to ensure the resin becomes and stays smooth. The probes are then taken out of the furnace after half an hour and put into 100% ethanol three times. Embedding For embedding, the probes are consecutively put into containers with epoxy propane and synthetic resin. These transition solvents differ in their epoxy propane/resin ratio. From the first container to the last one the ratio changes from 2:1 to 1:2 and the time probes have to stay inside rises from 15 minutes to an hour. Preparations will infiltrate with pure resin over night and put into molds containing fresh resin the next day. To be sectionable on Monday morning preparations are left inside the resin to harden over the weekend at 60 C. Sectioning Firstly, the exact place of the probe inside the resin is marked with a black dot. To be able to cut out the marked spot easily with a microtome, a small hole is drilled right next to the dot to pull the saw blade through. Secondly, without paying attention to the plastic mold inside of which the preparation is still fixed, the saw or microtome cuts around the dot. The result is a small circular piece of resin that contains the object to be examined. To get rid of the mold liquid nitrogen is used to separate them. Finally, the preparations are sectioned into ultrathin slices with a glass knife or a diamond knife and put on little platelets out of copper which have a diameter of 4 mm.

5 Staining For staining and thus contrasting the preparations radioactive uranyl acetate is used. This is another process we were allowed to perform ourselves, which was a lot of fun since we spent most of our time watching. Besides, staining preparations is pretty easy. At first one has to put a paraffin paper into a Petri bowl with cover, dribble a drop of radioactive uranyl acetate on the paper and carefully put the copper platelet on it with fine tweezers. Next you have to take it out after five minutes, dip it consecutively into three little glass bowls filled with double distilled water to clean it and dry it by dabbing it on special paper. In a final step you stain the preparation s proteins in lead citrate. Because the lead citrate tends to crystallize slightly, the air humidity inside the Petri bowl has to be lowered by little sodium platelets. The dried preparation is then finally ready for viewing under the electron microscope. For pictures taken with the TEM see two last pages Right hand side: Transmission Electron microscope Lower left hand side: TEM with computer and operating buttons

6 The transmission electron Microscope TEM, Viruses, HFF-cells First of all, this is a short explanation of how the TEM works: Both TEM and SEM keep the preparations in a vacuum. An electron gun inside the microscope emits an electron beam which transmits the specimen. On emerging, the beam carries the information on structure and shape of the preparation and creates an image on a monitor right below. Inside the apparatus there is a little LCD-camera that makes viewing the images and editing them on a computer screen possible. Using the microscope we obtained a very clear picture of cells and their content. Furthermore we were able to identify infected cells and check their viruses state of development. Below is an illustration of a naked, virus on the left hand side and a grown one on the right: For our studies we used the Humane Cytomegalovirus (HMCV). It can cause severe brain damage in newborns and result in death during transplantations because of its unusually rapid growth to a huge size. This fact also features as the prefix mega in the viruse s name. The cells we examined are human foreskin cells shortened HFF. During our examinations we learned that there is a lot more to a human cell than we have learned so far in our biology classes. For instance we learned now, that in a figurative Nucleus Tegument proteins sense, the mitochondrium forms the power station of the cell. It changes organic substances and releases energy. Mitochondria consist of an outer and an inner membrane and are to be found next to viruses and dense bodies (small vacuoles filled with tegument protein waste) outside the nucleus. According to what we were able to see, inside the nucleus there is the DNA, some capsids and tegument proteins. Outside the nucleus there is also the Golgi-Apparatus which is responsible for transporting and changing proteins.

7 Scanning electron microscope Preparation, examination, postgraduates Apart from the recently discussed TEM there is another electron microscope: The Scanning electron microscope shortened SEM. In contrast to TEM, which displays the inner structure of objects, the SEM uses impinging electrons to create an image of the preparation s surface, as if it were scanning it. Preparation Although there are several distinct procedures, we got to know and perform only one of them: the critical point method. The specimen, for instance a little flea, is carefully put into a small plastic box and put into a drying unit. The unit is cooled down and at a pressure of 50 to 60 bar introduces liquid CO 2. As a result, the water inside the animal is exchanged for Acetone and afterwards for CO 2. Temperature rises again and at the critical point of C the liquid CO 2 merges into gas. Consequently the animal is dry but best preserved and kept from shrivelling. At last the dried animal is put onto a small slice of silicium. A sticking layer on the slice clings onto the animal instantly. To increase electric conductivity of electrons the preparation is vapour coated with a thin layer of metal. In our cases either Platinum or Gold-Palladium was used. Examination We used the SEM to discover the subtleties of for example erythrocytes, mold fungi and little animals such as fleas, ticks and crustaceans. The SEM allows zooming several thousand times and thus gives a very clear vision of the objects which is highly fascinating. Moreover we were explained the reasons and effects of the animals build. What I liked most was the examination of a piece of wing of a butterfly. Unfortunately the screen does not show colors, but the structure is very well visible. The structure of a wing s scale resembles a ladder. Consequently a lot of air passes through when the butterfly flies which explains the relatively slow speed in comparison to a fly, whose wings come with a strong air resistance. Postgraduates A very interesting highlight in our week was the encounter with two postgraduates. One of these two young men is doing research on solar cells for his dissertation. His aim is to decrease the amount of energy needed in the cells production to make them cheaper in the end. The other one was proud to show us enthusiastically his 250 million years old crustaceans. At the university there are a lot of postgraduates who use the machines and microscopes to do research for their dissertations. In the virology department we were introduced to a young postgraduate who presented her dissertation in English. Although I am usually quite comfortable with the English language, it was hard to follow because of the amount of unfamiliar technical terms.

8 Miscellaneous In the course of the week I interviewed some of the staff and research assistants regarding their jobs. A technical assistant answered my question on jobs abroad there were enough and even better paid. Her job at the University was relatively secure; yet she was not sure about its consistency after she retires. Many others, mostly students, only come for single projects and after the projects are done so are they. Furthermore she told me there were hardly any mandatory skill enhancement trainings except for crash courses in operating new machines and gadgets which are paid by the department. A job offer there requires perfect English and a University degree in either Biology or Medicine, but there are also chemists or physicists (cf. the postgraduate experimenting with solar-cells). Very striking was the amount of toxic waste produced in laboratories. No longer required excesses of a used chemical must not be put back into their original cans and have to be disposed of separately. All this adds up to a remarkable amount of waste. During our free time we used a small light-optic microscope we know from school to examine little blossoms, our fingers and whatever else we could find. We were quite shocked of how much our hands had dried out from washing them so unusually often. Conclusion Unfortunately, the week passed way too quickly. We were often given the opportunity to help or to perform small steps ourselves which made learning vivid and gave us a chance to immediately make use of what we had just learned. I liked that very much. Although microscopy had not belonged to my fields of interests before, I found the career orientation utterly interesting and in the long run I can say that this week aroused an ongoing interest in both Biology and Microscopy in me. These days I am proud to call a small light-optical microscope my property which I frequently use to discover the subtleties of my environment. Last but not least I want to thank all members of the department who explained and showed us everything eagerly with remarkable patience. My biggest thanks go to my friend Alice and her grandmother Ms. Dr. Kleinschmidt who was willing to take care of us and together with Mr. Prof. Walther gave me this amazing opportunity. The second objective set by BOGY, i.e. helping students choose a career, was not fulfilled in my case. It rather made it even more difficult. I do not regret this, though. I am rather thankful for showing me new options.

9 ANNEX Pictures taken with TEM and SEM TICK The tick s mouthparts

10 discharged virus particles