Electron microscope ppt
- 1. ELECTRON MICROSCOPE MRS. R. NITHYA M. A., M. Sc., M. Phil., PGDCA., ASSISTANT PROFESSOR SRI ADI CHUNCHANAGIRI WOMEN’S COLLEGE CUMBUM - 625516
- 2. Electron microscope What are electron microscopes? Electron microscopes are scientific instruments that use highly energetic electron to examine objects on a very fine scale which yield the following information: 1. Topography: The surface features of an object (hardness, reflectivity… etc.) 2. Morphology: The shape and size of the particles 3. Composition: The elements and compounds that the object is composed of and the relative amount of them. 4. Crystallographic information: How the atoms are arranged in the object.
- 3. Why were the EMs advented? To study objects of < 0.2 micrometer For analysis of sub cellular structures Intra cellular pathogens – viruses Cell metabolism Study of minute structures in the nature. EMs have greater resolving power than light microscope An EM can magnify structure from 100 – 250000 times than light microscope.
- 4. TYPES OF ELECTRON MICROSCOPE There are two types of electron microscopes, namely Transmission Electron Microscope (TEM) – allows one the study of the inner surface. Scanning Electron Microscope (SEM) – used to visualize the surface of objects. Transmission Electron Microscope (TEM) The first TEM was built by Max Knoll and Ernst Ruska in 1931. The TEM was first made available in the market in 1939.
- 5. TRANSMISSION ELECTRON MICROSCOPE TEM is a microscopy technique where a beam of electrons is transmitted through an ultra thin specimen. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a CCD camera.
- 6. The TEM Components The TEM consists of Electron gun Condenser Lenses Objective Lenses Projector lenses and Fluorescent screen Electrons are emitted by the electron gun The condenser lenses focus the beam onto the specimen. The objective lens forms a focused image, which is enlarged by the projector lenses. The image is viewed on a fluorescent screen or a computer monitor.
- 7. Electron gun Electron gun is the source of electron beam. It is placed at the top of the TEM. It consists of V- shaped filament, Whenelt clinder and an anode plate. Whenelt cylinder acts as a cathode. It is a cup-like structure with a hole at the Centre. When high voltage is applied between filament and anode plate, the filament is heated up to incandescence for emitting electrons. Electrons are attracted towards the anode plate, they are forced out through the hole int the centre of the anode plate by the cathode shield.
- 8. Condenser lenses Two condenser (Electromagnetic coils) They collect and concentrate the electrons into a strong electron beam before focusing it onto the specimen. Specimen stage A thin section of specimen is placed on a thin plastic film mounted on a copper grid. The specimen mounted grid is placed in the path of the electron beam. Objective lens Placed below the specimen stage. It collects the image of the specimen and focuses towards the amplifier lens. Amplifier lens It just kept below the objective lens. It magnifies the image produced by the objective lens to several 1000 times.
- 9. Projector lens It collects the magnified image and focuses it onto fluorescent screen or photographic plate. Vacuum tube The entire setup is placed in a vacuum tube because electrons can move in a straight line only in a vacuum. Vacuum pressure of 10-7 to 10-9 Pa is applied in the vacuum tube. Cooling System While TEM is working, a large amount of heat is produced. To keep the apparatus at a low temperature cooling water is circulated through a cooling system around the TEM.
- 10. SAMPLE PREPARATION DEHYDRATION The wet sample is dehydrated by keeping in increasing concentration of ethanol or acetone FIXATION Fixation is done by immersing the specimen in chemical preservatives called fixative. Osmium tetroxide, glutaraldehyde, potassium permanganate, formalin, etc. are common fixatives. These fixatives form covalent bond with biological molecules like proteins and lipids. They stabilize the structural organization in the specimen.
- 11. EMBEDDING The specimen is embedded in a hard embedding medium like araldite vestopal-W or Epson-812 or Plastic medium The embedded specimen is cut into thin section of 50-100nm thickness using a glass or diamond knife fixed in an ultramicrotome. The thin section is mounted on a copper grid of 3mm diameter and covered with parlodoan. Ultramicrotome
- 12. STAINING The specimen is kept dipped in a solution containing heavy metal ions for metallic staining. Solutions of phosphotungstate, Lead acetate, Lead hydroxide Osmium tetroxide are useful for this staining. The specimen mounted grid is then placed on the specimen stage. The image of the selected section is then viewed on the fluorescent screen. Image of the specimen is viewed through optical system. Gamma phage
- 13. APPLICATIONS OF TEM Ideal tool for the study of ultra structure of cells Chloroplast Plant cell
- 14. Employed in the localization of nucleic acid, enzymes and protein in cells and cell organelles. Baculo virus release DNA Uncoiled DNA
- 15. Used in cancer research for the cytological observation of cancer cells. Colon cancer cells
- 16. Used in identification of plant and animal viruses Plant virus – tobacco mosaic virus Animal virus – Polio virus
- 17. ADAVANTAGES TEM offer the most powerful magnification, potentially over one million times or more TEMs provide information on element and compound structure Images are high- quality and detailed They are easy to operate with proper training
- 18. SCANNING ELECTRON MICROSCOPE SEM produces the image by scanning it with focus beam of electron. Electrons interact with electrons in sample and convey information in form of signals to detectors. SEM can achieve resolution better than 1nanometer.
- 19. SEM COMPONENTS SEM consists of an Electron gun – produces an e-beam Condenser lens – collect and concentrate the electrons into a strong electron beam before focusing it onto the specimen Deflection coil – change the direction of electron path helps for focussing the e- beam on the specimen Specimen stage – Specimen mounted grid is p[laced on the specimen stage in a 450 slanting position. When e- beam is focused on the specimen , it produces secondary electrons(SE), back- scattered electrons (BSE) and characteristic x-rays.
- 20. Secondary e- detector The emitted SE is collected by SED and converted into signal that is sent to a screen which produces the final image. Additional sensors – detect backscattered electrons and x-rays. Amplifiers Different kind of amplifiers(grid, scintillator and PMT) are used to measure the electrical signals. The electrical signals are converted into bright spots of varying density by scanning circuit to give the image of the specimen. Photographic plate The image may be captured on a photographic plate or computer monitor or hard disk.
- 21. Vacuum tube The entire setup is placed in a vacuum tube because electrons can move in a straight line only in a vacuum. Vacuum pressure of 10-7 to 10-9 Pa is applied in the vacuum tube. Cooling System While TEM is working, a large amount of heat is produced. To keep the apparatus at a low temperature cooling water is circulated through a cooling system around the TEM. Blood cells Head of bee
- 22. Specimen preparation - Dry sample Metallic Coating Dry materials such as wood, bone, feathers, insect’s wings and shells are coated with electronically conducting materials. They are metallic gold, platinum, tungsten, iridium, osmium, chromium and graphite. Metallic coating prevents the accumulation of electrostatic charges on the specimen. Penguin feather Peacock feather
- 23. Wet sample preparation Fixation Fixation is done by immersing the specimen in chemical preservatives. It stabilize the molecular organization. Common fixatives are Osmium tetraoxide, glutaraldehyde, potassium permanganate, formalin etc. Dehydration After fixation the specimen is dehydrated by keeping it in concentrated ethanol or acetone. Staining The specimen is then coated with an ultrathin layer of electroconductive alloy. Then it is placed in slanting position on the specimen holder.
- 24. APPLICATIONS OF SEM SEM is very useful to view the surface architecture of microscopic creatures like bacteria, diatoms, pollen grains, nematodes and others. SEM gives 3-D structure of objects. Pollen grains of sunflower
- 25. Hairs and scales on plant and animal surfaces are characterized with SEM. Plant trichome Hairs on the legs of palnt bug
- 26. SEM is employed in the analysis of structural features of compound eyes of insects. Eye of black garden ant
- 27. Mosquito eyes Eyes of yellow dung fly