Electron microscope

electron microscope transmission electron microscope scanning electon microscope

1. Electron Microscope Dr.P.Nithiya Assistant professor Department of Botany Seethalakshmi Ramaswami College Tiruchirappalli - 620002

2. Electron Microscope Microscopes that are use electrons as the light source and electromagnetic coils to direct the path of the e - are called as electron microscopes. ( The optical system is completely replaced by electromagnetic coils). The first electron microscope was designed by Knoll and Ruska (1931). ( Wavelength of e - = 0.05A  very short wavelength with very high magnification ). The magnification of electron microscope is 1000 times higher than the light microscope. ( Therefore the magnification of e - is 100 × 1000 = 1, 00,000 X.)

3. Types of Electron Microscope Transmission electron microscope ( TEM ) Scanning electron microscope ( SEM)

4. Transmission electron microscope (TEM): In this type e- are allowed to transmit through the specimen is called TEM. The first TEM was designed by Max Knoll and Ernst Ruska (1931).

5. Max Knoll and Ernst Ruska

6. Principle  The basic principle of electron microscope is similar to the compound microscopes but the e- beam is substituted for light source and electromagnetic coils to optical lens.  When high voltage current is passed through the cathode ray tube, e- beams are produced.  Electromagnetic coils direct the e- beams to pass through the specimen.  It is stained with gold or osmium and the image is collected by objective lens and amplifier (electromagnetic coils).  The image cannot be seen by our naked eye, so it is casted on a screen or photographic plate or camera.

7. Electron microscopes are kept in vacuum because 1. Electrons are easily absorbed in air. 2 . Electrons are move in a straight line only in vacuum.

8. Instrumentation: The TEM has an electron gun and condenser lens. A) Electron gun B) Condenser lens C) Objective lens D) Amplifier lens E) Projector lens F) Ancillary equipment

9. A) Electron gun:  Made up of cathode ray tube with tungsten filament (2mm long)  Located at the top of the microscope.  It generates e-

10. B) Condenser lens  Two condenser lens or electromagnetic coils are present below the e- gun.  They collect and direct the beams into the specimens on a stage.  A thin section of specimen is placed on a thin plastic film mounted on a copper gird ( 3 mm diameter).

11. C) Objective lens  It is an electromagnetic coil placed below the specimen stage.  It collects the specimen image and focus towards the amplifier lens.

12. D) Amplifier lens  It is an electromagnetic coil below the objective lens and magnifies the image several times.

13. E) Projector lens collects the magnified image and focused on a fluorescent screen or photographic plate.

14. F) Ancillary equipment 1 . The entire set up is placed in a vacuum tube. 2. TEM release large amount of heat during working hours, so cooling system is present 3. It needs high power supply

15. Preparation of specimen for TEM: Biological material contains low atomic weight elements like carbon, hydrogen, oxygen and nitrogen . They do not give high resolution. Therefore , the biological sample has to be loaded with heavy atoms like gold or osmium and these atoms protect the specimen from destruction.

16. It involves the following steps. The specimens are dehydrated by keep it in ethanol or acetone. Place it chemical fixative like Osmium tetra oxide. (These fixatives form covalent bonds with biological molecules). After fixation, the specimen is embedded in araldite or plastic medium .

17. It involves the following steps The embedded specimen is cut in to thin sections of 50-100nm thickness using a glass or diamond knife in an ultra microtome. The thin sections are mounted on the copper grid. The grid with specimen is mounted on the specimen stage. The image is viewed on fluorescence screen.

18. TEM image

19. Applications TEM is an ideal tool for the study of ultra structure of a cell. It is used to identify plant and animal virus. It is widely applied in various researches in oncology, pollution, biochemistry, molecular biology, etc.

20. Disadvantages: Very high cost. We cannot study 3 dimensional structures of the specimens. The specimens should be fixed properly and should take ultra thin sections, because an electron has limited penetrating power. We could not study live specimens. It is successful only under high vacuum condition.

21. Scanning Electron Microscope Dr.P.Nithiya Assistant professor Department of Botany Seethalakshmi Ramaswami College Tiruchirappalli - 620002

22. Scanning Electron Microscope In SEM , the surface of the specimen is scan by electron beam. This was first designed by Max Knoll (1935).

23. Principle : SEM use electron beam for illumination and electromagnetic coils for directing the path of e - beam. When e - is focused on the specimen, it produces secondary e - (SE ), back scattered e - (BSE) and characteristic X-rays . Secondary electrons are reflected due to the interactions between atoms in specimens and e - beam. Back scattered e - gives information about the distribution of different elements .

24. Characteristic X-rays are emitted by the sample when the e - beam removes e - from the inner shells of the atoms of the specimens. These three rays are detected by specialized detectors. Electronic amplifier like collectors, Scintillator and Photomultiplier tube (PMT ) are used to measure the e - signals. The e - signals are converted in to image which is focused on the monitor.

25. Instrumentation: Electron gun : it is the source of e - beam and located at the top of the microscope. It consists of cathode plate and anode plate .

26. Condenser lens: there are two condenser lenses just below the e- gun. They collect and concentrate the e- in to a strong beam.

27. C. Deflection coils: below the condensers, there is a deflection coil to direct the beam of e- in to the specimen stage .

28. D. Specimen stage: it is present in slanting position at the lower side of deflection coil.

29. E. Separate e- detectors ( scintillator& PMT ) are attached in the vacuum tube . Electronic amplifiers are connected with detectors. The electric signals are converted into bright spots of varying density by scanning circuit .

30. Additional things G. Image is displayed on a photographic plate or computer monitor. H. The entire set up should be placed in a vacuum tube. I. Power supply with high voltage. J. SEM releases huge amount of heat, so cooling system is present around it.

31. Preparations of specimens for SEM : Dry materials like wood, bone, feathers, insect’s wings and shells are coated with thin film of electro conductive materials like gold , platinum, tungsten, osmium, chromium and graphite . Then the specimens are placed on the stage.

32. Preparation of wet specimens involves the following steps. Specimens are fixed by fixatives like osmium tetroxide, potassium permanganate, formalin ,etc . which stabilize molecular organization of specimens. They are dehydrated by keeping in the increasing concentrations of ethanol or acetone . The specimens are coated with ultra thin layer of electro conductive alloy . Then they are put in the specimen stage. The e - beam is passing through the specimen and final image is produced on the computer screen .

33. SEM IMAGES

34. Advantages :  SEM is use full to view the surface of microorganisms (Bacteria, Diatoms), pollen grains, hairs and scales of plants and animals.  It is free of chromatic aberrations.  It produce 3D image.  SEM is used study archeological specimens and fossils.  It is used to analyze the compound eyes of insects.

35. Disadvantages :  Lower resolution than TEM.  High cost.  Complete vacuum is needed.  Factors limit the quality is uncontrolled emission of e- and scan faults.

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