1. THE ELECTRON MICROSCOPE

2. Introduction

12. Electron Microscope – Any class of microscopes that use electrons instead of light to form images of very small objects such as individual parts of small living things

13. Electron Microscope Uses a magnetic field to bend beams of electrons; – greater magnification & resolving power than light microscope The two types are: Scanning and Transmission

14. Which is the most powerful kind of microscope?

15. THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE fluorescent (TV) screen, photographic film Human eye (retina), photographic film Focussing screen VacuumAir-filledInterior MagnetsGlassLenses High voltage (50kV) tungsten lamp Tungsten or quartz halogen lamp Radiation source x500 000x1000 – x1500 Maximum magnification 0.2nm Fine detail app. 200nmMaximum resolving power Electrons app. 4nm Monochrome Visible light 760nm (red) – 390nm Colours visible Electromagnetic spectrum used ELECTRON MICROSCOPELIGHT MICROSCOPEFEATURE

16. THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE Copper gridGlass slideSupport Heavy metalsWater soluble dyesStains Microtome only. Slices  50nm Parts of cells visible Hand or microtome slices  20 000nm Whole cells visible Sectioning ResinWax Embedding OsO 4 or KMnO 4 AlcoholFixation Tissues must be dehydrated = dead Temporary mounts living or dead Preparation of specimens ELECTRON MICROSCOPE LIGHT MICROSCOPEFEATURE

17. Properties of electron Electron are used as a source of illumination They are negatively charged subatomic particles When the atoms of metal are excited by sufficient energy in the form of heat, the electron leave their orbit, fly off from space & are lost in atoms Metal tungsten is commonly used as a source of electron

18. The electron are readily absorbed & scattered by different form of matter So a beam of electron -> produced & sustained only in high vacuum Electron are like light waves-> So used in image formation Electron interact with the atoms of the biological specimens to form the image

19. Electron beam (A) Transmitted electron (B) Inelastically scattered electrons (C) Elastically scattered electrons (D) Back-scattered electrons (E) Secondary electrons X-rays Visible light

20. 1.Transmitted electrons (A) of the beam passes straight through the specimen on to the screen 2.Some electron (B) of the beam lose a bit of their energy while passing through the specimen & get deflected a little from their original axis of the beam  inelastically scattered electrons 3.Some electron (c) interact with atoms of specimen & get elastically scattered without losing energy. Electron deviate widely

21. 4. Some electron (D) get backscattered instead of getting transmitted through the specimen 5. In some cases the electrons get absorbed by the atoms of the specimen & instead low energy electron (E) are emitted. These electron are termed secondary electron. These are very useful for forming the image in the SEM 6. Some atom emit x-ray & light energy

22. Working & Image formation Working of EM is based on same plan as that of light microscope Electron are used for magnification & image formation Image formation occurs by electron scattering Electron strike the atomic nuclei & get dispersed This dispersed electron form image The electron image is converted in to visible form by projecting on a fluorescent screen

23. Electron in the form of a beam pass through the condenser coil & fall on the object They get scattered & transmitted through the object & pass through the objective coil, which magnifies the image of the object The projector coil further magnifies the image & projects on the fluorescent screen/film The image formation occurs when the energy of the electron is transformed in to visible light through excitation of the chemical coating of the screen

24. Those electron which reach the fluorescent screen form bright spot while the area where the electron do not reach the screen form dark spot The varying degree of intensity of electrons form the image with varying degree of grey. Electron scattering, however, is due to the atomic nuclei which consist of protons & neutrons Higher the atomic number, greater the scattering

25. Since biological materials generally have a low atomic number, the dispersion is poor Very poor dispersion means very poor contrast in the image formation In order to increase contrast, a number of salts with high atomic number are used. Such salts can be used during the process of fixation or staining

26. Magnification Objective & projector coil help in magnifying the image formed in EM. In order to have maximum magnification, an intermediate coil is fitted between the objective & projector coils. This coil further increase the magnification Magnification –> objective coil – 100, projector coil - 200, so net magnification= 20,000 EM fitted with intermediate coil can achieve magnification as high as 1,60,000

27. Resolution The resolving power of a microscope is limited by wavelength of illumination forming the image Shorter wavelength, smaller detail can be resolved This concept led to discovery of EM Resolution power of good EM is 4-10 Å

28. Transmission Electron Microscope (TEM) Electrons are passed through very thin specimens to see what is inside! Invented in1933 Magnification is 500,000x

29. 29 The TEM is a complex viewing system equipped with a set of electromagnetic lenses used to control the imaging electrons in order to generate the extremely fine structural details that are usually recorded on photographic film. Since the illuminating electrons pass through the specimens, the information is said to be a transmitted image. The modern TEM can achieve magnifications of one million times with resolutions of 0.1 nm.

30. 30 Basic Systems Making Up a Transmission Electron Microscope The transmission electron microscope is made up of a number of different systems that are integrated to form one functional unit capable of orienting and imaging extremely thin specimens. The illuminating system consists of the electron gun and condenser lenses that give rise to and control the amount of radiation striking the specimen. A specimen manipulation system composed of the specimen stage, specimen holders, and related hardware is necessary for orienting the thin specimen outside and inside the microscope.

31. 31 Basic Systems Making Up a Transmission Electron Microscope The imaging system includes the objective, intermediate, and projector lenses that are involved in forming, focusing, and magnifying the image on the viewing screen as well as the camera that is used to record the image. A vacuum system is necessary to remove interfering air molecules from the column of the electron microscope. In the descriptions that follow, the systems will be considered from the top of the microscope to the bottom.

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34. 34 Page 167 TABLE 6.3 Major Column Components of the TEM* ComponentSynonymsFunction of Components Illumination System Electron GunGun, SourceGenerates electrons and provides first coherent crossover of electron beam Condenser Lens 1C1, Spot SizeDetermines smallest illumination spot size on specimen (see Spot Size in Table 6.4) Condenser Lens 2C2, BrightnessVaries amount of illumination on specimen — in combination with C1 (see Brightness in Table 6.4) Condenser Aperture C2 ApertureReduces spherical aberration, helps control amount of illumination striking specimen

35. 35 Specimen Manipulation System Specimen ExchangerSpecimen Air LockChamber and mechanism for inserting specimen holder Specimen StageStageMechanism for moving specimen inside column of microscope Imaging System Objective Lens — Forms, magnifies, and focuses first image (see Focus in Table 6.4) Objective Aperture — Controls contrast and spherical aberration Intermediate LensDiffraction LensNormally used to help magnify image from objective lens and to focus diffraction pattern Intermediate ApertureDiffraction Aperture, Field Limiting Aperture Selects area to be diffracted Projector Lens 1P1Helps magnify image, possibly used in some diffraction work Projector Lens 2P2Same as P1

36. 36 Observation and Camera Systems Viewing Chamber — Contains viewing screen for final image Binocular Microscope Focusing ScopeMagnifies image on viewing screen for accurate focusing Camera — Contains film for recording

37. Scanning Electron Microscopy (SEM) Visualizes Surface Features

38. Extremely useful in studying surface structure The electron do not form the image by being transmitted, but by getting emitted from the surface of specimen Illuminating system of SEM similar to TEM The electron beam is, how ever compressed with 1 or more condenser coils, which result in the formation of an narrow pencil of electron

39. The electron which falls on specimen are primary electron The electron probe is focused on the surface of specimen The electron are emitted as secondary electrons from the surface The specimen is kept at inclined angle As the electron does not have to pass through specimen, its thickness is not important

40. The three dimensional image in SEM is formed by secondary electron But these electron does not have sufficient energy to excite the fluorescent screen and form the image as in TEM by primary electron In SEM secondary electron are first collected, amplified & then used for the formation of an image on the phosphor screen of cathode ray tube

41. Layout and performance of SEM 1-3Electron gun 4, 10Aperture 5-6Condenser lenses 7Scanning coils 8Stigmator 9Objective lens 11X-ray detector 12Pre-amplifier 13Scanning circuits 14Specimen 15Secondary electron detector 16-18Display/Control circuits

42. Specimen Preparation Specimens are coated with metals to deflect electrons from a beam scanned across the sample.

43. SEMSEM of Stereocilia Projecting from a Cochlear (inner ear) Hair Cell

44. Copper grid slides © 2007 Paul Billiet ODWSODWS

45. Higher Resolution Is Achieved by Viewing Sections of Fixed, Stained, and Embedded Samples A microtome cutting sections of an embedded sample.

46. Microtome knife © 2007 Paul Billiet ODWSODWS

47. Fig. 3-22