Electron microscope

Electron microscope ppt

1. ELECTRON MICROSCOPE BY : SANJAY SINGH BHOJ M.Sc 3 rd SEMESTER, ZOOLOGY

2. CONTENTS INTRODUCTION TRANSMISSION ELECTRON MICROSCOPE PARTS OF (TEM) SPECIMEN PREPARATION (TEM) SCANNING ELECTRON MICROSCOP (SEM) PARTS OF (SEM) SPECIMEN PREPARATION (SEM)

3. INTRODUCTION In the 1950s, researchers began to use the electron microscope which far surpassed the resolving power of the light microscope. Resolving power is inversely related to wavelength . Instead of light, electron microscope use electron beams which has much shorter wavelength than visible light. Enhanced resolution & magnification allowed researchers to clearly identify subcellular organelles & to study cell ultrastructure.

4. There are two types of electron microscopes : (1) Transmission electron microscope (TEM) (2) Scanning electron microscope (SEM) (A)Transmission electron microscope (TEM) : aims an electron beam at a thin section of specimen which may be stained with metals to absorb electrons & enhance contrast . Image is focused onto a viewing screen or film. Used to study internal cellular structure.

5. Parts of electron microscope (TEM) Electron gun : the light source of EM, in which tungsten filament emit electrons when high voltage is applied. Condenser magnet lens : focus the beam of electrons on the specimen. This electron beam passes through specimen , some of electrons are scattered while other are focused by projector magnetic lens onto fluorescent screen .

6. The electron gun is at the top of the central column. The image on the fluorescent screen may be viewed through a magnifier positioned over the viewing window. The camera is in a compartment below the screen. A modern Transmission Electron Microscope

7. Specimen Preparation for TEM Fixation : Glutaraldehyde to cross-link proteins & osmium tetroxide (OsO4) to fix & stain are most common fixatives. This process stabilize cell structure . Dehydration: in a series of alcohols to remove the water.

8. Embedding: in plastic such as epon for thin sectioning Plastic in vial polymerizes into a solid block at bottom edge of the block Block containing Tissue is trimmed to Prepare for sectioning Tissue is sliced into section approximately 100nm thick as block moves down across the sharp edge of a glass or diamond knife

9. The sections are then mounted onto copper or gold EM grids & subsequently stained with heavy metals for example, uranyl acetate & lead citrate. Sections are stained EM grid containing sections ready to be stained with heavy metals, placed in grid holder and examined in the electron microscope

10. Ultrathin Epon sections (60 nm thick) of developing rat sperm cells stained with uranyl acetate and lead citrate. Transmission electron microscopy (TEM).

11. (B) Scanning electron microscope (SEM) SEM is useful for studying the surface of a specimen. Electron beam scan the surface of the specimen usually coated with a thin film of gold. Scanning beam excites secondary electrons on the sample’s surface. These secondary electrons are collected & focused onto a viewing screen. SEM has great depth of field & produces a three-dimensional image .

12. Scanning Electron Microscope (SEM) Electron gun, and condenser lenses are same as in TEM. But here electron beam strike the metal coated surface of specimen & bounce , these bounced electrons are called secondary electrons. These secondary electrons entering the detector & then to photomultiplier. Where they converted to electric current. Which produce image in cathode-ray tube.

13. Specimen preparation for (SEM) Samples are fixed in glutaraldehyde, then dehydrated through a series of solvent. Then dried completely either in air or by critical point drying: this method removes all water from specimen instantly. Then specimen are then mounted onto a special metal holder or stub & coated with a thin layer of gold before viewing in SEM.

14. Scanning Electron Microscopy (SEM ) (A) Drosophilia melanogaster head. (B) T4 bacteriophage.

15. REFERENCES Karp. G. Cell and Molecular Biology. John Wiley & Sons.Inc. 6 th ed. 2010 Wilson & Walker. Biochemistry & Molecular Biology. Cambridge University Press. 7 th ed. 2010.