scanning electron microscope

oral pathology
laboratory

1. GOOD MORNING ! ‹#›

2. Scanning Electron Microscope ‹#›

3. CONTENTS: Introduction History Applications Principle Parts Preparation of sample Advantages and disadvantages References ‹#›

4. Introduction : The scanning electron microscope is one of the most versatile instruments available for examination and analysis of the microstructural characteristics of solid objects. The primary reason for the SEMs usefulness is the high resolution which can be obtained when bulk objects are examined. Another important feature of SEM images is the three dimensional appearance of specimen , which is a result of large depth of focus. ‹#›

5. SEM can provide useful information about the composition at the specimen surface. The user can obtain high magnification images, with a good depth of field, and can also analyse individual crystals or other features. A high-resolution SEM image can show detail down to 25 Angstroms, or better. When used in conjunction with the closely-related technique of energy-dispersive X-ray microanalysis (EDX, EDS, EDAX), the composition of individual crystals or features can be determined. ‹#›

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7. The area to be examined is irradiated with a finely focused beam of electrons which may be static or swept in a raster across the surface of the specimen. They are obtained from specific emission volumes within the sample and are used to measure many characteristics of the sample (composition, surface topography , crystallography,) ‹#›

8. The resultant images are 3 dimensional and have better depth of field. ‹#›

9. History of microscope Zacharias Jansen Anton van Leeuwenhoek ‹#›

10. The first operational electron microscope was presented by Ernst Ruska and Max Knoll in 1932, and 6 years later Ruska had a first version on the market. In 1986 Ruska received a Nobel Prize in physics for his "fundamental work in electron optics and for the design of the first electron microscope". ‹#›

11. The invention of the electron microscope by Max Knoll and Ernst Ruska at the Berlin Technische Hochschule in 1931 finally overcame the barrier to higher resolution that had been imposed by the limitations of visible light. Since then resolution has defined the progress of the technology. The ultimate goal was atomic resolution - the ability to see atoms. To avoid the effects of objective lens chromatic aberration with thick samples in TEM , SEM was invented. ‹#›

12. It was Manfred von Ardenne in 1937 invented SEM. Further work was reported by Zworykin's group, followed by the Cambridge groups in the 1950s and early 1960s headed by Charles Oatley , all of which finally led to the marketing of the first commercial instrument by Cambridge Scientific Instrument Company as the "Stereoscan" in 1965, which was delivered to DuPont . ‹#›

13. Applications of SEM: Scanning Electron Microscopy - SEM - is a powerful technique in the examination of materials. It is used widely in metallurgy, geology, biology and medicine, to name just a few. ‹#›

14. APPLICATIONS Biomedical engineering Cell & Tissue morphology Microbiology Pharmaceuticals Plant and animal biology Subcellular analysis Environmental and Food Sciences ‹#›

15. BIOMEDICAL ENGINEERING SEMs combine the latest concepts from medicine, biotechnology and engineering for designing a variety of technologies such as support matrices for cell growth, artificial tissue and implantable biomedical device. Growth of osteoblasts on zirconia ceramics ‹#›

16. CELL & TISSUE MORPHOLOGY SEMs are the ideal instruments for investigating cellular and tissue structure with high resolution. Typical applications involve observing shape changes of grooves, pores, blebs or microvilli on the cellular in response to the changes in the extracellular environment. Detail of a fibroblast cell ‹#›

17. MICROBIOLOGY High resolution imaging of the microbial surface using SEM helps to have a better understanding of the morphology of microbial populations, bacteria communication and biofilm formation. SEMs help researchers visualize microbial populations with great focal depth and high resolution. S.mutans on dental filling ‹#›

18. SUBCELLULAR ANALYSIS The SEM technology is becoming more popular in this field, due to emerging techniques which are available to scanning electron microscopy. SEM offers several solutions for scientists interested in the subcellular investigations of biological samples. Osteoblast layer in a mouse tooth. ‹#›

19. Principle In a scanning electron microscope, the specimen is exposed to a narrow electron beam from an electron gun, which rapidly moves over or scans the surface of the specimen. This causes the release of a shower of secondary electrons and other types of radiations from the specimen surface. The intensity of these secondary electrons depends upon the shape and the chemical composition of the irradiated object. These electrons are collected by a detector, which generates electronic signals. These signals are scanned in the manner of a television system to produce an image on a cathode ray tube (CRT). ‹#›

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22. Construction of SEM : Electron operating system Specimen stage Secondary electron detector Image display unit Operation system Electron gun Condenser lens Objective lens Scanning coil ‹#›

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24. E lectron Gun : Electron gun produces an electron beam. Thermo electrons are emitted from a filament(cathode) made of thin tungsten wire by heating the filament to a high temperature. These thermo electrons are gathered as an electron beam flowing into the metal plate(anode) by applying positive voltage to anode. If a hole is made at the centre of the anode, the electron beam flows through it. An electrode is placed between cathode and anode to adjust the current of electron beam. The electron beam is finely focused by the action of wehnelt electrode. ‹#›

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26. Construction of lens : A direct electric current is passed through a coil wound electric wire a rotationally symmetric magnetic field is formed and a lens action is produced on electron beam. The surrounding of the coil is enclosed by yoke so that part of magnetic field leaks out from a narrow gap. A portion with narrow gap is called pole piece. When the current passing through the coil changes, the strength of coil is also changed which is not possible in light microscope. ‹#›

27. Lenses ‹#›

28. Condenser lens and objective lens Two stage lenses which combine the condenser and objective lenses are located below the electron gun. The electron beam from the electron gun is focused by the 2 stage lenses and a small electron probe is produced. Placing a lens below the electron gun enables you to adjust the diameter of electron beam. A fine electron beam is required for SEM. The aperture is placed between condenser and objective lens. The aperture is made of thin metal plate and has a small hole. ‹#›

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30. The electron beam, which passed through the condenser lens illuminates this aperture plate. The aperture allows a part of electron beam to reach the objective lens. The objective lens is used for focusing. It determines the final diameter of electron probe. If the objective lens is not good, an optimally fine electron probe cannot be produced . ‹#›

31. Specimen stage In general, the specimen is observed at higher magnification in an electron microscope. Thus a specimen stage which stably supports the specimen and moves smoothly is required. The specimen stage in SEM can perform following movements: Horizontal movement Vertical movement Specimen tilting Rotation Change of image resolution Selection of field of view ‹#›

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33. Most SEMs use eucentric specimen stage. By the use of this stage the specimen does not change after shifting the field of view when the specimen is tilted. In addition to manual drive stage, the use of motor driven stage has increased. In computer controlled specimen stage, the stage can be moved to selected point by simply clicking mouse and restore the stage to desired observed point. ‹#›

34. Secondary electron detector The secondary electron detector detects secondary electrons emitted from specimen invented by Everhart and Thornley. Also called E-T detector. A scintillator (fluorescent) substance is coated on the tip of the detector and a high voltage of 10kv is applied to it. The secondary electrons from the specimen are attracted to this high voltage and then generate light when they hit the scintillator. This light is directed to a photomultiplier tube (PMT) through a light guide. Then the light is converted to electrons and these electrons are amplified as an electric signal. This is present in sample chamber nearer to objective lens. ‹#›

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39. Preparation of sample Treatment of biological specimens involves following procedure: Removing and cleaning of tissues Fixation Dehydration Drying Mounting and coating Coating is same as for other non conductive specimens. It is coated with a thin metal film so that surface has conductivity. ‹#›

40. ‹#› Coating The dried specimen is mounted on an aluminium stub. A simple adhesive like durofix may be used. The specimen is placed in the chamber of a sputter coating unit and air is evacuated by purging the chamber with argon for 2 mins. When vacuum of 0.1torr is attained gold is discharged from a target situated above the specimen and deposited on the surface of sample. The thickness of the layer of gold is determined by the distance of the specimen from the target, period of time taken to sputter and strength of current applied to target.

41. ‹#› Cryopreservation: Eliminates the artefacts associated with fixation, dehydration and embedding. There are now cryosystems which interface directly to SEM which allow fresh tissue to be frozen rapidly with liquid nitrogen, sputtered with gold and then examined in SEM while it is still frozen. These cryosystems consists of Slushing chamber to freeze specimen Low temperature preparation chamber in which specimens may be fractured have the ice sublimed from their surface and be coated. This preparation chamber can be fitted directly to SEM or may be a separate unit.

42. Differences between TEM and SEM SEM TEM Scattered electrons T ransmitted electrons Larger samples can be examined. Sample has to be cut into thinner sections. Large amount of sample can be examined at a time. Only small amount of sample can be examined at a time. Comparatively lesser resolution. Resolution is greater than SEM. Effective Instrument Resolution - 1nm Effective Instrument Resolution - 0.5nm ‹#›

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53. ‹#› References : Cullings handbook of histopathological and histochemical techniques - 3rd edition. Bancroft book of histopathology - 7th edition. Various internet sources

54. Thanks! ‹#›