Scanning Electron Microscope (SEM)
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Electron microscopes use a beam of electrons to examine objects on a very fine scale. There are two main types: transmission electron microscopes (TEM) allow study of inner structures by passing electrons through thin samples, while scanning electron microscopes (SEM) are used to visualize surfaces by scanning a focused electron beam over the sample. SEMs detect signals from electron interactions to construct digital images, and require vacuum and conductive samples mounted on stubs. They provide three-dimensional topographical and compositional information at high magnifications.
Scanning Electron Microscope (SEM)
- 2. Electron Microscope Electron Microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield information about the: • Topography • Morphology • Composition • Crystallographic information
- 5. Mainly 2 types: • Transmission Electron Microscope (TEM) - allows one the study of the inner structures. • Scanning Electron Microscope (SEM) - used to visualize the surface of objects.
- 6. Differences between SEM and TEM TEM SEM Electron beam passes through thin sample. Electron beam scans over surface of sample. Specially prepared thin samples are supported on TEM grids. Sample can be any thickness and is mounted on an aluminum stub. Specimen stage halfway down column. Specimen stage in the chamber at the bottom of the column. Image shown on fluorescent screen. Image shown on TV monitor. Image is a two dimensional projection of the sample. Image is of the surface of the sample
- 7. Compound microscope image TEM image Budding yeast cell E. coli bacteria Compound microscope image TEM image SEM image SEM image
- 8. First Electron Microscope Invented by Ernst Ruska Year-1933 He was awarded the Nobel Prize for physics for his invention in 1986
- 9. History of sem First SEM – debuted in 1938 by Manfred Von Ardenne In 1965, Cambridge Scientific Instrument (UK) & JOEL (Japan) first commercialized SEM individually.
- 11. SEM Produces images of a sample by scanning it with a focused beam of electrons in a raster scan pattern Electrons interact with atoms -- produces various signals that contain information about the sample's surface topography and composition.
- 15. 1.Electron cannon. 2. Electro-magnetic lenses to focus the electron beam .3. Vacuum pumps system . 4.Opening to insert the object into the high- vacuum observation chamber. 5. Operation panel with focus, alignment and magnification tools and a joystick for positioning of the sample. 6. Screen for menu and image display 7.Cryo-unit to prepare frozen material before insertion in the observation chamber in Cryo-SEM mode PARTS OF SEM
- 16. Electron gun consisting of cathode and anode. The condenser lens controls the amount of electrons travelling down the column The objective lens focuses the beam into a spot on the sample. Deflection coil helps to deflect the electron beam. SED attracts the secondary electrons. Additional sensors detect backscattered electrons and X-rays.
- 17. SEM SAMPLE PREPARATION Appropriate size samples --- fit in the specimen chamber Mounted rigidly on a specimen holder-- specimen stub aluminiumstubs
- 18. SEM SAMPLE PREPARATION For imaging in the SEM, specimens must be Electrically conductive Electrically grounded
- 19. SEM SAMPLE PREPARATION 1. Cleaning the surface of the specimen 2. Stabilizing the specimen 3. Rinsing the specimen 4. Dehydrating the specimen 5. Drying the specimen 6. Mounting the specimen 7. Coating the specimen
- 20. SEM SAMPLE PREPARATION Cleaning the surface of the specimen Very important Surface contains many unwanted deposits, such as dust, mud, soil etc
- 21. SEM SAMPLE PREPARATION Stabilizing the specimen Hard, dry materials such as wood, bone, feathers, dried insects, or shells can be examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms usually require chemical fixation to preserve and stabilize their structure. Stabilization is typically done with fixatives.
- 22. SEM SAMPLE PREPARATION Fixation -- performed by incubation in a solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in combination with formaldehyde and other fixatives. Fixatives that can be used are:- 1. Aldehydes. 2. Osmium tetroxide. 3. Tanic acid. 4. Thiocarbohydrazides.
- 23. SEM SAMPLE PREPARATION Rinsing the specimen Sample must be rinsed -- remove excessive fixatives.
- 24. SEM SAMPLE PREPARATION Dehydrating the specimen Water must be removed Air-drying causes collapse and shrinkage, this is commonly achieved by replacement of water in the cells with organic solvents such as ethanol or acetone. Dehydration -- performed with a graded series of ethanol or acetone.
- 25. SEM SAMPLE PREPARATION Drying the specimen Specimen should be completely dry Otherwise the sample will be destroyed
- 26. SEM SAMPLE PREPARATION Mounting the specimen Specimen has to be mounted on the holder Mounted rigidly on a specimen holder called a specimen stub Dry specimen -- mounted on a specimen stub using an adhesive such as epoxy resin or electrically conductive double-sided adhesive tape.
- 27. Charge-up
- 29. This charge-up phenomenon can be prevented by coating the non-conductor sample with metal (conductor).
- 30. Sample coating is intended to prevent charge-up phenomenon by allowing the charge on the specimen surface go to ground through the coated conductive film.
- 31. SEM SAMPLE PREPARATION Coating the specimen To increase the conductivity of the specimen and to prevent the high voltage charge on the specimen Coated with thin layer i.e., 20nm-30nm of conductive metal. All metals are conductive and require no preparation before being used.
- 32. SEM SAMPLE PREPARATION Coating the specimen Non-metals need to be made conductive Done by using a device called a "sputter coater.” Conductive materials Gold Gold-palladium Alloy Platinum Osmium Iridium Tungsten Chromium Graphite
- 33. “Sputter Coater”
- 34. A spider coated in gold
- 35. Electron beam-sample interactions The incident electron beam is scattered in the sample, both elastically and inelastically This gives rise to various signals that we can detect Interaction volume increases with increasing acceleration voltage and decreases with increasing atomic number Images: Smith College Northampton, Massachusetts
- 36. So now we have a beam that is scanning across the sample surface and this beam is synched to the beam of a CRT.
- 40. Detectors Image: Anders W. B. Skilbred, UiO Secondary electron detector Backscattered electron detector
- 42. • A secondary electron detector attracts the scattered electrons and, depending on the number of electrons that reach the detector, registers different levels of brightness on a monitor.
- 44. A low atomic weight area of the sample will not emit as many backscattered electrons as a high atomic weight area of the sample. In reality, the image is mapping out the density of the sample surface.
- 46. How do we get an image? 156 electrons! Image Detector Electron gun 288 electrons!
- 48. Scanning electron micrographs of the early human embryo
- 49. This form of image processing is only in gray scale which is why SEM images are always in black and white. These images can be colorized through the use of feature-detection software, or simply by hand editing using a hand graphic editor. This is usually for aesthetic effects, for clarifying structure, or for adding a realistic effect to the sample.
- 50. Pollen and Stamens Wool fibers
- 51. So how does a SEM change the magnification of an image? By reducing the size of the area scanned by the scan coils, the SEM changes the magnification of the image.
- 52. Vacuum Chamber SEMs require a vacuum to operate. Without a vacuum, the electron beam generated by the electron gun would encounter constant interference from air particles in the atmosphere. Not only would these particles block the path of the electron beam, they would also be knocked out of the air and onto the specimen, which would distort the surface of the specimen.
- 53. BIOLOGICAL APPLICATIONS OF SEM Virology - for investigations of virus structure Cryo-electron microscopy – Images can be made of the surface of frozen materials. 3D tissue imaging - – Helps to know how cells are organized in a 3D network – Their organization determines how cells can interact. Forensics - SEM reveals the presence of materials on evidences that is otherwise undetectable SEM renders detailed 3-D images – extremely small microorganisms – anatomical pictures of insect, worm, spore, or other organic structures
- 54. SEM Advantages Gives detailed 3D and topographical imaging and the versatile information garnered from different detectors. Works very fast. Modern SEMs allow for the generation of data in digital form. Most SEM samples require minimal preparation actions.
- 55. SEM Disadvantages SEMs are expensive and large. Special training is required to operate an SEM. Preparation of samples can result in artifacts. Limited to solid samples. Carry a small risk of radiation exposure associated with the electrons that scatter from beneath the sample surface.
- 56. Thank you
Editor's Notes
- #10: The development of a SEM began a few years after the invention of a TEM by Ruska in 1931, but the commercialization of the SEM required about 30 yrs.
- #11: M. von Ardenne's first SEM
- #12: SEM is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons in a raster scan pattern. The electrons interact with atoms in the sample, producing various signals that contain information about the sample's surface topography and composition.
- #18: All samples must be of an appropriate size to fit in the specimen chamber and are generally mounted rigidly on a specimen holder called a specimen stub
- #19: For conventional imaging in the SEM, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge at the surface.
- #21: The proper cleaning of the surface of the specimen is important, because the surface contains many unwanted deposits, such as dust, mud, soil etc. depending upon the source of the sample/specimen.
- #22: Hard, dry materials such as wood, bone, feathers, dried insects, or shells can be examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms usually require chemical fixation to preserve and stabilize their structure. Stabilization is typically done with fixatives. Fixation cab be achieved by :- Perfusion or microinjection. Immersions. With vapours.
- #23: Fixation is usually performed by incubation in a solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in combination with formaldehyde and other fixatives. Fixatives that can be used are:- Aldehydes. Osmium tetroxide. Tanic acid. Thiocarbohydrazides.
- #24: After the fixation step, the sample must be rinsed in order to remove excessive fixatives.
- #25: All water must be removed from the samples because the water would vaporize in the vacuum. The fixed tissue is then dehydrated. Because air-drying causes collapse and shrinkage, this is commonly achieved by replacement of water in the cells with organic solvents such as ethanol or acetone. Dehydration is performed with a graded series of ethanol or acetone.
- #26: For SEM, a specimen is normally required to be completely dry, since the specimen chamber is at high vacuum. Otherwise the sample will be destroyed in the electron microscope chamber.
- #27: After the specimen has been cleaned, fixed, rinsed, dehydrated and dried, using an appropriate protocol, specimen has to be mounted on the holder that can be inserted into the scanning electron microscope. All samples must also be of an appropriate size to fit in the specimen chamber and are generally mounted rigidly on a specimen holder called a specimen stub. The dry specimen is usually mounted on a specimen stub using an adhesive such as epoxy resin or electrically conductive double-sided adhesive tape.
- #32: Coating the specimen. The idea of coating the specimen is to increase the conductivity of the specimen and to prevent the high voltage charge on the specimen by conducting charge to the ground. These specimen are coated with thin layer ie.20nm-30nm of conductive metal. All metals are conductive and require no preparation before being used.
- #33: All non-metals need to be made conductive by covering the sample with a thin layer of conductive material. This is done by using a device called a "sputter coater.” Conductive materials in current used for specimen coating includes gold, gold-palladium alloy , platinum, osmium , iridium, tungsten, chromium and graphite.
- #38: Specimen interaction is what makes Electron Microscopy possible. The energetic electrons in the microscope strike the sample and various reactions can occur as shown below. The reactions noted on the top side of the diagram are utilized when examining thick or bulk specimens (SEM) while the reactions on the bottom side are those examined in thin or foil specimens (TEM). In TEM electrons have very high energy of about 100s of KeV. They are moving so fast that sometimes they just go through the matter without even noticing and they don’t interact. Some of them which are very important for generating the image, go through and bounce of the nucleaus of the atom and bent and this is the process of elastic scattering, in which the electrons don’t change energy, don’t change speed but change direction and this is what we call a good scattering and is used to generate the image in TEM. Unfortunately there is another process of scattering, the bad scattering and these are the electrons in your primary beam generated in the microscope that goes through the atom and interacts with the electrons in the sample and in doing so they loss some energy of their own and they pass it to the sample. These inelastically scattered electrons are going to contribute in the noising of the image and at the same time really damaging the sample.
- #39: After absorbance of energythey give their own electrons
- #42: SE are shallow but very imp for providing surface features
- #44: Are used to determine crystal structures and orientations of minerals
- #46: For elemental analysis
- #55: It gives detailed 3D and topographical imaging and the versatile information garnered from different detectors. This instrument works very fast. Modern SEMs allow for the generation of data in digital form. Most SEM samples require minimal preparation actions.
- #56: SEMs are expensive and large. Special training is required to operate an SEM. The preparation of samples can result in artifacts. SEMs are limited to solid samples. SEMs carry a small risk of radiation exposure associated with the electrons that scatter from beneath the sample surface.