Scanning Electron Microscope (SEM)

Scanning Electron Microscope (SEM)

1. 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: T opography Morphology Composition C rystallographic information

3. Scale

4. Types of EM 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.

5. 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

6. Compound microscope image TEM image Budding yeast cell E. coli bacteria Compound microscope image TEM image SEM image SEM image

7. First Electron Microscope Invented by Ernst Ruska Year-1933 He was awarded the Nobel Prize for physics for his invention in 1986

8. History of sem First SEM – debuted in 1938 by Manfred Von Ardenne In 1965, Cambridge Scientific Instrument (UK) & JOEL (Japan) first commercialized SEM individually.

9. 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. 

10. 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

11. 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.

12. SEM SAMPLE PREPARATION Appropriate size samples --- fit in the specimen chamber M ounted rigidly on a specimen holder-- specimen stub aluminium stubs

13. SEM SAMPLE PREPARATION For imaging in the SEM, specimens must be  E lectrically conductive E lectrically grounded 

14. SEM SAMPLE PREPARATION Cleaning the surface of the specimen Stabilizing the specimen Rinsing the specimen Dehydrating the specimen Drying the specimen Mounting the specimen Coating the specimen

15. SEM SAMPLE PREPARATION Cleaning the surface of the specimen Very important S urface contains many unwanted deposits, such as dust, mud, soil etc

16. 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.

17. 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:- Aldehydes. Osmium tetroxide. Tanic acid. Thiocarbohydrazides .

18. SEM SAMPLE PREPARATION Rinsing the specimen Sample must be rinsed -- remove excessive fixatives.

19. SEM SAMPLE PREPARATION Dehydrating the specimen W ater must be removed A ir-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.

20. SEM SAMPLE PREPARATION Drying the specimen Specimen should be completely dry Otherwise the sample will be destroyed

21. SEM SAMPLE PREPARATION Mounting the specimen S pecimen has to be mounted on the holder M ounted rigidly on a specimen holder called a specimen stub D ry specimen -- mounted on a specimen stub using an adhesive such as epoxy resin or electrically conductive double-sided adhesive tape .

22. Charge-up

23. This charge-up phenomenon can be prevented by coating the non-conductor sample with metal (conductor).

24. 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.

25. SEM SAMPLE PREPARATION Coating the specimen To increase the conductivity of the specimen and to prevent the high voltage charge on the specimen C oated with thin layer i.e., 20nm-30nm of conductive metal . All metals are conductive and require no preparation before being used.

26. SEM SAMPLE PREPARATION Coating the specimen N on-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

27. “Sputter Coater”

28. A spider coated in gold

29. 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

30. So now we have a beam that is scanning across the sample surface and this beam is synched to the beam of a  CRT.

31. Detectors Image: Anders W. B. Skilbred, UiO Secondary electron detector Backscattered electron detector

32. 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.

33. 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.

34. How do we get an image? 156 electrons! Image Detector Electron gun 288 electrons!

35. SCANNING ELECTRON MICROSCOPIC IMAGE OF THE TONGUE

36. Scanning electron micrographs of the early human embryo

37. 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.

38. Pollen and Stamens Wool fibers

39. 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.

40. 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.

41. 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

42. 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.

43. 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.

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