SCANNING ELECTRON MICROSCOPy- theory and intrumentation.pptx

This is an in-depth set of slides on the theory, working principle, and installation of a scanning electron microscope. It differentiates electron microscopes and ight microscopes and shows the unique perspective that is given by a scanning electron microscope. This also explains the difference between SEM and TEM and why SEM is only applicable for surface observation. This also includes sample preparation for SEM.

1. SCANNING ELECTRON MICROSCOPE BY ANJISHNU MUKHOPADHYAY CC13 PRESENTATION 1

2. CONTENT INTRODUCTION HISTORY PRINCIPLE CONSTRUCTION IMAGE SIGNAL MAGNIFICATION RESOLUTION SAMPLE PREPARATION COLOUR IN SEM 3D IN SEM ADVANTAGES DISADVANTAGES APPLICATIONSS 2

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4. INTRODUCTION A scanning electron microscope (SEM) is a type of  electron microscope  that produces images of a sample by scanning the surface with a focused beam of  electrons . The electrons interact with  atoms  in the sample, producing various signals that contain information about the surface  topography  and composition of the sample. 4

5. HISTORY The first scanning electron microscope (SEM) debuted in 1938 ( Manfred Von Ardenne ) with the first commercial instruments around 1965. Its late development was due to the electronics involved in "scanning" the beam of electrons across the sample. 5

6. M. von Ardenne’s  first SEM 6

7. PRINCIPLE and SCANNING PROCESS The SEM uses electrons instead of light to form an image. A beam of electrons is produced at the top of the microscope by heating of a metallic filament. The electron beam follows a vertical path through the column of the microscope. It makes its way through electromagnetic lenses which focus and direct the beam down towards the sample. Once it hits the sample, other electrons ( backscattered or secondary ) are ejected from the sample. Detectors collect the secondary or backscattered electrons, and convert them to a signal that is sent to a viewing screen similar to the one in an ordinary television, producing an image. 7

8. CONSTRUCTION Electron gun (Filament) Lenses Scan coils Chamber Detectors Computer hardware and software 8

9. Electron gun Electron guns are typically one of two types.  Thermionic guns , which are the most common type, apply thermal energy to a filament (usually made of tungsten, which has a high melting point) to coax electrons away from the gun and toward the specimen under examination.  Field emission guns , on the other hand, create a strong electrical field to pull electrons away from the  atoms  they're associated with. Electron guns are located either at the very top or at the very bottom of an SEM and fire a beam of electrons at the object under examination. These electrons don't naturally go where they need to, however, which gets us to the next component of SEMs . 9

10. Thermionic gun 10

11. Lenses J ust like optical microscopes, SEMs use lenses to produce clear and detailed images. The lenses in these devices, however, work differently. For one thing, they aren't made of glass. Instead, the lenses are made of  magnets  capable of bending the path of electrons. By doing so, the lenses focus and control the electron beam, ensuring that the electrons end up precisely where they need to go. 11

12. EM lens 12

13. Scan coils After the beam is focused, scanning coils are used to deflect the beam in the X and Y axes so that it scans in a raster fashion over the surface of the sample. 13

14. Sample chamber The sample chamber of an SEM is where researchers place the specimen that they are examining. Because the specimen must be kept extremely still for the microscope to produce clear images, the sample chamber must be very sturdy and insulated from vibration. In fact, SEMs are so sensitive to vibrations that they're often installed on the ground floor of a building. The sample chambers of an SEM do more than keep a specimen still. They also manipulate the specimen, placing it at different angles and moving it so that researchers don't have to constantly remount the object to take different images. 14

15. SEM with opened sample chamber 15

16. Detectors When the electron beam interacts with a sample in a scanning electron microscope (SEM), multiple events happen. In general, different detectors are needed to distinguish secondary electrons, backscattered electrons, or characteristic x-rays. Depending upon the accelerating voltage and sample density, the signals come from different penetration depths. 16

17. Detectors used in SEM 17

18. IMAGE SIGNALS 18 Cathodoluminesence

19. IMAGE SIGNALS 19 Cathodoluminesence

20. Secondary electrons (se) Generated from the collision between the incoming electrons and the loosely bonded outer electrons Low energy electrons (~10-50 eV) Only SE generated close to surface escape (topographic information is obtained) Detected by Everhart–Thornley detector 20

21. Backscattered electrons ( Bse ) A fraction of the incident electrons is retarded by the electro- magnetic field of the nucleus and if the scattering angle is greater than 180° the electron can escape from the surface High energy electrons Fewer BSE than SE Detected by D irectional BSE detector 21

22. Characteristic x rays Characteristic X-rays  that are produced by the interaction of  electrons  with the sample may also be detected in an SEM equipped for  energy-dispersive X-ray spectroscopy  or  wavelength dispersive X-ray spectroscopy . Analysis of the x-ray signals may be used to map the distribution and estimate the abundance of elements in the sample 22

23. Cathodoluminescence Cathodoluminescence, the emission of light when atoms excited by high-energy electrons return to their ground state, is analogous to  UV -induced  fluorescence , and some materials such as zinc sulfide and some fluorescent dyes, exhibit both phenomena. In the SEM, CL detectors either collect all light emitted by the specimen or can analyse the wavelengths emitted by the specimen and display an emission  spectrum  or an image of the distribution of cathodoluminescence emitted by the specimen in real color. 23

24. MAGNIFICATION Magnification in an SEM can be controlled over a range of about 6  orders of magnitude  from about 10 to 3,000,000 times.   Unlike optical and transmission electron microscopes, image magnification in an SEM is not a function of the power of the  objective lens . 24

25. MAGNIFICATION SEMs may have  condenser  and objective lenses, but their function is to focus the beam to a spot, and not to image the specimen.. In an SEM, magnification results from the ratio of the raster on the display device and dimensions of the raster on the specimen. Magnification is therefore controlled by the current supplied to the x, y scanning coils, or the voltage supplied to the x, y deflector plates, and not by objective lens power. 25

26. RESOLUTION SEM is not a  camera  and the detector is not continuously image-forming like a  CCD  array or  film . Unlike in an optical system, the  resolution  is not limited by the  diffraction limit , fineness of lenses or mirrors or detector array resolution. The spatial resolution of the SEM depends on the size of the electron spot, which in turn depends on both the wavelength of the electrons and the electron-optical system that produces the scanning beam. 26

27. RESOLUTION The resolution is also limited by the size of the interaction volume, the volume of specimen material that interacts with the electron beam. The spot size and the interaction volume are both large compared to the distances between atoms, so the resolution of the SEM is not high enough to image individual atoms, as is possible with a  transmission electron microscope  (TEM). . 27

28. RESOLUTION The SEM has compensating advantages, though, including the ability to image a comparatively large area of the specimen; and the variety of analytical modes available for measuring the composition and properties of the specimen. Depending on the instrument, the resolution can fall somewhere between less than 1 nm and 20 nm. As of 2009, The world's highest resolution conventional (≤30 kV) SEM can reach a point resolution of 0.4 nm using a secondary electron detector. . 28

29. RESOLUTION The SEM has compensating advantages, though, including the ability to image a comparatively large area of the specimen; and the variety of analytical modes available for measuring the composition and properties of the specimen. Depending on the instrument, the resolution can fall somewhere between less than 1 nm and 20 nm. As of 2009, The world's highest resolution conventional (≤30 kV) SEM can reach a point resolution of 0.4 nm using a secondary electron detector. . 29

30. SAMPLE PREPARATION 30

31. Cleaning the surface of the specimen The proper cleaning of the surface of the sample is important because the surface can contain a variety of unwanted deposits, such as dust, silt, and detritus, media components, or other contaminants, depending on the source of the biological material and the experiment that may have been conducted prior to SEM specimen preparation. 31

32. Stabilizing the specimen Stabilization is typically done with fixatives. Fixation can be achieved, for example, by perfusion and microinjection, immersions, or with vapours using various fixatives including aldehydes, osmium tetroxide, tannic acid, or thiocarbohydrazide 32

33. Rinsing the specimen After the fixation step, samples must be rinsed in order to remove the excess fixative. 33

34. Dehydrating the specimen The dehydration process of a biological sample needs to be done very carefully. It is typically performed with either a graded series of acetone or ethanol. 34

35. Drying the specimen The scanning electron microscope (like the transmission electron microscope) operates with a vacuum. Thus, the specimens must be dry or the sample will be destroyed in the electron microscope chamber. Many electron microscopists consider a procedure called the Critical Point Drying (CPD) as the gold standard for SEM specimen drying. Carbon dioxide is removed after its transition from the liquid to the gas phase at the critical point, and the specimen is dried without structural damage. 35

36. Mounting the specimen After the sample have been cleaned, fixed, rinsed, dehydrated, and dried using an appropriate protocol, specimens must be mounted on a holder that can be inserted into the scanning electron microscope. Samples are typically mounted on metallic (aluminum) stubs using a double-sticky tape. It is important that the investigator first decides on the best orientation of the specimen on the mounting stub before attaching it. A re-orientation proves difficult and can result in significant damage to the sample. 36

37. Coating the specimen The idea of coating the specimen is to increase its conductivity in the scanning electron microscope and to prevent the build-up of high voltage charges on the specimen by conducting the charge to ground. Typically, specimens are coated with a thin layer of approximately 20 nm to 30 nm of a conductive metal (e.g., gold, gold-palladium, or platinum). 37

38. A spider sputter-coated in gold 38

39. ADVANTAGES Advantages of a Scanning Electron Microscope include its wide-array of applications, the detailed three-dimensional and topographical imaging and the versatile information garnered from different detectors. SEMs are also easy to operate with the proper training and advances in computer technology and associated software make operation user-friendly. Although all samples must be prepared before placed in the vacuum chamber, most SEM samples require minimal preparation actions. 39

40. DISADVANTAGES The disadvantages of a Scanning Electron Microscope start with the size and cost. SEMs are expensive, large and must be housed in an area free of any possible electric, magnetic or vibration interference. Maintenance involves keeping a steady voltage, currents to electromagnetic coils and circulation of cool water. SEMs are limited to solid, inorganic samples small enough to fit inside the vacuum chamber that can handle moderate vacuum pressure. 40

41. APPLICATIONS SEMs have a variety of applications in a number of scientific and industry-related fields, especially where characterizations of solid materials is beneficial. In addition to topographical, morphological and compositional information, a Scanning Electron Microscope can detect and analyze surface fractures, provide information in microstructures, examine surface contaminations, reveal spatial variations in chemical compositions, provide qualitative chemical analyses and identify crystalline structures. In addition, SEMs have practical industrial and technological applications such as semiconductor inspection, production line of miniscule products and assembly of microchips for computers. SEMs can be as essential research tool in fields such as life science, biology, gemology, medical and forensic science, metallurgy. 41

42. COLOUR IN SEM FALSE COLOUR WITH SINGLE DETECTOR FALSE COLOUR WITH MULTIPLE DETECTOR 42

43. False colour with single detector The easiest way to get color is to associate to this single number an arbitrary color, using a  color look-up table  (i.e. each grey level is replaced by a chosen color). This method is known as  false color . On a BSE image, false color may be performed to better distinguish the various phases of the sample. 43

44. Falsely coloured Kidney stone 44

45. False colour with multiple detectors S econdary electron and backscattered electron detectors are superimposed and a color is assigned to each of the images captured by each detector with a result of a combined color image where colors are related to the density of the components. This method is known as density-dependent color SEM (DDC-SEM). Micrographs produced by DDC-SEM retain topographical information, which is better captured by the secondary electrons detector and combine it to the information about density, obtained by the backscattered electron detector. 45

46. Falsely coloured cardiovascular calcification 46

47. 3D IN SEM P hotogrammetry  is the most metrologically accurate method to bring the third dimension to SEM images. Contrary to photometric methods (next paragraph), photogrammetry calculates absolute heights using  triangulation  methods. The drawbacks are that it works only if there is a minimum texture, and it requires two images to be acquired from two different angles, which implies the use of a tilt stage. 47

48. An SEM stereo pair of  microfossils  of less than 1 mm in size ( Ostracoda ) produced by tilting along the longitudinal axis. 48

49. REFERENCES   Faulkner, Christine; et al. (2008).  "Peeking into Pit Fields: A Multiple Twinning Model of Secondary Plasmodesmata Formation in Tobacco" . Plant Cell. 20 (6): PMID   18667640 . ^   Wergin , W. P.; Erbe , E. F. (1994).  "Snow crystals: capturing snow flakes for observation with the low-temperature scanning electron microscope" . Scanning. 16 (Suppl. IV): IV88. ^  Barnes, P. R. F.; Mulvaney, R.; Wolff, E. W.; Robinson, K. A. (2002). "A technique for the examination of polar ice using the scanning electron microscope". Journal of Microscopy. 205 (2): 118–124.  PMID   11879426 . . ^  Hindmarsh, J. P.; Russell, A. B.; Chen, X. D. (2007). "Fundamentals of the spray freezing of foods—microstructure of frozen droplets". Journal of Food Engineering. 78 (1): 136–150.  49

50. GALLERY 50 SOYABEAN CYST NEMATODE AND EGG COMPOUND EYE OF ANTARTIC KRILL HUMAN BLOOD CELLS

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