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Scanning Electron Microscopy (SEM) lecture

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a brief lecture on SEM; principles, working, instrumentation, and its forensic applications.

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Scanning Electron Microscopy (SEM) lecture

  1. 1. 8/30/2016 bhargava 1 SEM Saurabh Bhargava
  2. 2. Objectives of this lecture • At the end of the lecture one should be able to answer the questions related to- – Basic principles of SEM – Instrumentation of SEM – Image generation/ formation by SEM – Collection & interpretation of images of SEM – Forensic applications of SEM 8/30/2016 bhargava 2
  3. 3. 8/30/2016 bhargava 3 M A G N I F I C A T I O N C O M PL E XI T Y & C O S T NEED..?
  4. 4. Resolution • Resolution is defined as the ability to distinguish two very small and closely-spaced objects as separate entities. • Resolution is best when the distance separating the two tiny objects is small. • Resolution is determined by certain physical parameters that include the wavelength of light, and the light-gathering power of the objective and condenser lenses. 8/30/2016 bhargava 4
  5. 5. Conti.. • A simple mathematical equation defines the smallest distance (dmin) separating the two very small objects: dmin = 1.22 x wavelength N.A. objective + N.A. condenser 8/30/2016 bhargava 5
  6. 6. Electron Microscopy • An electron microscope is a microscope that uses accelerated electrons as a source of illumination. • Electron microscopes were developed due to the limitations of Light Microscopes which are limited by the physics of light. • In the early 1930's there was a scientific desire to see the fine details of the interior structures of organic cells (nucleus, mitochondria...etc.). • This required >10000x magnifications which could not be achieved by simple light/optical microscopy. • Because the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, the electron microscope has a higher resolving power than a light microscope and can reveal the structure of smaller objects. 8/30/2016 bhargava 6
  7. 7. Historical Aspects of Electron Microscopy – The development of a SEM began a few yrs after the invention of a TEM by Ruska in 1931, but the commercialization of the SEM required about 30 yrs. – In 1935, the original prototype of the SEM, which scans the specimen with an e- beam to obtain an image, was made by Knoll(Germany). – In 1942, Zworykin (USA), developed a SEM for observing a bulk specimens. – In 1965, Cambridge Scientific Instrument (UK) & JOEL (Japan) first commercialized SEM individually. 8/30/2016 bhargava 7
  8. 8. 8/30/2016 bhargava 8 Fig: SEM image of pneumonia pathogen Fig: SEM image of a ‘diatom’
  9. 9. 8/30/2016 bhargava 9 Fig: SEM image of pollen grains Fig: SEM image of house fly compound eye surface @450x Fig: SEM image of kidney stone (Calcium Oxalate Dihydrate crystals)
  10. 10. 8/30/2016 bhargava 10 Fig: SEM image of the tip of a needle @50x & @750x magnifications
  11. 11. Information Retrieved • Topography : – The surface features of an object (hardness, reflectivity...etc.) • Morphology: – The shape and size of the particles (ductility, strength, shape ...etc.) • Composition: – The elements and compounds that the object is composed of and the relative amounts of them. • Crystallographic Information: – How the atoms are arranged in the object. 8/30/2016 bhargava 11
  12. 12. SEM • Scanning electron microscopy (SEM) uses a focused electron probe to extract structural and chemical information point-by-point from a region of interest in the sample. • The high spatial resolution of a SEM makes it a powerful tool to characterise a wide range of specimens at the nanometre to micrometre length scales. 8/30/2016 bhargava 12
  13. 13. Scanning Electron Microscope (SEM) • A SEM is a type of electron microscope that images a sample by scanning it with a high- energy beam of electrons in a raster scan pattern. • The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition, and other properties such as electrical conductivity. 8/30/2016 bhargava 13
  14. 14. What Happens When e- Beam Strikes The Specimen..? • When the specimen is bombarded with the electron beam, electrons are ejected from the atoms of the specimen surface. • Inelastic scattering, place the atom in the excited state. The excited atom “wants ” to return to a ground or unexcited state. Hence the atoms will relax giving off the excess energy. • X-rays, Cathod luminescence are some of the ways of relaxation for an excited atom. • A resulting electron vacancy is filled by an electron from a higher shell, and an X-ray is emitted to balance the energy difference between the two electrons. 8/30/2016 bhargava 14
  15. 15. 8/30/2016 bhargava 15 Electron Beam & Sample Interactions
  16. 16. 8/30/2016 bhargava 16 Secondary Electrons Generation Secondary Electron Generation -sample electrons ejected by the primary beam [green line] -low energy -surface detail & topography
  17. 17. 8/30/2016 bhargava 17 Back Scatter Electron Generation Backscattered Electron Generation -SEM-BSE -primary beam electrons -high energy -composition and topography [specimen atomic number]
  18. 18. 8/30/2016 bhargava 18 X-Rays Generation •X-Rays are produced to balance the difference of energy when an electron from outer shell replaces the one from inner shell.
  19. 19. Instrumentation 1. Electron optical system (to produce electrons) – Electron gun, condenser lens, objective lens, deflection coil. 2. Specimen stage (to place the specimen). 3. Secondary e- detector (to collect secondary e-). 4. Image display unit. 5. Operating system – The electron optical system and a space surrounding the specimen are kept at vacuum. 8/30/2016 bhargava 19
  20. 20. Electron Gun • It produces an electron beam. • The electrons are emitted from a filament (cathode) made of a thin tungsten wire, (about 0.1mm ) by heating the filament at high temperature (about 2800k). • These e- flow as an e- beam, towards the metal/anode (positive voltage). • If the hole is made at the centre of the plate the e- beam flows through this hole. 8/30/2016 bhargava 20
  21. 21. Magnetic Lens System • Condenser lens is a lens placed right below the electron gun and is used to adjust the width of the electron beam as per requirement. • The condenser lens controls the intensity of the electron beam reaching the specimen. • The objective lens brings the electron beam into focus on the specimen. • The scanning coils deflect the electron beam horizontally and vertically over the specimen surface. This is also called rastering. – A fine e- beam is required for SEM, for better performance. 8/30/2016 bhargava 21
  22. 22. Specimen Stage/Chamber • It receives and supports the specimen. • Specimen stage should be able to perform the horizontal and vertical movements smoothly. • The horizontal movement is used for selection of the field while the vertical movement is used to change the image resolution. 8/30/2016 bhargava 22Fig: SEM sample chamber (opened)
  23. 23. Secondary e- Detector • It is used for detecting the secondary e- emitted from the specimen. • The secondary e- are attracted to the high voltage (10 kV) and then generate light when they hit the scintillator (fluorescent substance). • This light is directed to a photon- multiplier tube (PMT) through a light guide. • The light is converted to the electrons, and these are amplified as an electric signal. 8/30/2016 bhargava 23
  24. 24. Image Display Unit • The output signals from the secondary electron detector are amplified through PMT and then transferred to the display unit forming a SEM image. 8/30/2016 bhargava 24
  25. 25. 8/30/2016 bhargava 25
  26. 26. Vacuum System • The electron optical system and the specimen chamber must be kept at a high vacuum of 10-3 to 10-4 Pa. • High vacuum that minimises scattering of the electron beam before reaching the specimen. • This is important as scattering of the electron beam will increase the probe size and reduce the resolution. • Thus , these components are evacuated by diffusion pump. 8/30/2016 bhargava 26
  27. 27. Working of SEM • The electron gun produces an electron beam when tungsten wire is heated by current. • This beam is accelerated by the anode. • The beam travels through electromagnetic fields and lenses, which focus the beam down toward the sample. • A mechanism of deflection coils guide the beam so that it scans the surface of the sample in a rectangular frame (raster pattern). • When the beam touches the surface of the sample, it produces: – Secondary electrons (SE) – Back scattered electrons (BSE) – X - Rays... • The emitted SE is collected by SED and converted into signal that is sent to a screen which produces final image. • Additional detectors collect these X-rays, BSE and produce corresponding images. 8/30/2016 bhargava 27
  28. 28. Secondary Electron (SE) Images – For routine scanning electron microscope images, secondary electrons (SE) form the usual image of the surface. – Secondary electrons are low energy electrons formed by inelastic scattering and have energy of less than 50eV. – The low energy of these electrons allows them to be collected easily. – This is achieved by placing a positively biased grill on the front of the SE detector. – The positive grill attracts the negative electrons and they go through it into the detector. This is the case for the Everhart-Thornley detector which is most commonly used. – To increase the yield of SE emitted from the specimen, heavy metals such as gold or platinum are routinely used to coat specimens (sputter). An extremely thin layer is applied (~10 nm). – This coating is applied for two main reasons: 1. Non-conductive specimens are often coated to reduce surface charging that can block the path of SE and cause distortion of signal level and image form; and 2. Low atomic number (Z) specimens (e.g. biological samples) are coated 8/30/2016 bhargava 28
  29. 29. 8/30/2016 bhargava 29
  30. 30. Detectors • Secondary electron detector (SED) – – Everhart-Thornley Detector – Due to the low energies of secondary electrons (SE) (~2 to 50 eV) they are ejected only from near-surface layers. – Therefore, secondary electron imaging (SEI) is ideal for recording topographical information. – To attract (collect) these low- energy electrons, usually around +200 to 300V is applied to the cage at the front end of the detector. – A higher kV (7 to 12kV) is applied inside the cage i.e. to the scintillator, to accelerate the electrons into the scintillator screen. 8/30/2016 bhargava 30
  31. 31. 8/30/2016 bhargava 31 SE image formation
  32. 32. Backscattered electron (BSE) images • Backscattered (BS) electrons are high-energy electrons (>50 eV) ejected back out from the sample. These BSE are used to produce a different kind of image. • Such an image uses contrast to tell us about the average atomic number of the sample. • The higher the average atomic number, the more primary electrons are scattered (bounced) back out of the sample. This leads to a brighter image for such materials. • For example, a grain of sand (in a mixture of mineral sand) that is made up of a titanium mineral looks whiter than a grain made of a silicon material (Ti versus Si). In the image (next slide), the left picture is taken using backscattered electrons. Here there is a difference in contrast between the grains labelled Si and Ti whereas in the right image, taken using secondary electrons, there is no difference in contrast between these grains. 8/30/2016 bhargava 32
  33. 33. 8/30/2016 bhargava 33
  34. 34. Backscattered electron detector (BSED) • The BSED is mounted below the objective lens pole piece and centred around the optic axis. • As the specimen surface is scanned by the incident electron beam, backscattered electrons (BSE) are generated, the yield of which is controlled by the topographical, physical and chemical characteristics of the sample. • Both compositional or topographical backscattered electron images (BEI) can be recorded depending on the window of electron energies selected for image formation. 8/30/2016 bhargava 34
  35. 35. 8/30/2016 bhargava 35 Light’ region is made up predominantly of Fe. (i.e. the heaviest element) ‘Grey’ region is made up predominantly of Ca. ‘Dark’ region is made up predominantly of Si and Al. (i.e. the lightest elements)
  36. 36. Types 1. Conventional (high vacuum) scanning electron microscopy (SEM) – This is the most common type of machine. – It requires a dry, conductive sample (often achieved by applying a thin layer of metal to the surface with a technique called sputtering). – The sample must be able to withstand a high vacuum. 2. Variable Pressure or Low Vacuum scanning electron microscopy (LVSEM) – This type of machine is basically like a conventional SEM but has the advantage in low vacuum (LV) mode that the pressure can be adjusted in the sample chamber. – This means LVSEM can be used to image the surface of non-conductive samples (no metal needs to be added to the surface of such samples). – It is particularly useful for viewing polymers, biological samples, and museum samples that cannot be changed in any way, particulate samples, and geological materials. 8/30/2016 bhargava 36
  37. 37. 8/30/2016 bhargava 37 3. Cryo-scanning electron microscope (Cryo-SEM) – Cryo stands for frozen. A cryo-scanning electron microscope is a conventional SEM that has been fitted with specific equipment that allows samples to be viewed in the frozen state. – This is particularly useful for directly viewing hydrated (wet) samples, delicate biological samples, hydrogels, food, bio-films, foams, fats, and waxes, suspensions, pharmaceuticals and nano-particles. 4. Environmental scanning electron microscope (ESEM) – This machine is designed to view a sample in its natural state, without the need for desiccation. – Sample temperature and specimen chamber vapour pressure can both be controlled, allowing samples to be heated, cooled, wetted or dried. – Imaging of biological processes, for example pollen tube growth in real time through wetting of pollen.
  38. 38. Applications • SEM is one of the most versatile technique, and can be used to- I. Image morphology of samples II. Image compositional and some bonding differences III. Examine wet and dry samples while viewing them (only in an ESEM) IV. View frozen material (in a SEM with a cryostage) V. Generate X-rays from samples for microanalysis (EDS) VI. View/map grain orientation/crystallographic orientation and study related information like heterogeneity 8/30/2016 bhargava 38
  39. 39. X- Ray Analysis 8/30/2016 bhargava 39 •An incoming high-energy electron dislodges an inner-shell electron in the target, leaving a vacancy in the shell •An outer shell electron then “jumps” to fill the vacancy •A characteristic x-ray (equivalent to the energy change in the “jump”) is generated or •The energy released from an electron replacement event produces a photon with an energy exactly equal to the drop in energy. 1. Elemental identification 2. Chemical characterization 3. Quantitative analysis
  40. 40. 8/30/2016 bhargava 40 •When an electron from a K-shell is replaced by one from the next closest shell (L), it is designated as a Kα event •When an electron from a K-shell is replaced by one from the second closest shell (M), it is designated as a Kβ event
  41. 41. 8/30/2016 bhargava 41 Lα - When an electron from a L-shell is replaced by one from the next closest shell (M). The K shell will never donate its electron as this would require an increase in energy, not a drop.
  42. 42. 8/30/2016 bhargava 42 •Certain events such as Mα, Lβ, and Kγ are only possible in atoms of sufficient atomic weight. •Each element has a family of characteristic X-rays associated with it
  43. 43. 8/30/2016 bhargava 43
  44. 44. 8/30/2016 bhargava 44 Instrumentation
  45. 45. Gunshot Residue (GSR) Analysis • GSR is often found on criminals and also on victims if shot at close range. • Particles are very characteristic, therefore presence of these particles forms evidence of firing a gun. • Particles normally consist of Pb (lead), Sb (antimony) and Ba (barium). • The proportion of elements present in GSR differ slightly and databases of GSR from different manufacturers can be used to identify what ammunition was used in a crime. • New ammunition: environmentally friendly (no Sb). 8/30/2016 bhargava 45
  46. 46. Gunshot Residue (GSR) Analysis 8/30/2016 bhargava 46
  47. 47. 8/30/2016 bhargava 47 GSR Analysis on cloth fibre •Bullet fragments (blue) can be identified on cloth fibres and distinguished from other metal pieces by their elemental composition
  48. 48. Analysis • Qualitative Analysis: – Peak energy gives qualitative information about the constituent elements • Quantitative Analysis: – Peak intensity gives information about the element composition to find the changes in concentration of elements. 8/30/2016 bhargava 48
  49. 49. FORENSIC APPLICATIONS • In forensic science, police laboratories use the SEM to examine and compare evidence, such as 1. Metal fragments, 2. Paint, 3. Inks, 4. Hair, 5. Fibers – To prove a person's guilt or innocence. Through careful examination, detectives are able to determine if samples collected from a crime scene have properties that match the scenario developed by detectives. 8/30/2016 bhargava 49
  50. 50. Entomological applications • In the field of biological science, large objects such as insects and animal tissues and small objects such as bacteria and viruses are studied by SEM. • There are many applications for the SEM in this field and one of them is entomological taxonomy. 8/30/2016 bhargava 50
  51. 51. Soils & Rocks • Investigation of soils and geological samples is common place in scanning electron microscopy. • Analysis of morphology can inform about weathering processes. • Compositional differences can be seen with the help of backscattered electron imaging (image). • Microanalysis can provide details on elemental composition. 8/30/2016 bhargava 51
  52. 52. Sample preparation • The correct preparation is vital to getting good quality information from your sample. A poorly mounted or incorrectly processed sample can lead to viewing artefacts. • For non-conductive samples that need coating to make them conductive, consider whether compositional information is required (e.g. BSE imaging, X-ray spectroscopy), and use a carbon coating if this is the case. • Gold or platinum metal coatings will interfere with the compositional signal from the sample. 8/30/2016 bhargava 52

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