Electron microscopy

1. Electron Microscopy

2. The Central player - (e) The electron “e” is an elementary particle Also called corpuscle carries a negative charge. the electron was discovered by J. J. Thompson in 1897 e is a constituent of the atom 1000 times smaller than a hydrogen atom. the mass of the electron 1/1836 of that of a proton.

3. The milestones

4. The Wave Properties In 1924, the wave-particle dualism was postulated by de Broglie ( Nobel Prize 1929 ). All moving matter has wave properties with the wavelength λ being inversely related to the momentum p by λ = h / p = h / mv (h : Planck constant; m : mass; v : velocity)

5. The Wavelength Resolving power of EM is from Wave properties of electrons Limit of resolution is indirectly proportional to the wavelength of the illuminating light ie , longer the wavelength, lesser is the resolution λ = √150 / V, where λ – wavelength in Angstroms, V – accelerating voltage in volts

6. The electron wave The generation of a monochromatic and coherent electron beam is important Design of modern electron microscopes is based on this concept

7. Scheme of electron-matter interactions arising from the impact of an electron beam onto a specimen. A signal below the specimen is observable if the thickness is small enough to allow some electrons to pass through

8. Elastic Electron Interactions no energy is transferred from the electron to the sample. These signals are mainly exploited in Transmission Electron Microscopy and Electron diffraction methods.

9. Inelastic Electron Interactions Energy is transferred from the electrons to the specimen The energy transferred can cause different signals such as X-rays, Auger electrons secondary electrons, plasmons , phonons, UV quanta or cathodoluminescence . Used in Analytical Electron Microscopy … SEM

10. What is Electron Microscopy? Electron microscopy is a diagnostic tool with diversified combination of techniques …… that offer unique possibilities to gain insights into structure, topology, morphology, and composition of a material.

11. What is an Electron Microscope ? A special type of microscope having a high resolution of images , able to magnify objects in nanometres , which are formed by controlled use of electrons in vacuum captured on a phosphorescent screen

12. Why were the EMs advented ? To study objects of < 0.2 micrometer For analysis of sub cellular structures Intra cellular pathogens - viruses Cell metabolism Study of minute structures in the nature Greater resolving power of the EMs than light microscope An EM can magnify structures from 100 – 250000 times than light microscopy

13. The novelty of EMs from others Beam of Electrons …… instead of a beam of light Electro-magnetic lens ………..instead of Ground glass lenses Cylindrical Vacuum column - Electrons should travel in vacuum to avoid collisions with air molecules that cause scattering of electrons distorting the image

14. Comparison of lens system of Light & Electron Microscope

15. TYPES OF Electron Microscopy Transmission Electron Microscopy (TEM) Bright Field (BF) / Dark Field (DF) High-Resolution Transmission Electron Microscopy (HRTEM) Energy Filtered Transmission Electron Microscopy (EFTEM) Electron Diffraction (ED)   Scanning Transmission Electron Microscopy (STEM) Bright Field (BF)/ Dark Field (DF) High-Angle Annular Dark Field (HAADF-STEM)

16. TYPES OF Electron Microscopy Analytical Electron Microscopy (AEM) X-ray spectroscopy Electron Energy Loss Spectroscopy (EELS) Electron Spectroscopic Imaging (ESI)   Scanning Electron Microscopy (SEM) Secondary Electron Imaging (SE) Back-scattered Electron Imaging (BSE)

17. Commonly used EMs in biology Transmission Electron Microscope Scanning Electron Microscope “ mainly for various life forms and microbes” Scanning tunneling microscope Atomic Force Microscope “ Actual visualisation of molecules and individual atoms, also in motion”

18. Transmission Electron Microscopy The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this group developing the first TEM with resolving power greater than that of light in 1933 and the first commercial TEM in 1939.

19. TEM - Definition TEM is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen , interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film , or to be detected by a sensor such as a CCD camera .

20. Applications TEMs are capable of imaging at a significantly higher resolution than light microscopes, owing to the small de Broglie wavelength of electrons. to examine fine detail —even as small as a single column of atoms, which is tens of thousands times smaller than the smallest resolvable object in a light microscope. application in Biological sciences like cancer research , virology , materials science as well as pollution , nanotechnology , and semiconductor research. Application in chemical & physical sciences like in chemical identity, crystal orientation, electronic structure and sample induced electron phase shift as well as the regular absorption based imaging .

21. The TEM components vacuum system in which the electrons travel, an electron emission source for generation of the electron stream ( tungsten filament, or a lanthanum hexaboride ( LaB 6 )), Voltage source 100 – 300 kV a series of electromagnetic lenses, and electrostatic plates. The latter two allow the operator to guide and manipulate the beam as required. Also required is an Insertion device to allow the insertion into, motion within, and removal of specimens from the beam path. Imaging devices are subsequently used to create an image from the electrons that exit the system on Phosphor screen having Zinc sulphide

22. Different types of TEM Bright Field (BF) - BFTEM Dark Field (DF) - DFTEM High-Resolution Transmission Electron Microscopy (HRTEM) Energy Filtered Transmission Electron Microscopy (EFTEM) Electron Diffraction (ED)

23. Bright Field Imaging The most common mode of operation for a TEM is the bright field imaging mode. concept of “mass-thickness contrast” is “As the thickness of the specimen increases, the contrast also increases” Thicker regions of the sample, or regions with a higher atomic number will appear dark, regions with no sample in the beam path will appear bright – hence the term "bright field". The image is in effect assumed to be a simple two dimensional projection of the sample ( modelled via Beer's law ) A TEM image of the polio virus. The polio virus is 30 nm in size.

24. Diffraction Contrast Imaging Electrons passed through Crystal with equidistant lattice planes With regular spacing between the scattering centers , coherent constructive interference of the scattered electron in certain directions happens and thereby three-dimensional secondary wavelets or diffracted beams are generated This phenomenon is called Bragg diffraction . These beams are captured on image screen Crystalline diffraction pattern from a twinned grain of FCC Austenitic steel

25. Electron Energy Loss Spectroscopy electrons can be rejected based upon the voltage using magnetic sector based devices known as EELS spectrometers. These devices allow for the selection of particular energy values, EELS spectrometers can be operated in both spectroscopic and imaging modes, allowing for isolation or rejection of elastically scattered beams. EELS imaging can be used to enhance contrast in observed images, including both bright field and diffraction.

26. High resolution TEM - HRTEM Crystal structure can also be investigated by high-resolution transmission electron microscopy (HRTEM), HRTEM is also known as phase contrast. In a specimen of uniform thickness, the images are formed due to differences in phase of electron waves , which is caused by specimen interaction. Image formation is given by the complex modulus of the incoming electron beams. The image is dependent on the number of electrons hitting the screen, it can be manipulated to provide more information about the sample as in complex phase retrieval techniques.

27. 3D Imaging By taking multiple images of a single TEM sample at differing angles , typically in 1° increments, a set of images known as a "tilt series" can be collected. This methodology was proposed in the 1970s by Walter Hoppe . Under absorption contrast conditions, this set of images can be used to construct a three-dimensional representation of the sample. [36] Three dimensional imaging -A TEM image of a parapoxavirus

28. Scanning TEM (STEM) Modified type of TEM by the addition of a system that rasters the beam across the sample to form the image , combined with suitable detectors. The STEM uses magnetic lenses to focus a beam of electrons The image is formed not by secondary electrons as in SEM but by primary electrons coming through the specimen

29. Scanning Transmission Electron Microscopy

30. High angular annular dark field – STEM HAADF- STEM Also called “Z- contrast imaging” In this method small clusters, single atoms of heavy atoms (e.g. in catalysts) can be recognized in a matrix of light atoms Because the contrast produced is high Eg : In the TEM image, the overlapping of specimen leads to a darker contrast whereas in the HAADF-STEM image the contrast in the intersection becomes brighter than in the specimen. Comparison of TEM & STEM (a) TEM and (b) HAADF-STEM image of Pd balls on silica. The overlap regions are relatively dark in (a) and bright in (b), respectively.

31. Cryoelectron microscopy Cryoelectron Tomography ( cryo -ET) Nano sized intra cellular structures are studied In unfixed , unstained, hydrated , flash frozen cells It bridges the cellular & molecular research This technique facilitate visualisation of molecular structure of proteins and large molecules . Cryoelectron microscopy involves vitrification of the macromolecular assemblies 3D images can also be generated

32. Limitations of TEM Many materials require extensive sample preparation Difficult to produce a very thin sample relatively time consuming process with a low throughput of samples. The structure of the sample may change during the preparation process. Small field of view may not give conclusive result of the whole sample. sample may be damaged by the electron beam, particularly in the case of biological materials.

33. Biological Sample preparation Sample thickness – less than 1 mm 3 Rapid fixation with least cell damage – Gluteraldehyde and Osmium tetroxide Gluteraldehyde has aldehyde groups which bonds with amino groups of proteins, forming insoluble complexes OsO4 binds to cell membranes containing fatty acids Dehydration – alcohol series Embedding – Epoxy resins ( Epon or araldite) Section – 0.1 micrometre using ultramicrotome (Ideal – 70-90 nm thickness) Staining – Uranyl acetate , Lead citrate

34. Cryofixation By rapid freezing – ultra freezing methods “ Done to avoid ice crystal formation which damage the fragile intra cellular str.” Water becomes frozen in liquid state , so that ice is not formed -- VITRIFICATION Liquid propane (-42 deg C) Liquid Helium (-273 deg C) High pressure freezing with jets of Liq N Adv: Less time consuming, enzymology possible

35. Different techniques for TEM Negative staining Shadow casting Freeze fracture replication Freeze etching

36. 1. Negative Staining tq Heavy metal deposits are collected onto specimen grid, except where the particle is present The specimen appears bright on the view screen Human papilloma virus by negative staining

37. 2. Shadow Casting tq Viruses, DNA, RNA visualisation The grids with specimen is placed in a sealed chamber Vacuum is created Heated platinum+carbon filament deposits metal onto the surface directly in line , creating shadow Areas of shadow appear bright on view screen The image in photographs are reversed – objects appear bright with a dark shadow

38. 3. Freeze Fracturing Tq Step 3 Step 4 Step 5

39. Definition of SEM An electron microscope that produces images of a sample by scanning over it with a focused beam of electrons . The incident electrons interact with electrons in the sample , producing various signals that can be detected and contain information about the sample's surface topography and composition.

40. The electron beams The types of signals produced by a SEM include secondary electrons , back-scattered electrons (BSE), X-rays , light rays ( cathodoluminescence ), A standard SEM uses Secondary electrons & Back scattered electrons

41. Salient features Electrons are used to create images of the surface of specimen - topology Resolution of objects of nearly 1 nm Magnification upto 500000 x (250 times > light microcopes ) secondary electrons (SE), backscattered electrons (BSE) are utilized for imaging specimens can be observed in high vacuum, low vacuum and In Environmental SEM specimens can be observed in wet condition. Gives 3D views of the exteriors of the objects like cells, microbes or surfaces

42. 3D data measurement 3D data can be measured in the SEM with different methods such as: photogrammetry (2 or 3 images from tilted specimen) photometric stereo (use of 4 images from BSE detector) i nverse reconstruction using electron-material interactive models [31][32] Possible applications are roughness measurement, measurement of fractal dimension, corrosion measurement and height step measurement.

43. Biological sample preparation Chemical fixation with Gluteraldehyde , optionally with OsO4 – for soft tissues No fixation needed for dry specimen like bones, feathers etc Dehydration by replacement of water in the cells with organic solvents such as ethanol or acetone , and replacement of these solvents in turn with a transitional fluid such as liquid carbon dioxide by critical point drying . The carbon dioxide is removed while in a supercritical state, so that no gas-liquid interface is formed within the sample during drying. The dry specimen is mounted on a specimen stub using epoxy resin ultrathin coating done by low-vacuum sputter coating or by high-vacuum evaporation . Conductive materials in current use for specimen coating include gold , gold/ palladium alloy, platinum , osmium , [12] iridium , tungsten , chromium , and graphite .

44. SEM - Cryo -imaging SEM is equipped with a cold stage for cryo -microscopy, C ryofixation is used and low-temperature scanning electron microscopy performed on the cryogenically fixed specimens. Cryo -fixed specimens are cryo -fractured under vacuum in a special apparatus to reveal internal structure, sputter-coated, and transferred onto the SEM cryo -stage while still frozen. Low-temperature scanning electron microscopy is also applicable to the imaging of temperature-sensitive materials such as ice and fats. Freeze-fracturing, freeze-etch or freeze-and-break is a preparation method particularly useful for examining lipid membranes and their incorporated proteins

45. SEM - Cathodolumiscence Cathodoluminescence means the “ emission of light occurs when atoms are excited by high-energy electrons” It is analogous to UV -induced fluorescence , Eg : Cathodoluminescence is most commonly experienced in cathode ray tube in television sets and computer CRT monitors. In the SEM, CL detectors display an emission spectrum or an image of the distribution of cathodoluminescence emitted by the specimen “ in real colour ” very powerful probe for studying nanoscale features and defects.

46. SEM - X-ray microanalysis X-rays , which are produced by the interaction of electrons with the sample, may be detected in an SEM equipped for energy-dispersive X-ray spectroscopy (EDXS) or wavelength dispersive X-ray spectroscopy (WDXS) .

47. Environmental SEM ( ESEM) Environmental SEM (ESEM) in the late 1980s samples are observed in low-pressure gaseous environments and high relative humidity (up to 100%). ESEM is especially useful for non-metallic and biological materials because coating with carbon or gold is unnecessary. Uncoated Plastics and Elastomers & uncoated biological samples . ESEM makes it possible to perform X-ray microanalysis on uncoated non-conductive specimens. ESEM may be the preferred tool for electron microscopy of unique samples from criminal or civil actions , where “ forensic analysis ” may need to be repeated by several different experts.

48. Scanning tunneling microscopy 1980 – Gerd Bennig and Heinrich Rohrer invented STM Also called Scanning Probe microscopes Object resolution 0.1-0.01 nm Thin wire probe made of platinum - iridium is used to trace the surface of the object Electrons from the probe overlap with electron from the surface tunnel into one another’s clouds tunnels create a current as the probe moves on the uneven surface of the specimen

49. The applications The STM can be used in ultra high vacuum, air, water, and various other liquid or gaseous environments and at temperatures ranging from near zero to a few hundred degrees Celsius First movie made using STM – “individual fibrin molecule forming a clot” Live specimen examination – as in “Virus infected cells exploding and releasing new viruses” Visualisation of intra cellular changes

50. Atomic force microscope Advanced type of EM Three dimensional imaging Measurement of structures at the level of an atom To study DNA, especially the base pairs by detecting differences in density Used under water to study chemical reactions at living cell surfaces Cell wall chemical composition visualisation Used to measure forces – as in protein unfolding, polysaccharide flexibility etc

51. Low Voltage Electron Microscope- LVEM The low-voltage electron microscope (LVEM) is a combination of SEM, TEM and STEM in one instrument operated at relatively low electron accelerating voltage of 5 kV. Low voltage increases image contrast which is especially important for biological specimens. This increase in contrast eliminates the need to stain . Sectioned samples need to be thinner than they would be for conventional TEM (20–65 nm).

52. Recent advances The effect of metallic nanoparticles on cells was probed by treating two cell lines with C-coated Cu nanoparticles . The up-take of these nanoparticles was shown by HAADF-STEM that reveal them as bright patches inside the cells (s. image). Nanoparticle Cytotoxicity Depends on Intracellular Solubility: Comparison of Stabilized Copper Metal and Degradable Copper Oxide Nanoparticles A. M. Studer , L. K. Limbach , L. Van Duc , F. Krumeich , E. K. Athanassiou , L. C. Gerber, H. Moch , and W. J. Stark Toxicology Lett . 197 (2010) 169-174 DOI

53. Inner Shell Ionisation – X-ray or Auger electrons When an electron drops down from a higher level to fill the vacancy in an atom. By this process, the atom can relax but the excess energy has to be given away . This excess energy of the electron, causes difference between the energy levels. The process of getting rid of the additional energy generates either of a characteristic X-ray or of an Auger electron .