Electron microscope - SEM and TEM
- 1. Gaurav Raja Parmar M.Sc. 1st sem Department of Biotechnology, Barkatullah University, Bhopal
- 3. What is microscopy ? Microscopy is the technology of making very small things visible to the human eye. Therefore microscope is a major tool of the microbiologist historically. It was the microscope that first revealed the secrets of microbial structure and even today. It remains a powerful tool in microbiological studies.
- 4. Microscope create a visible detailed image of such objects that are otherwise too small to be observed by the human eye. There are different type of electcron microscope but all of them depend on the following three factors to produce a clear image. Magnification Contrast Resolution Principle of Microscopy
- 5. The primary function of microscope is magnification . That is enlargement of the image of an object. Any convex lens ( thicker in the center than at the edge) can magnify the image of object. By refraction. It bends parallel rays of light from an object so that they meet at a single point . MAGNIFICATION
- 6. Contrast In addition to the factor of magnification . Two other factor contrast refers to a difference in light intensity. In order to be perceived through the microscope. There are few types of microscopes which can even increase contrast.
- 7. Resolution Resolution is the ability of the lens to separate or distinguish between small objects that are close together. Resolution is described mathematically by an equation developed in the 1870s by Ernst Abbe, a German physicist responsible for much of the optical theory underlying microscope design. Resolution is described by Resolving power of a microscope determines by three factors The size of the objective lens- The wavelength of the light passing through the specimen- The refractive index of the material between the objective lens and the specimen-
- 9. The electron microscope is a type of microscope that uses a beamof electrons to create an image of the specimen. It is capableof much higher magnifications and has a greater resolving power than a light microscope, allowing it to seemuch smaller objects in finer detail. They are large, expensive pieces of equipment, generally standingalone in a small, specially designedroomand requiring trained personnel to operate them. WHAT IS ELECTRON MICROSCOPE ? https://www.jic.ac.uk
- 10. Why use electrons instead of light ? In the 1920s it was discovered that accelerated electrons (parts of the atom) behave in vacuum just like light. They travel in straight lines and have a wavelength which is about 100 000 times smaller than that of light. Furthermore, it was found that electric and magnetic fields have the same effect on electrons as glass lenses and mirrors have on visible light.
- 11. Main features of Electron Microscope In the Electron microscope the wavelength of electrons are 1,00,000 times shorter than light microscope. At 60,000 volts a typical accelerating voltage used in Electron microscope. The wavelength of electron is approximately 0.005 nm, permitting a theoretical resolution of approximately 0.2 nm. The useful magnification for an electron microscope is well over 1,00,000x
- 12. History of electron Microscope Dr. Ernst Ruska (often abbreviated to TEM) at the University of Berlin and Max Knoll combined built the first electron microscope in 1931. For this and subsequent work on the subject, Ernst Ruska was awarded the Nobel Prize for Physics in 1986.
- 13. There are two types of electron microscope Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM)
- 14. The Transmission Electron Microscope The Transmission Electron Microscope (TEM) is the original form of electron microscopy. The TEM was developed in 1931 and is based on transmitted electrons. Here, a high voltage electron beam is emitted by a cathode and controlled by magnetic lenses. This electron beam is then partially transmitted through a very thin specimen. So thin that electrons can pass through it. The eyepiece or ocular is replaced by a fluorescent screen
- 15. Structure of Transmission Electron Microscope (a) Electron gun: It consists of a tungsten filament or cathode that emits electrons on receiving high voltage electric current (50,000-100,000 volts). (b) Ray tube (Microscope Column): It is a high vacuum metal tube (2mt. high) through which electrons travel. (c) Condense lens: It is the electromagnetic coil which focuses the electron beam in the plane of the specimen. (d) Objective lens: It is the electromagnetic coil which produces the first magnified image formed by the objective lens and produces the final image. (e) Projector lens: It is also an electromagnetic coil which further magnifies the first image formed by the objective lens and produces the final image. (f) Fluorescent Screen or Photographic Film: Since unaided human eye cannot observe electrons, therefore, a fluorescent screen is used for viewing the final image of the specimen http://www.biologydiscussion.com
- 16. The Electron Gun The Electron gun refers to the top region of the TEM and SEM that generates a beam of electrons. Types of electron guns Heated tungsten, Lanthanum hexaboride (LaB6), Tungsten field emission gun (FEG) www.ammrf.org.au
- 17. Structure of TEM
- 18. Working of TEM
- 19. Sample Preparation for TEM Ultra-thin Sectioning Electron Staining Metal Shadowing (Shadow Casting) Surface Replicas Freeze Fracture (freeze Etching)
- 20. Scanning Electron microscope The scanning electron microscope (SEM)works in a different manner. It produces an image from electrons released from atoms on an object’s surface. The SEM has been used to examine the surfaces of microorganisms in great detail; many SEMs have a resolution of 7 nm or less.
- 21. Structure of SEM Essential components of all SEMs include the following like TEM Electron Source (Gun) Electron Lenses Sample Stage Detectors for all signals of interest Display / Data output devices Vacuum system Cooling system
- 22. Working of SEM
- 23. Sample preparation for SEM Fixation and dehydration Critical point drying Drying with hexamethyldisilazane (HMDS) and t‐Butanol Coating with Gold/Palladium using Sputter coater Image processing (software Scandium) www.ntnu.edu
- 24. Name of some companies who has manufactured Electron microscope Hitachi- Nikon FEI/ Philips Zeiss Indian Manufacturer and supplier Lead india laboratory equipment company – Vadapalani Chennai Scitech Sciebtifies- Mumbai Jupiter Scientific company Ammapet salem
- 25. Difference between SEM and TEM Features Scanning Electron Microscope Transmission Electron Microscope Classification of electrons (e-) used Scattered (backscattered or secondary) e- Transmitted e- beam Image formation process Image is produced after microscope collects and counts the secondary e- Internal or beyond e- that directly pass through the sample are the ones illuminated in the image macrom Focus of analysis Surface of sample (e.g. fine hairs, eye of a housefly, head of a pin) Internal or beyond surface (e.g. viruses, organelles, atoms, macromolecules) Image presentation 3-D 2-D Special sample preparation Sample must be “stained” by element that captures secondary e- Sample must be cut extremely thin Special sample preparation Sample must be “stained” by element that captures secondary e- Sample must be cut extremely thin
- 26. Similarity between SEM and TEM Type of Object - Non living Source of Radiation - Electron Medium - High Vacuum Nature of lens - One Electrostatsati and few electromagnetic lens
- 27. Applications Of Electron microscope Electron microscopy is being used today in research laboratories around the world to explore the molecular mechanisms of disease, to visualize the 3D architecture of tissues and cells, Structural biology– 3D techniques, and single particle analysis, allow researchers to derive important information regarding protein domain arrangements and, in some cases, to trace individual polypeptide chains. The combination of electron microscopy reconstruction with X-ray crystallography and NMR spectroscopy Cellular biology– High-resolution cryo microscopy avoids the alterations caused by conventional preparation techniques to allow imaging of cell membrane structures and sub-cellular morphology in fully hydrated conditions. Tissue biology– An electron microscope’s ability to provide high resolution ultrastructural imaging over large areas and volumes of tissues or cells is invaluable in discerning critical relationships among components of biological systems across large differences in spatial scale. Biomaterials– The properties of biomaterials and nanoparticles are highly dependent on structural characteristics that are readily observed using electron microscopy
- 28. Forensics– Forensic science uses electron microscopy to analyze criminal evidence such as gunshot residue, clothing fibers, handwriting samples, and soil. 3D Nano characterization– Nano characterization moves to a new level with SEM, TEM, and DualBeam tomography affording 3D visualization at the nanoscale. Analytical techniques such as electron backscatter diffraction (EBSD), X-ray microanalysis (EDSX), and energy-filtered TEM (EFTEM) can also be extended to three dimensions, In situ nano processes– The electron microscope becomes a lab in a chamber with ESEM and ETEM technologies allowing dynamic control over temperature, pressure, and gas type for in situ nano process investigations. Researchers can visualize and correlate the structure, property, and function of materials undergoing chemical and physical processes such as catalysis, oxidation, reduction, polymerization, deformation and thermally induced phase transformations
- 29. Limitations of Electron Microscopy Electron microscopes are very expensive to buy and maintain. They are dynamic rather than static in their operation: requiring extremely stable high voltage supplies, extremely stable currents to each electromagnetic coil/lens, continuously-pumped high/ultra-high vacuum systems and a cooling water supply circulation through the lenses and pumps. As they are very sensitive to vibration and external magnetic fields A significant amount of training is required in order to operate an electron microscope successfully and electron microscopy is considered a specialised skill. The samples have to be viewed in a vacuum, as the molecules that make up air would scatter the electrons. https://www.jic.ac.uk
- 30. Recent Research In-Situ High-Resolution Transmission Electron Microscopy Investigation of Overheating of Cu Nanoparticles Chunlin Chen, Ziyu Hu, Yanfen Li, Limin Liu, Hirotaro Mori, Zhangchang Wang January 20,2016 Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba- ku, Sendai 980-8577, Japan
- 31. Sumbali G.,Mehrotra R.S. (2009); Microscopy and staining; In Principle of Microbiology; The McGraw Hill Education Private limited, 7 West Patel Nagar ,New Delhi, India;Ed.4th; pp 41-65. Willey J.M., Sherwood L.M. ,Woolverton C.J.(2008); The Study of Microbial structure: Microscopy and specimen preparation; Prescott, Harley, klein’s Microbiology ;McGraw-Hill,1221 Avenue of the Amerias, New York ;Ed.7th ; pp 28-34. Wilson K., Walker J. (2010); Microscopy: Principle and Techniques of Biochemistry and Molecular Biology; Cambridge university press, New York ;Ed.7th ; pp- 100-136 REFERENCES