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.
The Wave Properties• In 1924, the wave-particle dualism waspostulated 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)
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
The electron wave• The generation of a monochromatic and coherent electron beam is important• Design of modern electron microscopes is based on this concept
Scheme of electron-matter interactions arisingfrom 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
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.
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
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.
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
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
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
Comparison of lens system of Light & Electron Microscope
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)
TYPES OF Electron MicroscopyAnalytical 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)
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”
Transmission Electron MicroscopyThe 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.
TEM - DefinitionTEM is a microscopy technique wherebya 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.
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.
- vacuum system in which the electrons travel, - an electron emission source for generationThe TEM components of the electron stream (tungsten filament, or a lanthanum hexaboride (LaB6)), - 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
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)
• The most common mode of operation for a TEM is the bright field imaging mode. • concept of “mass-thickness contrast”Bright Field Imaging isA TEM image of the polio virus. Thepolio virus is 30 nm in size. • “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 Beers law)
• Electrons passed through CrystalDiffraction Contrast with equidistant lattice planesImaging • With regular spacing betweenCrystalline diffraction pattern from a the scattering centers ,twinned grain of FCC Austenitic steel • 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
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.
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.
• By taking multiple images of a single TEM sample at differing3D Imaging angles,Three dimensional imaging -A TEM • typically in 1° increments,image of a parapoxavirus • 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.
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
Scanning Transmission Electron Microscopy
• HAADF- STEM • Also called “Z- contrast imaging”High angular annular darkfield – STEM • In this method small clusters, single atoms of heavy atoms (e.g. in catalysts) Comparison of TEM & STEM can be recognized in a matrix of light (a) TEM and (b) HAADF-STEM image of atoms Pd balls on silica. • Because the contrast produced is high The overlap regions are relatively dark in (a) and bright in (b), respectively. • 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.
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
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.
Biological Sample preparation• Sample thickness – less than 1 mm3• 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
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
Different techniques for TEM• Negative staining• Shadow casting• Freeze fracture replication• Freeze etching
• Heavy metal deposits are collected onto specimen grid,1. Negative Staining tqHuman papilloma virus by negative except where the particle isstaining present • The specimen appears bright on the view screen
• Viruses, DNA, RNA visualisation2. Shadow Casting tq • 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
3. Freeze Fracturing TqStep 3Step 4Step 5
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 samples surface topography and composition.
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
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
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) - inverse reconstruction using electron-material interactive models• Possible applications are roughness measurement, measurement of fractal dimension, corrosion measurement and height step measurement.
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, iridium, tungsten, chromium, and graphite.
SEM - Cryo-imaging• SEM is equipped with a cold stage for cryo-microscopy,• Cryofixation 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
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.
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).
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.
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
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
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
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).
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
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.