Electron Microscopes are scientific instruments that use a beam of extremely energetic electrons to study objects on a précised scale. This examination can produce information about the topography, morphology, composition and crystallographic information about the object. Light Microscopes had certain limitations as the physics of light to 500x or 1000x magnification and a resolution of 0.2 micrometers instigating thereby the development of Electron Microscopes. In the early 1930’s this theoretical limit had been reached and there was a scientific aspiration to comprehend the fine details of the interior structures of organic cells like nucleus, mitochondria, etc. This required 10,000x and above magnification which was just not imaginable using Light Microscopes.
The Transmission Electron Microscope (TEM) was the first type of Electron Microscope to be developed and is designed accurately on the Light
Transmission Microscope except that a focused beam of electrons is used as a replacement for of light to “see through” the specimen. Max Knoll and Ernst Ruska developed the instrument in Germany in 1931. However, the first Scanning Electron
Microscope (SEM) came out in 1942 with the first commercial instruments around 1965. Its delayed development was because of the electronics behind the “scanning” the beam of electrons through the sample.
This type of electron microscope uses a beam to create an image, with electromagnets acting as lenses. The limit of resolution is increased by a factor of 1000 over the light microscope. The Transmission Electron Microscope (TEM) produces a two-dimensional (2D) image of an ultra-thin section by capturing electrons that have passed through the specimen. The degree of interaction between the electrons and the heavy metal stain influence the kinetic energy of the electrons, which are collected by a fluorescent plate.
The light of fluctuating intensity produced is proportional to the electron’s kinetic energy and is employed to develop the image. The Transmission Electron Microscope is useful for studying a cell’s interior and its ultra-structure. A Scanning Electron Microscope (SEM) used to make a three-dimensional image of the specimen surface.
PRINCIPLE OF ELECTRON MICROSCOPE
The beam of high energy electrons interacts with the specimen within a particular area of cross section depending upon the electron beam energy. An accelerated electron penetrates into the atom and its path is deflected causing it to scatter. In some cases, even complete backscattering can occur producing the back-scattered electrons (BSE). Electron microscope utilizes all such interactions occurring between the matter and the highly accelerated electron incident on it. These transmitted electrons are focused
with assistance of electromagnetic lenses and their very short wavelength allows the specimen to be imaged with a very high spatial resolution as compared to the light microscope.
Highly accelerated electrons are ready to transmit through a skinny specimen and are used for the formation of image in Transmission Electron Microscopy. These electrons can transmitted unscattered through the specimen, or can be scattered elastically or in elastically.
In elastic scattering, the bombarded electrons are deflected from their original path and scattered by atoms in the sample or specimen without any loss of energy. Such electron leave the sample with almost same k.E. and velocity as was possessed by it initially. However, the path of electron may change after interaction with the specimen. An inelastic scattering interaction involves transfer of energy from the incident electrons to the sample with the loss of energy. This pattern can then give information about the structure, atomic arrangements and phases present within the area being examined.
Thick specimen doesn’t allow incident electrons to transmit through, instead result into interactions surface generating electrons with diverse energies. These electrons are used for imaging in SEM.
Some of the electrons and radiations generated as results of matter-electron interaction and are used in electron microscopy imaging are discussed below.
i) Backscattered Electrons:
Electrons reflected back after interacting with the sample by elastic scattering through an angle greater than 90 degree are called backscattered electrons (BSE). These electrons are often distinguished on the idea of their high K.E. as they escape from the sample with energies quite like the primary-beam energy.
The fraction of primary electrons that escape as BSE is given because the backscattering coefficient η which varies directly with the specimen’s number. The higher atomic number elements produce more BSEs and appear brighter than lower atomic number elements. Because of this, BSE images show contrast in different parts of the specimen having different average number.
ii) Secondary electrons:
During inelastic interactions the incident electrons may transfer their energy to the valence electrons. These electrons are weakly bound to the nucleus and need only a small amount of energy to overcome the binding force and to eject out the atom. These are known as Secondary Electrons (SE). Most SEs eject with a small k.E. (<50 eV) therefore only those which are close to the surface i.e. generated only within very small depth (< 2 nm) are able to escape into the vacuum. Due to this reason SEs are utilized by SEM imaging to study topography of the specimen. The secondary electron coefficient (δ) is calculated as
the average number of secondary electrons produced per primary electron.
iii) Inner-Shell Ionization:
The incident electron passing very close to an atom might transfer a part of its energy to an inner shell electron. However for ejection, these electrons require large amount of energy as they are strongly bound to the atom.
If the accelerated electron possesses enough of the energy for its ejection, the inner shell electron is ejected as high kinetic energy secondary electrons.
v) Characteristic X-rays:
In the process of ejection of an inner shell electron, a vacancy is made within the lower
energy shell. In order to maintain the energy imbalance, electrons from a higher energy level fill the electron hole during a lower level. This is usually accompanied by emission of an X-ray photon having characteristic energy depending upon the difference between the 2 energy levels involved in electron transition.
v) Auger electrons:
Auger electron emission is a mechanism of stabilizing the atom after inner-shell ionization, analogous to X-ray emission. Emission of a core electron occurs by acquiring high amount of energy from incident electron, leaving the ion during a highly excited state. This ion rapidly tends to come back to a lower energy state by emission of an auger electron, ionizing the atom. The void formed in an inner shell is filled by an electron tumbling from a higher shell and therefore the energy liberated in this process is transferred to another electron, which emit from the atom in form of Auger electron.
When a sample or specimen is bombarded by high energy electrons, one or more electrons from
the lower-energy level are promoted to the higher-energy conduction level while returning to the ground state valence state, they may be temporarily trapped for short while into some extrinsic or intrinsic traps made due to impurities or structural defects in lattice. The loss of energy occur while leaving this usually in UV, Visible or IR region which give rise to luminescence.
COMPARISON BETWEEN SEM AND TEM ELECTRON MICROSCOPE
|OPERATIONAL MODE||Only surface (morphology)||Internal stud ( anatomy)|
|SOURCE OF DETECTION||Transmitted electrons||Reflected and backscattered electrons, x- rays|
|VOLTAGE APPLIED FOR ELECTRON GENERATION||10-12.5 Kv||100Kv|
|MEDIUM||Low vacuum requirement||High vacuum requirement|
|IMAGE FORMATION||2-D image||3-D image|
|SPECIMEN THICKNESS||Typically less than 150nm||Any|
APPLICATIONNS OF ELECTRON MICROSCOPE
There is no crime without clues; even the foremost expert criminals tend to leave behind traces of crimes they committed. Forensic investigations aim at examining the evidences left at the crime
scene and analyzing them.
There are various applications in electron microscope:
- Gunshot residue analysis
- Firearms identification – bullet marking comparison
- Investigation of gemstones and jewellery
- Examination of paint particles and fibers
- Filament bulb investigation at traffic signals
- Handwriting and print examination/forgery
- Counterfeit bank notes
- Trace comparison
- Examination of non-conducting materials
- High resolution surface imaging.
For scanning electron microscope, go through this link.
For transmission electron microscope, go through this link.