ELECTRON MICROSCOPY

BY NEHA SAROHA

ANDRAMANJOT KAUR

A BRIEF AND A BASIC SET UP OF ELECTRON MICROSCOPE

An ordinary light microscope consists of light source, a condenser for focusing light on or near the the object, an object holder,, an objective lens for focusing the image and an eyepiece for projecting the image formed by the objective into eye.

This is also true for an electron microscope except that light is replaced by an electron beam, the sample holder is wire screen called a GRID, and the lenses are electromagnets rather than glass.

PRINCIPLE OF THE ELECTRON MICROSCOPE There is a limit to the resolution that one

can obtain in microscopes that operate with visible light. The resolution increases with increasing frequency of the light beam, and electron microscopes use a beam of electrons to examine specimens on a much smaller scale than a light or optical microscope. The interaction of the electron beam with the specimen gives information concerning its morphology, topography, crystallographic arrangement and elemental composition

WORKING The optical system of an electron

microscope:

The illumination source consists of a white hot tungsten filament, which emits electron. The potential of the anode to which electrons re drawn is normally from 40-100kv greater than that of the filament. The filament and the anode together constitute the

electron gun. The anode contains small orifice through which

some of the fastest electrons pass. This whole plus a small aperture just below it collimate the electron to form a beam. The beam is slightly divergent because the electrons are deflected toward the edge of the orifice of the anode owing to its positive potential. The divergent beam is then made to converge onto the specimen by an

electromagnetic condenser lens. The beam is rarely focused sharply on the sample because an intense beam could destroy it.

The image is formed by what is often called THE SUBSTRACTIVE ACTION OF THE SAMPLE, that is some of the electrons are scattered from the atoms of the object.

The pattern of this loss of electrons generates the image pattern.

The objective lens, which Is adjusted so that the sample is precisely at its focal point, then refocuses the beam to produce an image.

This image is then magnified in several stages by 3 electromagnetic lenses called the diffraction , intermediate and projector lenses.

The final projector lens forms the image on either on a fluorescent screen or photographic plate.

TYPES OF ELECTRON MICROSCOPE

Transmission Electron microscope : MAX KNOLL and ERNST RUSKA in 1931.

Scanning Electron Microscope: by VON ARDENNE IN 1938.

TRANSMISSION ELECTRON MICROSCOPY Transmission Electron

Microscopy (TEM) is a well known technique for imaging solid materials at atomic resolution.

Structural information can be acquired both by (high resolution) imaging as well as by electron diffraction.

BASIC SYSTEM OF TEM IMAGING

ELECTRON DIFFRACTION

CHEMICAL ANALYSIS

IMAGING Image contrast is obtained by interaction of the

electron beam with the sample. Several contrast effects play a role. In the resulting TEM image denser areas and areas containing heavier elements appear darker due to scattering of the electrons in the sample. In addition, scattering from crystal planes introduces diffraction contrast.

This contrast depends on the orientation of a crystalline area in the sample with respect to the electron beam. As a result, in a TEM image of a sample consisting of randomly oriented crystals each crystal will have its own grey-level

In this way one can distinguish between different materials, as well as image individual crystals and crystal defects. Because of the high resolution of the TEM, atomic arrangements in crystalline structures can be imaged in large detail

ELECTRON DIFFRACTION In case of a crystalline material, electron

diffraction will only occur at specific angles, which are characteristic for the crystal structure present.

As a result, a diffraction pattern of the irradiated area is created that can be projected onto the CCD camera.

In this way, electron diffraction can provide crystallographic information from thin films, bulk materials as well as from nanometer sized particles.

CHEMICAL ANALYSIS As a result of the

interaction of the electron beam with the specimen, some energy is transferred from the electrons to the sample.

The excitation and de-excitation of atoms and molecules in the sample allow (local) chemical analysis.

This analysis can either be performed using the broad beam used for normal imaging, or by focusing the beam size down to 0.2 nm.

WORKING OF TEM

The "Virtual Source" at the top represents the electron gun, producing a stream of monochromatic electrons.

This stream is focused to a small, thin, coherent beam by the use of condenser lenses 1 and 2. The first lens, largely determines the "spot size"; the general size range of the final spot that strikes the sample. The second lens,usually controlled by the "intensity or brightness knob" ,actually changes the size of the spot on the sample; changing it from a wide dispersed spot to a pinpoint beam.

The beam is restricted by the condenser aperture, knocking out high angle electrons (those far from the optic axis, the dotted line down the center).

The beam strikes the specimen and parts of it are transmitted.

This transmitted portion is focused by the objective lens into an image.

APPLICATIONS OF TEM

The TEM has its primary uses in metallurgy ,and the biological sciences, especially in the study of cells at the molecular level.

TEMs have been particularly useful in metallurgy, especially in terms of developing images of crystals and metals at the molecular level - allowing scientists to study their structure, interactions and identify flaws.

DISADVANTAGES OF TEM Sample preparation for TEM generally

requires more time and experience than for most other characterization techniques.

A TEM specimen must be approximately 1000 Å or less in thickness in the area of interest.

The entire specimen must fit into a 3mm diameter cup and be less than about 100 microns in thickness. A thin, disc shaped sample with a hole in. the middle, the edges of the hole being thin enough for TEM viewing, is typical.

Hence limiting the dimensions of the sample.

Electron beam passes through the specimen.

SCANNING ELECTRON MICROSCOPE

Uses electrons reflected from the surface of a specimen to create image.

Produces a 3-dimensional image of specimen’s surface features.

WORKING OF SEM1) The "Virtual Source" at the top represents

the electron gun, producing a stream of monochromatic electrons.

2) The stream is condensed by the first condenser lens (usually controlled by the "coarse probe current knob"). This lens is used to both form the beam and limit the amount of current in the beam. It works in conjunction with the condenser aperture to eliminate the high-angle electrons from the beam.

3) The beam is then constricted by the condenser aperture, eliminating some high-angle electrons.

4) The second condenser lens forms the electrons into a thin, tight, coherent beam and is usually controlled by the "fine probe current knob".

5) A user selectable objective aperture further eliminates high-angle electrons from the beam.

6) A set of coils then "scan" or "sweep" the beam in a grid fashion (like a television), dwelling on points for a period of time determined by the scan speed (usually in the microsecond range).

7) The final lens, the objective, focuses the scanning beam onto the part of the specimen desired.

8) When the beam strikes the sample (and dwells for a few microseconds) interactions occur inside the sample and are detected with various instruments.

9) Before the beam moves to its next dwell point these instruments count the number of e- interactions and display a pixel on a CRT whose intensity is determined by this number (the more reactions the brighter the pixel).

10) This process is repeated until the grid scan is finished and then repeated, the entire pattern can be scanned 30 times/sec.

APPLICATIONS OF SEM1) Reveal topographical surface

details.2) Detect sub-surface information.3)Detect compositional differences. But the major problem lie with this

technique is that the detector on SEM can not detect very light elements (H, He, Li ) and many instrument can not detect elements with atomic number less than 11 (Na)

ADVANTAGES OF SEM OVER OM

The SEM has a large depth of field, which allows a large amount of the sample to be in focus at one time and produces an image that is a good representation of the three-dimensional sample.

The combination of higher magnification, larger depth of field, greater resolution, compositional and crystallographic information makes the SEM one of the most heavily used instruments in academic/national lab research areas and industry.

Mag Depth of Field Resolution OM: 4x – 1400x 0.5mm ~ 0.2mm

SEM: 10x – 500Kx 30mm 1.5nm

SAMPLE PREPARATION The great capability of all the matter for

scattering electrons require that a sample should be very thin- otherwise no beam will get through to form an image.

In practice maximum thickness is approximately 0.1 micron for 100 armstong resolution.

This poses no real problem in observing viruses, fibrils or macromolecules but for most cells (1-50 thick) it is necessary to make thin sections.

Hence for the sample preparation various methods have been employed:

1) Embedding, Sectioning and Staining.

2) Replica Formation 3) Freeze Etching 4) Freeze Fracture 5) Shadow Casting

EMBEDDING, SECTIONING AND STAINING If the material under observation is too

thick for the passage of electrons, a thin slice or section must be made. To prepare this section the sample must be made rigid so that it can be cleanly cut. This process is called as EMBEDDING. It consists of gradual replacement of the aqueous material of the sample with an organic monomer that can be hardened by polymerization. The usual procedure is to place the sample in a sol. of a fixative which is usually a dilute formaldehyde sol.

The fixative denatures and often cross links proteins and other structures and presumably fixes all structures in place so that they ll not be moved or disrupted by further handling. The fixed sample is then transferred to ethanol water mixtures. This is called DEHYDRATION. The sample is then transferred to an alcoholic solution of the monomer which is then stimulated to polymerize.

After the sample has become solid the plastic containing the undisrupted sample is sliced with an ultra microtome into layers.

The sections are then stained by exposure to solutions of salts of Mo, tunsgsten, Pb, or U or to the vapor of osmium tetraoxide.

REPLICA FORMATION The method used for observing the

surface of an electron-opaque or easily destroyed specimen is called REPLICA FORMATION. The specimen is coated first with a thin layer of platinum and then with a supporting layer of carbon, both deposited by vacuum evaporation. The bilayer is then float off onto water and picked up on a grid.

This method has been used to study the surfaces of viruses. Membranes and certain protein crystals.

FREEZE ETCHING In replica formation water in the sample

must be removed. This presents a problem as structures usually collapse during air drying as a result of surface tension effects accompanying the phase changes that occurred during evaporation of the solvent. It avoids the production of artifacts due to drying. In this sample is rapidly frozen, sectioned or fractured and placed in a vacuum with conditions of pressure and temperature such that the water sublimes from the surface of sample. A replica of this is then prepared by evaporating a platinum or carbon while it is still in vacuum.

FREEZE FRACTURE The replica method and freeze etching

have been combined in a technique that allows visualization of the internal structure of extended objects consisting of two or more layers.

A sample containing membrane is frozen and then fractured by the impact of microtome blade. Often the cleavage plane of the membrane which consist of two layers lying along the middle of the bilayer. The ice is then sublimed away and replica is made by successive coating with platinum and carbon.

Best to study biological membrane.

SHADOW CASTING This procedure is mainly used for small

particles like viruses, phages etc. The particles are applied by spraying a

suspension of the particles onto a grid overlaid by a support film. The liquid quickly evaporates, the sample is placed in vacuum and a heavy metal is applied by evaporation.

The amount of metal deposited on the sample affects contrast.

The current amount of metal is determined empirically by using metal wire of a particular diameter and counting the number of turns of this wire around the tungsten.

The magnitude of vacuum during this also effect the picture quality.

VARIOUS STAINING METHODS USED IN EM

Negative staining

Positive staining

NEGATIVE CONTRAST TECHNIQUE Developed by Brenner and Horne. It consists of a embedding small particles or

macromolecules in an electron –opaque film or continuous stain.

the strain penetrates the interstices of the particle but not the particle itself. Hence the image is a result of the relative intensity of the beam at every point which is further proportional to the thickness of the opaque film.

here the smple is either mixed with the strain and sprayed on the grid.

Used for a wide variety of phages and viruses

DISADVANTAGESi. the interpretation of negative stained

samples is sometimes difficult because various patterns can be observed depending on:

1. the thickness of the stain 2. whether it has the interstices of the particle 3. whether it has adsorbed specifically to

the sample. For this reason it is usually necessary to look

at a large number of preparations and particles.

POSITIVE STAINING When heavy atoms of phosphotungstic

acid and uranyl acetate are used to stain the sample, so as to increase density of biological structure is called as positive staining.

It is used to the study the structures of ribosomes, DNA,RNA polymerase and collagen.

It’s most important application is that it is used in kleinchmidt spreading.

A BRIEF COMPARISON

LIMITATIONS OE EM Sample must be solid and must fix into

microscopic chamber. Sample must be stable at vacuum

pressure of 10-5 to 10-6 torr. Sample can not be examined in a living

state because of high vacuum. Thin sections are used to reveal inner

information because of penetration power of electron beam.

VARIOUS OTHER MODIFICATION OF EM Reflection Electron Microscope(REM)

Reflection High Energy Electron Diffraction(RHEED)

Spin Polarized Low Energy Electron Microscopy(SPLEEN)

Scanning Transmission Electron Microscope(STEM)

REM

In reflection electron microscope images obtained using electron beams diffracted at a small angle from the surfaces of bulk specimens, it is observed that the magnification in directions almost parallel to the incident beam appears to increase rapidly with distance from the in-focus position in both directions. An explanation for this effect is offered in terms of the curvature of the lines of energy flow around the cross-over of the electron beam formed by the condenser action of the fore-field of the objective lens.

RHEEED A high energy beam (3-100keV) is directed at

the sample surface at a grazing angle. The electrons are diffracted by the crystal structure of the sample and then impinge on a phosphor screen mounted opposite to the electron gun. The resulting pattern is a series of streaks. The distance between the streaks being an indication of the surface lattice unit cell size. The grazing incidence angle ensures surface specificity despite the high energy of the incident electrons. If a surface is atomically flat, then sharp RHEED patterns are seen. If the surface has a rougher surface, the RHEED pattern is more diffuse.

SPLEEM Probing the spin of matter with slow

electron is based on their strong spin –spin interaction, in contrast to fastest electron that experience a strong spin interaction.

In addition to strong spin interaction slow electron have a short inelastic mean free path in solids. Therefore their penetration depth into the solid is small and the spin can be probed only in reflection experiments.

Electron with energy tend to 10eV and about 100eV are used, at lower energy spin- spin interaction is stronger.

STEM A special TEM-technique in which an electron

transparent sample is bombarded with a finely focused electron beam which can be scanned across the specimen or rocked across the optical axis and transmitted, secondary, back scattered and diffracted electrons as well as the characteristic X-ray spectrum can be observed.

STEM essentially provides high resolution imaging of the inner microstructure and the surface of a thin sample, as well as the possibility of chemical and structural characterization of micrometer and sub-micrometer domains through evaluation of the X-ray spectra and the electron diffraction pattern.