Objective

 

To familiarize the technique of sample preparation for transmission electron microscopy.

 

Principle

 

Electron beams  are used in electron microscope to illuminate the specimen and thus creates an image. Since the wavelength o f electrons are 100,000 times shorter than visible light the electron microscopes have greater resolving power. They can achieve a resolution of 0.2nm and magnifications upto 2,000,000 x. Light microscopes show limited resolution than electron microscopes. Light microscopes have a resolution of 200nm and can magnify upto 2000x. Electron microscopes use a beam of electrons to illuminate the specimen instead of light as in light microscopy. The electron microscopes are of the following types:

 

1. Transmission electron microscope
2. scanning electron microscope
3. scanning tunneling electron microscope

In transmission electron microscope (TEM), the source of illumination is a beam of electrons of very short wavelength, emitted from a tungsten filament at the top of a cylindrical column of about 2 m high. The whole optical system of the microscope is enclosed in vacuum. Air must be evacuated from the column to create a vacuum so that the collision of electrons with air molecules and hence the scattering of electrons are avoided. Along the column, at specific intervals magnetic coils are placed. Just as the light is focused by the glass lenses in a light microscope, these magnetic coils in the electron microscope focus the electron beam. The magnetic coils placed at specific intervals in the column acts as an electromagnetic condenser lense system. The specimen stained with an electron dense material and is placed in the vacuum. The electron beams are  passes through the specimen and   scattered by the internal structures.

 

 

The heated filament emits electrons which are then accelerated by a voltage in the anode. A higher anode voltage will give the electrons a higher speed. Thus the electrons will have a smaller de Broglie wavelength according to the equation, λ = h/mv.  The resolving power of a microscope is directly related to the wavelength of the irradiation, which used to form an image. The faster the electrons travel, the shorter their wavelength. As the wavelength is reduced, the resolution is increased. Therefore, the resolution of the microscope is increased if the accelerating voltage of the electron beam is increased.

 

Transmission electron microscopy  involves a high voltage   beam of electron emitted by a cathode and formed by magnetic lenses. The beam of electron that has been partially transmitted through the very thin  specimen carries information about the structure of the specimen. The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor  like CCD (charge-coupled device) camera. The image detected by the CCD may be displayed in real time on a monitor or computer.

The TEM has the ability  ability to determine the positions of atoms within materials which has made an indispensable tool for nano-technologies research and development in many fields, including heterogeneous catalysis and the development of semiconductor devices for  photonics and electronics.  In the life sciences, it is still mainly the specimen preparation which limits the resolution of what we can see in the electron microscope, rather than the microscope itself.

There are four parts for a transmission electron microscope:

•    Electron source
•    Electromagnetic lens system
•    Sample holder
•    Imaging system

The electron source is an electron gun which consists of a tungsten filament. This filament emits electrons when it is heated. The beam of electrons are the focused on the specimen by the condenser which consists of electromagnets called magnetic lenses. The sample holder consists of a mechanical arm which holds the specimen. The imaging system also consists of electromagnetic lens system and a screen which has a phosphorescent plate. The plate glows when hit by the electrons after passing through the specimen.

 

 ELECTRON GUN

 

The function of an electron gun is to emit an intense beam of electrons into the vacuum which accelerates the between the cathode and the anode. There are two main types of electron gun: thermionic electron gun and field emission gun. The metals contain free electrons.  The valence are free electrons  electrons, which are loosely bound in  the nucleus. Those  electrons cannot escape from the metal surface . The positively charged nucleus will try to pull back the free electrons when they try to escape from the surface. Hence the electrons have to overcome the  potential barrier in order to escape from the surface of the metals. The energy required to overcome this potential barrier is called work function.

Work function, φ, is the minimum energy in electron volts required to remove an electron from the metal surface. If the electrons in metals are to be emitted from the cathode they have to overcome the work function.
Electrons are emitted from a metal by two methods:

1. Thermionic emission: In this method the electrons are emitted from the metals by heating them.
2. Field emission: In this method the electrons are emitted from metals, under strong electric fields.

Thermionic electron gun

The filament is made from a high melting point material or low work function, in order to emit many electrons. Tungsten filament is most commonly used  in thermionic electron gun. Tungsten wire used as thermionic cathodes are of 0.1-0.2mm in diameter bent like a hairpin and soldered on contacts. The wire is heated by a current of a few amperes.

Field emission electron gun

In fleld emission electron gun, a very strong electric field is used to extract electrons from a metal filament. Temperatures are lower than that needed for thermionic emission. This gives much higher source brightness than thermionic guns, but requires a very good vacuum.

 

SAMPLE PREPARATION

Sample preparation is important for electron microscopy. There are three main steps for sample preparation: Processing, embedding and polymerization.
Processing
This includes: fixation, rinsing, post fixation, dehydration and infiltration.

1) Fixation

This is done to preserve the sample and to prevent further deterioration so that it appears as close as possible to the living state, although it is dead now. It stabilizes the cell structure. There is minimum alteration to cell morphology and volume. Glutaraldehyde is often used as the fixative in TEM. As a result of glutaraldehyde fixation the protein molecules are covalently cross linked to their neighbors.

2) Rinsing

The samples should be washed with a buffer to maintain the pH. For this purpose, sodium cacodylate buffer is often used which has an effective buffering range of 5.1-7.4. The sodium cacodylate buffer thus prevents excess acidity which may result from tissue fixation during microscopy.

 

3) Post fixation

A secondary  fixation  with  osmium  tetroxide (OsO₄),  which is to  increase  the  stability  and contrast  of  fine structure.  OsO₄ binds phospholipid head regions, which creating contrast with the neighbouring protoplasm (cytoplasm). OsO₄ helps in the stabilization of  many proteins by transforming them into gels without destroying the structural features. Tissue proteins, which are stabilized by OsO₄ and does not coagulated by alcohols during dehydration.

For  imaging electrons scatterring ,heavy metals like uranium and lead are used  and thus give contrast between different structures. Thus we add more electron density to the internal structures.

4) Dehydration

The water content in the tissue sample should be replaced with an organic solvent since the epoxy resin used in infiltration and embedding step are not miscible with water.

5) Infiltration

 

Epoxy resin is used to infiltrate the cells. It penetrates the cells and fills the space to give hard plastic material which will tolerate the pressure of cutting.

 

6) Embedding:

After processing the next step is embedding. This is done using flat molds.

7) Polymerization

Next is polymerization step in which the resin is allowed to set overnight at a temperature of 60 degree in an oven.

8) Sectioning

The specimen must be cut into very thin sections for electron microscopy so that the electrons are semitransparent to electrons. These sections are cut on an ultramicrotome which is a device with a glass or diamond knife. For best resolution the sections must be 30 to 60 nm.

The resin block can be made ready for the sectioning by trimming it at the tip with a razor blade or black trimmer so that the smallest cutting face is available. Fix the block to a microtome and cut the sections.  Sections float onto a surface of liquid held in trough and remain together in a form of ribbon. Freshly distilled water is generally used to fill the trough. These sections are then collected onto a copper grid and viewed under the microscope.

Scanning Electron Microscope

SEM is used to examine the surfaces of cells and microorganisms and thus give contrast between different structures. SEM also uses a beam of electrons to create an image. It uses the electrons emitted by the surface of the specimen to produce the image. So the surface view of the specimen is obtained.