Microscopy-Electron microscope

Electron microscope is the magnifying instruments that utilizes the beam of high-speed electrons to illuminate the object. Electron microscopy was discovered for visualizing the ultra-structure of the microorganisms, cells, etc. It gives high magnification (up to 1000000X) and incredibly high resolution (dependent upon the wavelength of radiations). Electron microscopes use signals arising from the interaction of an electron beam with a sample to obtain information about structure, morphology, and composition. The first transmission electron microscope was developed by Ernst Ruska and Max Knoll of Germany in 1931.

Principle- Use of electron beams to analyze the specimen.

Electron microscope can be of two types-

1.      Transmission Electron Microscope

2.      Scanning Electron Microscope

 

ü  Transmission Electron Microscope (TEM)

Transmission electron microscope is a microscopy technique in which a 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, or  layer of photographic film, or to be detected by a sensor.

 

Components of a Transmission Electron Microscope

1.      Electron gun- produces high energy, large current, high coherence electron beam necessary for generating diffraction patterns and high spatial resolution images.

2.      Condenser lenses- doughnut-shaped electromagnets called magnetic lenses are used to focus the beam on the specimen. Electromagnetic lenses that consist of coils of wires with electricity running through them. They create a magnetic field that focuses and consists of the electrons into a thin beam.

3.      The electron beam then enters the image producing system. At the beginning of this system is the sample being imaged. The electrons are transmitted through the sample and into another series of electromagnetic lenses that focus the electrons onto a fluorescent or phosphorescent screen to create the image.

Specimen preparation- Soaking with solutions of heavy metal salts such as lead citrate and uranyl acetate. The lead and uranium ions bind to structures in the specimen and make them more electron opaque, thus increasing contrast in the material.

 

Principle and Working

·         A modern Transmission Electron Microscope (TEM) is complex, but the basic principles behind its operation can be readily understood.

·         A heated tungsten filament in the electron gun generates a beam of electrons that is focused on the specimen by the condenser.

·         Since electrons cannot pass through a glass lens, doughnut-shaped electromagnets called magnetic lenses are used to focus the beam.

·          The column containing the lenses and specimen must be under high vacuum to obtain a clear image because electrons are deflected by collisions with air molecules.

·         The specimen scatters some electrons, but those that pass through are used to form an enlarged image of the specimen on a fluorescent screen.

·         A denser region in the specimen scatters more electrons and therefore appears darker in the image since fewer electrons strike that area of the screen, these regions are said to be “electron-dense”. In contrast, electron transparent regions are brighter.

·         The image can be captured as a photograph for a permanent record.

 

 

 

Advantages

·         It offers very powerful magnification and resolution.

·         Have a wide range of applications and can be utilized in a variety of different scientific, educational and industrial fields (study the structure of virus particles bacteria etc).

·         Provide information or element and compound structure.

·         Images are high qualified and detailed.

           

Disadvantages

·         Large and very expensive.

·         Laborious sample preparation.

·         Operation and analysis require special training.

·         Samples are limited to those that are electron transparent.

·         Images are black and white.

 

ü  Scanning Electron Microscope

 

·         The Scanning Electron Microscope (SEM) 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 7nm or less.

·         Specimen preparation for SEM  is relatively easy and in some cases, air-dried material can be examined directly.

·         However, microorganisms usually must first be fixed, dehydrated, and dried to preserve the surface structure and prevent the collapse of the cells when they are exposed to the SEM’s high vacuum.

·         Before viewing, dried samples are mounted and coated with a thin layer of metal to prevent the buildup of an the electrical charge on the surface and to give a better image.

·         To create an image, the  SEM scans a narrow, tapered electron beam back and forth over the specimen.

·         When the beam strikes a particular area, surface atoms discharge a tiny shower of electrons called secondary electrons, and these are trapped by a detector strike a scintillator, causing to emit light flashes that a photomultiplier converts to an electric current and amplifies.

·         The signal is sent to a cathode-ray tube and produces an image that can be viewed or photographed.

·         The number of secondary electrons reaching the detector depends on the nature of the specimen’s surface.

·         When the electron beam strikes a raised area, a large number of secondary electrons enter the detector, in contrast, fewer electrons escape a depression in the surface and reach the detector.

·         Thus raised areas to appear lighter on the screen and depressions are darker.

·         A realistic three-dimensional image of the microorganism's surface results.

 

Application

The actual in situ location of microorganisms in ecological niches such as the human skin and the lining of the gut also can be examined.

Advantages

·         It gives detailed 3D and topographical imaging and versatile information.

·         This works very fast.

·         Modern SEMs allow for the generation data in digital form.

·         Most SEM samples require minimal preparation actions.

Disadvantages

·         SEMs are expensive and large.

·         Special training is required to operate an SEM.

·         SEMs are limited to solid samples.

·         SEMs carry a small risk of radiation exposure associated with the electrons that scatter from beneath the sample surface.