SCANNING ELECTRON MICROSCOPE
The SEM is used for examining the surface details of microorganisms, cells and tissues. The image is obtained by scanning the specimen surface with a narrow electron beam, collecting and amplifying the generated signals and feeding them to the cathode ray tube for display. SEM consists two main parts, viz. the probing system and the display system.
1 The Probing system
The electron beam is generated by an electron gun (or probe), which consists of a V-shaped hairpin tungsten filament. When the filament is heated, electrons are emitted and accelerated through the anode.
The diameter of the initial focused spot of electrons, called the Spot size, is 50 µm. To increase resolution at higher magnifications, electron source is demagnified (i.e., the spot size is decreased) to 2 nm by using electromagnetic condenser lenses.
2 The display system
As the electron beam strikes the specimen, many signals are generated, each of which gives different information about the specimen, when signals are collected by specific detectors.
· Secondary electrons provide information on surface morphology
· X-rays are used for elemental detection and analysis
· Backscattered electrons are used for surface topography
2. 1 Surface morphology
Secondary electrons emitted by the sample are attracted by a grid (at positive potential) located in front of the detector. The detector collects all electrons coming towards it. These electrons are accelerated towards a scintillator by applying strong positive voltage (10-12.5 kV). When electrons hit the scintillator, they generate photons, which are guided by the light pipe to the photomultiplier where photoelectrons are produced. These photoelectrons are amplified and fed to the cathode ray tube (display screen).
The probe scans the specimen in a raster pattern synchronous with the electron beam of the cathode ray tube. A point-to-point correspondence between the specimen and the displayed image is thus achieved. Variations in secondary electron yield are then used to modulate the intensity of the electron beam in the cathode ray tube thus forming an image. The probe scans line by line on the specimen surface. The number of lines producing the image can be varied from 100 to 2000 thus giving different resolving power. To obtain a good photograph slow scan rate is important so that the probe can stay at one spot for longer time thus increasing the number of emitted secondary electrons. For viewing, fast TV scan rate can be used. As the angle between probe and specimen surface varies, the signal strength and contrast of the image is changed. The detector is placed to one side of the specimen, this enhances the specimen contrast because one specimen area prevents signals from other area from reaching the detector.
SEM has two different cathode ray tubes. One is for visualizing the specimen and the other for photographic recording.
Specimen preparation for SEM
The surface of an object to be studied by SEM must have the following characteristics:
· It must be free from foreign particles
· It must be stable when put under high vacuum
· It should remain stable after exposure to electron beam. It should develop as few surface charges as possible.
· It must emit sufficient number of secondary electrons.
Biological specimens need special attention because most of them are soft, full of extracellular fluids and poor conductors. There are number of preparative techniques depending on the nature of the tissue and type of studies to be carried out. Preparation of specimen for SEM studies involves the following steps:
1 Specimen size
Specimen size depends upon (a) stage capacity (x- y movement) of the SEM, (b) type of fixation and (c) drying process used. It should be smallest in size possible, in order to reduce the preparation artifacts. Tissue culture, single cell or microorganisms can be processed in suspension or attached to a suitable base (coverslip) before going through preparative procedures.
As extracellular products such as mucus, blood, other body fluids and tissue fragments may obscure the surface to be examined, they must be removed by gentle washing with cold isotonic buffer. Microorganisms and cell suspensions can be separated from their liquid environment by centrifugation.
2 Stabilization of specimen
Apart from hard objects such as teeth, bone and wood, all other biological materials must be stabilized in order to prevent from undergoing structural changes, which is done by chemical fixation or physical fixation (cryo-fixation). The fixative and fixation protocol is same as in the case of TEM specimen preparation.
3 Washing and dehydration
To remove the unreacted fixative, the specimen is washed in buffer (which was used for preparing the fixative), especially after secondary fixation with OSO4. The latter may be reduced by dehydrating agents, causing precipitates to form on the specimen, thus obscuring the surface morphology.
Chemically fixed objects must be dehydrated before drying. The purpose of dehydration is to remove water before drying. The most commonly used dehydrating agents are ethanol and acetone. To avoid initial osmotic damage to specimen, starting solvent (acetone or ethanol) should be low (30% or lower). Then specimens are dehydrated using increasing concentrations of the dehydrating agent up to dry absolute acetone stage.