Scanning Electron Microscope (SEM)

An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects.

Types of Electron Microscopes

Transmission Electron Microscope (TEM)
Scanning Electron Microscope (SEM)

Scanning Electron Microscope (SEM)

The scanning electron microscope (SEM) is useful in microbiology to reveal morphological features of microorganisms.

The Scanning electron microscope works on the principle of applying kinetic energy to produce signals on the interaction of the electrons, these electrons are secondary electrons, backscattered electrons and diffracted backscattered electrons which are used to view surface structure and atomic density of any specimen.

Secondary and backscattered electrons are used to produce an image.

Electron microscopes have very short wavelengths of electrons in comparison to the light microscope which enables better resolution power.

Working Parts of the Scanning Electron Microscope

This microscope is the assembly of various parts, works togather to produce image.

Electron Gun

The electron gun produces an electron beam, and this is a thermionic emission gun (TE Gun). Thermo-electrons are emitted from a filament made of thin tungsten wire (works as Cathode). A high voltage was applied to anode (about 1-30 kv) to generate the electrons. There are 3 commonly used types of electrons sources:

Common electron sources:

Tungsten filament
Solid state crystal (CeB₆ or LaB₆)
Field Emission Gun (FEG)

Vacuum System

Inside the electron optical system and in specimen chamber, high vacuum is created (about 10^-3 – 10^-4 Pa.) with the help of diffusion pump (generally used), Roughing pump and Turbomolecular pumps.

Presence of air molecules and other particles inside the column can interfere with electrons and can change the direction and energy of electrons and distort the quality of specimen image.

Anode Plate

The electrons leaving the filament have a low energy and, therefore, need to be accelerated to the desired speed before entering the electron column.

A high voltage between the electron source (cathode) and an anode plate is applied leading to an electrostatic field through which the electrons are guided and accelerated.

Condenser and Objective Lenses

In electron microscope electromagnetic lenses are used to control the path of electron beam and divert its path to focus them on at their focal point. The strength of electromagnetic lens (focusing power) can be change by changing the electric current passing through them.

A fine electron beam is required to produce the image of specimen, which is achieved by the combination of condenser and objective lenses.

Scanning Coils

After the beam is focused, scanning coils are used to deflect, so that it scans in a rectangular pattern from side-to-side in lines from top to bottom to create an image that is projected on a cathode-ray tube and displayed on a screen.

Specimen Holder

The specimen stage is a place to hold the specimen for visualization. For better observation of every part of specimen, the stage is constructed with the ability to move horizontally, vertically, rotationally and also in tilting position.

Secondary Electron Detector

A secondary electron detector is sued for detecting the secondary electrons emitted from the specimen. For construction of this, a fluorescent material is coated over a detector and high voltage (about 10 KV) is applied to it.

Due to this it attracts the scattered electrons and when these secondary electrons hits the detector, an electric current is generated, which further amplify and reaches to TV screen / Display unit to generate image of specimen.

Backscatter Electron Detector

This is a detector in the SEM to detect and collect the backscattered electrons. The contrast of the backscattered electron image depends on multiple factors, including the atomic number of the sample material, the acceleration voltage of the primary beam and the specimen angle (tilt) with relation to the primary beam.

Image Formation in SEM

In SEM, mainly secondary electrons are used for imaging of biological specimens. These electrons have a very low energy (around 50 eV) compared to the energy of the primary electrons (up to 30 keV). Due to the low energy, these electrons can escape only from the surface area of the specimen and therefore provide information about the surface topography.

The number of electrons which are backscattered from a certain spot of the specimen depends on the atomic density of the specimen. Hence, backscattered electrons provide a “density image” and information about the molecular composition.

The SE then hit a scintillator which converts the electrons to photons. The photons are guided by a light conducting tube on the photomultiplier tube, where the photons again are converted to electrons that are amplified finally leading to an electrical signal. The current is displayed on the screen as image on the monitor.

Interaction of Electrons with Specimen

When electron beam hits the specimen, two separate type of interaction occurs between the specimen and primary electrons. Due to this interaction secondary and backscattered electrons are produced from the surface of specimen.

Secondary electrons are the result of inelastic collision, whereas backscattered electrons are the result of elastic collision.

Backscattered Electrons

When the incident electrons (primary electrons) hits the specimen, and interacts with high atomic weight atoms inside the specimen. These electrons typically bounce back (due to elastic collision) just like a ball hits a wall and bounce back. These electrons are called backscattered electrons.

Secondary Electrons

Secondary electrons are produced when an incident electron excites an electron in the sample and loses some of its energy in the process.

The excited electron moves towards the surface of the sample and if still has sufficient energy, it exits from the sample surface and termed as secondary electron.

Secondary electrons are generated from the surface of the specimen, and thus provide information topography (surface structure).

Sample Preparation for SEM

Electron microscopes are run under high vacuum conditions that is not suitable environment for biological specimens like cells and tissues. The specimen for SEM investigation should have following properties:

Large biological specimens (cells, tissues) do not fulfil any of these requirements and have to be treated in order to be investigated in the electron microscope. Typically, the specimen is turned into a solid state to make it suitable for vacuum and resistant in the electron beam.

Fixation: During fixation, the native biological specimen is chemically stabilized with chemical fixatives like glutaraldehyde and osmium tetroxide. All biological processes are arrested during fixation.
Dehydration: The fixed specimen is ready for dehydration with a sequence of different ethanol concentrations until it is completely dehydrated.
Chemical Exchange: After dehydration, the ethanol is exchanged with acetone. In a dedicated device (critical point dryer) the acetone is exchanged with liquid pressurized carbon dioxide.
Sample Solidification: Subsequently, the temperature and the pressure in the chamber of the critical point dryer are increased until they reach the critical point of CO2 (31°C, 74 bar). The CO2 in the critical state (neither gas nor liquid) is released from the chamber very slowly providing a gently dried specimen.
Coating: The critical point dried specimen is coated with a thin layer of heavy metal (e.g. 2 nm). The heavy metal layer renders the specimen conductive which is required for yield of electrons and creating contrast for imaging.