Structural Biochemistry/Lipids/Techniques to Study Membranes/Electron Microscopy

Electron microscopy is a technique used for gaining a molecular picture of matter. The resulting picture of a compound is called an electron micrograph. Depending on the resolution of the device, micrographs can show images of particles and macromolecules on the nanometer scale. This is particularly effective in biochemistry because electron micrographs can give images of a particular protein at different angles and allow a three dimensional image of the outside of a protein to be created. This image gives an insight into the structure, and thus the function, of the protein in question.

Electron microscopy works on the basic principle of shooting a beam of electrons at a particular material (often under vacuum to avoid electron collisions with air molecule) and "reading" the result. The particular way a result is "read" gives different types of images for different materials and classifies the different types of electron microscopy. These different types are Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Scanning Transmission Electron Microscopy (STEM), Reflection Electron Microscopy (REM), and Low Voltage Electron Microscopy (LVEM).

Scanning Electron Microscopy (SEM)


SEM is a type of electron microscopy that images a sample's surface by scanning it with a high-energy beam of electrons in a rectangular formation of parallel scanning lines that is similar to the guidance of an electron beam on a television screen or a computer monitor. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition and other properties such as electrical conductivity. Scanning electron microscopes usually have a magnification range from 15x to 200,000x and a resolution of around 5 nanometers.

Just recently, in December 2012, the Italian scientist, Enzo Di Fabrizio, has obtained the first real photo of the DNA double - helix structure. In the image, one can see seven strands of DNA wrapped into a cord. Di Frabrizio used electron microscopy, which is a very useful technique to obtain a molecular picture at a nanometer scale. What he did was "developed a technique that pulls strands of DNA between two miniscule silicone pillars, then photographs them via an electron microscope"(Grenoble). This is a great example of how well electron microscopy works to obtain images of particles and macromolecules.

Transmission Electron Microscopy (TEM)
TEM uses a high voltage electron beam to create an image. An electron gun is then used to emit these electrons, which is usually fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode/cathode mechanism, which is then focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is transparent to electrons and in part scatters them out of the beam. Upon leaving the specimen, the electron beam contains information about the structure of the specimen that is visually enhanced by the microscope. The image is viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide.



Scanning Transmission Electron Microscopy (STEM)
STEM is a type of transmission electron microscope, where the electrons pass through the specimen, but, with scanning electron microscopy, the electron optics focus the beam into a narrow area which is scanned over the sample in a raster. This process makes these microscopes suitable for analysis techniques such as mapping by energy dispersive X-ray (EDX) spectroscopy, electron energy loss spectroscopy (EELS) and annular dark-field imaging (ADF). These signals can be obtained simultaneously, allowing direct correlation of image and quantitative data. By using this process, it is possible to form atomic resolution images where the contrast is directly related to the atomic number.

Reflection Electron Microscopy (REM)
In REM, an electron beam is incident on a surface, but instead of using the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons is detected. This technique is usually coupled with Reflection High Energy Electron Diffraction (RHEED) and Reflection high-energy loss spectrum (RHELS). This method improves clarity by increasing the resolution and increasing light scattering for a greater sensitivity in structural differences. This method is particularly effective in identifying molecular structure since electrons reflected can have their distance measured and calculated through geometric manipulation. However, reflective methods may not be as effective in certain environments since the index of refraction may not be constant if performed outside a vacuum.

Low Voltage Electron Microscopy (LVEM)
The low voltage electron microscope (LVEM) is a new type of microscope that is used to observe biological specimens. LVEM is divided into 2 parts: a miniature transmission electron microscope and conventional optical microscope. The miniature transmission electron microscope has maximum magnification 500 times by using emitter source of Schottky type, magnetic lens (for image formation), and electrostatic lens (for controlling magnification). The single crystal YAG fluorescent screen converts the electron image to light image. The miniature transmission electron microscope only uses 5kV source for accelerating voltage. The electron microscope is kind of small in size (around 20cm) and fits into a conventional optical microscope which has maximum magnification 400 times. The ability of having that maximum magnification is due to the aid of CCD camera (for image recording). The low accelerating voltage 5kV helps LVEM having imaging contrast twenty times higher than for 100kV. This seems to be an advantage of LVEM. However, the disadvantage of LVEM is a low transmittable thickness of the sample below 20 nm which limits only for small objects with the size of 20 nm.