Biological Low-Voltage Scanning Electron Microscopy
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In figure 1 we see a parasite within the parasitophorous vacuole PV displaying structural interactions with its host cell which would be difficult to capture in thin-section TEM. Figure 2 shows more elaborate structural interactions of the PV with the host cell.
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These structural interactions are thought to serve nutritional exploitation of host cells by Toxoplasma. The technique was excellently applied by the late Dr. Hans Ris to analyse nuclear pores in Xenopus oocytes which is displayed in figure 3. Figure 4 represents a different view of thoracic flight muscle from Drosophila and shows a de-embedded sample in which the hexagonal arrangement of thick and thin filaments are clearly visible. While only a few examples are depicted here, this technique can be applied to a variety of other cells or tissues in which structural detail is being studied.
Concluding Remarks and Future Directions LVSEM has allowed imaging of delicate biological material with only minimal coating requirements and it has generated a wealth of new data that could not have been obtained with any other microscopy method available to date. Future directions will undoubtedly include continuous improvements in increasing resolution and imaging of native structures with no or only minimal chemical fixation.
New directions include more complex applications such as the use of tools within SEMs to dissect and analyse biological material in more native conditions. Dissection of whole embryos has already been performed and excellently presented by Boyde . References:  McMullen D. Schatten H. The arrow depicts newly-resolved structure on the parasite surface connecting to the host cell.
From Schatten and Ris, .
From Schatten and Ris, . Seen here is the cytoplasmic surface with numerous nuclear pore complexes NPCs. Overview of using the environmental chamber with water, humidity and gaseous vapors in Polymer research. PDF 5. Applications of single-pass KFM showing that the mode complements phase imaging in compositional mapping of complex materials. PDF 8. Overview of the high resolution imaging for MEMS devices without the need for metal coating to dissipate charge buildup. Review of the excellent imagaing capabilities of low-voltage SEM for morphology invertigation of graphene.
PDF 3. Product Support Center Technical Support manuals, drivers, application notes, firmware, software, …. Legal Privacy Terms Trademark Acknowledgements. The wavelength of an electron is determined by the de Broglie wavelength and is linked to the accelerating voltage AV used to form the electron beam. For example, an AV of , volts results in an electron wavelength of 0. While it is possible to achieve sub-atomic imaging with an electron microscope, it is not possible to image biological samples at this resolution.
As we will see, the biological tissue is always the limiting factor! There are several different types of EM. These can be split into two main categories, transmission electron microscopes TEM and scanning electron microscopes SEM. The main differences between these are in the optics Fig. Figure 1. Both types of EM have an electron gun, which contains an electron source a filament that produces a cloud of electrons , a Wehnelt cylinder to form the beam and an anode to accelerate the beam. There are three main types of electron source; a tungsten filament, a lanthanum hexaboride LaB 6 crystal and a field emission filament.
Differences among the filaments are shown in table 1. Electrons travel along the magnetic field and can be focused in the same way that light is focused using glass lenses. Apertures are used in an EM to control the coherence of the beam, which affects resolution, and the amount of contrast in the signal. It has a large number of lenses. The condenser lenses depending on the microscope are responsible for the amount of illumination that reaches the sample and control beam intensity or brightness.
The objective lens focuses the beam of electrons onto the sample and applies a small amount of magnification. The intermediate and projector lenses magnify the beam and project it onto the camera CCD or film or screen to form an image. It takes only a few seconds to obtain a micrograph microscope image. The image is a result of the projected beam intensity: Transmitted electrons are detected as light areas in the micrograph; darker areas occur where electrons have been scattered or absorbed by the sample, thus reducing the number of electrons reaching the camera or screen.
This is known as bright field imaging and is the most common type of imaging for biological samples. TEMs are often classified based on the accelerating voltage AV they are capable of.
Most thin-section TEM will be conducted using kV. Advanced TEM techniques may require instruments capable of an AV between kV and 3 MV, which represent a resolution , to 3 million times smaller than light microscope resolution.
Scanning Electron Microscopy (Molecular Biology)
A SEM focuses the beam of electrons into a small spot that scans across the surface of a sample Fig. The condenser lens assembles the electrons into a fine beam. The objective lens focuses the beam onto the sample. Deflection coils cause the beam to move in a rectangular X and Y direction, producing a raster scan across the surface of the sample. The signal is transmitted to a computer screen.
Reducing the area being scanned results in an increase in magnification. Figure 2. Specimen-beam interaction at an atomic level. The main signals that are relevant for the TEM are transmitted and scattered electrons. The scattering of electrons creates contrast in the final image. For the SEM, the main signals are the secondary electrons and backscattered electrons.
Low-voltage electron microscope
An SEM image is formed from signals that are emitted from the sample as a result of the specimen-beam interaction Fig. Most biological SEM will generate images using two types of electrons.
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Secondary electrons SE are low energy electrons produced by small energy transfers between electrons from the beam and electrons orbiting atoms in the sample. The energy transfer causes the orbiting electron to leave the atom and become a secondary electron. An outer orbiting electron will then release some energy in order to jump into the gap left by the secondary electron.
In addition, there are a few applications that require the detection of characteristic X-rays energy dispersive X-ray spectroscopy or photons cathodeluminescence.