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Microscopes are some of the most common and well-known scientific instruments around. They are used to make small objects, invisible to the human eye, easier to see.
There are several different classes of microscopes. However, the most common way to categorize these devices is based on what interacts with the sample, be it light (or photons), electrons, or a probe. These classes are referred to as optical, electron, and scanning probe microscopes, respectively.
That said, you often see microscopes categorized according to the wavelength used to magnify an image as well. Common wavelengths include visible light, ultraviolet light, x-rays, and electrons. For example, if a microscope uses visible light to illuminate a sample, it’s typically referred to as a light microscope. If fluorescence is used, the microscope is referred to as a fluorescence microscope.
The most common types used across laboratories today are light microscopes, or optical microscopes, which utilize visible light, along with a series of lenses, to achieve magnification.
Electron microscopes, which employ beams of electrons as the light source, often serve as an alternative to light microscopes because, compared to visible light, electrons have significantly shorter wavelengths. Around 100,000 times shorter, to be more exact.
The use of shorter wavelengths increases resolving power and magnification, allowing for extremely high resolution of objects too small to be seen by standard microscopy methods like light microscopy. For this reason, electron microscopy is a powerful tool in several fields and industries where highly detailed imaging is required to understand a biological or nonbiological sample’s ultrastructure (fine structure, especially within a cell, that cannot be seen with standard light microscopes).
Areas within the life sciences that rely on electron microscopy include structural biology, drug discovery and development, materials science, microelectronics, nanotechnology, and forensics science.
Electron microscopes use electron-optical lens systems to obtain high-resolution images of biological and non-biological specimens. An electron gun is used to aim and release the electron beam towards the sample, which must be kept in a vacuum-sealed chamber. The beam is then controlled and focused at a specific location using electrostatic and electromagnetic lenses.
Electron microscopes suffer from poor contrast despite their numerous benefits, so staining is often used to achieve better clarity. Various electron microscope types provide different functions. They have been used to open up new fields of study in nanotechnologies and more. We’ll cover the most common types below.
Using transmission electron microscopy to obtain an image, a TEM is one of the most frequently used types of electron microscopes. Its main components include:
This column houses an electron-beam generator, or what is commonly referred to as an electron gun. It acts as an electron source and is typically a tungsten filament cathode. The beam generator is typically connected to a condenser system that is used to focus the electron beam onto the object for imaging and analysis.
Vacuum System
Since electrons are tiny and easily deflected by hydrocarbons or gas molecules, it is necessary to use the electron beam in a vacuum environment. When electrons are in a vacuum, they behave like light.
Different vacuum pumps are used to create and maintain vacuum pressures as low as 10–8 Pa (atmospheric pressure). These pumps include rotary, oil diffusion, turbomolecular, and ion getter pumps.
Furthermore, many airlocks and separation valves are used to avoid the need to evacuate the whole column every time a specimen or photographic material or a filament is exchanged.
Image Producing System
The image-producing system of a TEM is made up of electromagnetic lenses that typically consist of coils. When an electric current is passed through these coils, an electromagnetic field is created between the pole pieces, creating a gap in the magnetic circuit. By varying the current through the coils, the strength of the field and lens power can be varied.
In general, a TEM consists of three lensing stages: condenser lenses, objective lenses, and projector lenses. The condenser lenses are responsible for forming the electron beam generated by the electron gun, and the objective lenses focus the beam. Lastly, the projector lenses expand the beam onto a viewing screen with a phosphor coating, or other imaging devices, such as a digital camera (e.g., charge-coupled devices (CCDs).
A TEM’s magnification is due to the ratio of the distances between the specimen and the objective lens’ image plane.
Imaging Devices
Image recording was traditionally performed using a fluorescent viewing screen that emitted light when impacted by the transmitted electrons, which provided real-time imaging. A film camera was used to record permanent images.
Modern TEMs, on the other hand, rely on digital imaging devices, such as CCD (charge-coupled device) cameras to capture and record images. Some models may still include a viewing screen.
Similarities
Most other types of electron microscopes, such as scanning electron microscopes (SEMs) and scanning transmission electron microscopes (STEMs), have the same main components as a TEM:
SEMs are the second most common type of electron microscope and are used to look at the surface layer of nanoscale objects.
In this technique, a beam of electrons is scanned across specific areas on the surface of a sample to produce an image. This particular scanning technique is referred to as raster scanning.
Unlike TEM, where the electrons go through the sample, scanning electron microscopes have electrons bounce off the material’s surface. These electrons are called secondary electrons, and a screen is placed precisely to catch them as they come off the specimen.
The electron gun shoots the beam at the subject, and a series of electromagnets move the beam back and forth over its surface until the entire surface has been scanned. The results are commonly referred to as SEM images, and they show, in high-resolution detail, a sample’s surface composition and topography.
Though the image may have less resolution than TEM, SEM sample preparation time is much shorter.
A subtype of TEM, scanning transmission electron (STEM) microscopes rely on electrons passing through a thin sample.
In scanning transmission electron microscopes, the electron beam is focused into a fine point, around .05-.2 nanometers, before it is scanned over the entire object. Electron signals are collected point by point until a complete image of the surface is constructed.
Also unique to scanning transmission electron microscopes is the aberration corrector. This serves to correct the electron signal’s aberrations that occur due to them going through electromagnetic lenses. In short, it increases the resolution and clarity of the image. An electron energy loss spectrometer then analyzes the electrons. It examines the energy that the electrons lose when they bounce off the sample.
A subtype of scanning electron microscopes, environmental scanning electron microscopes differentiate themselves by being able to image both uncoated and wet specimens. This is important because it allows for samples to be viewed in their natural environment without much prior preparation.
Most ESEM procedures are the same as scanning electron microscopy. However, key differences exist in electron detection, beam transfer, and environmental condition management.
Of particular note in ESEM is a differential pumping system and gaseous detection device that are used. The differential pumping system provides a high vacuum environment for the electron beam column but leaves a separate high-pressure specimen chamber. Gaseous detection devices are employed to amplify the detection of secondary electron signals by utilizing gas ionization.
Unlike TEM and scanning electron microscopes, reflection electron microscopes do not analyze the transmission or the secondary electrons; instead, they look to analyze electron scattering. Electron scattering occurs when electrons bounce off of objects but do not lose any energy in the process.
This technique is commonly coupled with reflection high energy electron diffraction and reflection high energy loss spectroscopy.
In 2017, the Nobel Prize in Chemistry was awarded to researchers Jacques Dubochet, Joachim Frank, and Richard Henderson “for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution.” Their developments allowed electron microscopes to view biological material without damaging or drastically altering it.
Previously due to the powerful electron beams required for electron microscopy, any biological material being viewed would need to be dead. Joachim Frank developed the general method in the 1970s and 1980s, and Jacques Dubochet was able to image biological samples in water in a vacuum without the water evaporating. Finally, Richard Henderson proved the technology’s potential by imaging a 3D rendering of a protein on the atomic scale.
These innovations have had a severe impact on the study of biological materials. For example, in the 2010s, cryo-EM was employed to understand the Zika virus outbreak in Cambodia better.
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