We just learned all about light microscopy, which was the standard until the middle of
the 20th century, and remains tremendously useful today.
But as we began to learn more about the microscopic world, using light microscopes that could
visualize individual cells, we started to become curious about structures that are smaller
than a cell, some details of which were difficult to visualize and study under standard light
microscopes.
Fortunately, electron microscopy, developed in the 1930s and 40s, proved to be much more
powerful and allowed scientists to view these smaller structures.
Some electron microscopy techniques employ a resolution of 0.2 nanometers, which is more
than 1,000 times better than light microscopy.
With this higher resolution, electron microscopes are able to magnify their specimen up to 1,000,000
times their original size, allowing researchers to study proteins inside of cells, and the
molecular structure of viruses, all of which led to a better understanding of the microscopic
world.
Of course we have to ask, how do they do this?
Well the answer is in the name, they use beams of electrons, which interact with the sample
as the beam is transmitted through the specimen.
As we recall from our study of chemistry, electrons are stable, subatomic particles
that carry a negative charge.
Electron beams have a much shorter wavelength than the visible light that light microscopes
use, which we refer to as the de Broglie wavelength, as we learned in the modern physics series.
This shorter wavelength allows for a higher level of detail and resolution in imaging.
One type of electron microscroscopy is called Transmission Electron Microscopy, or TEM.
For TEM, specimens are fixed on a support grid and either embedded in resin or stained
with a material like uranyl aceate or compounds of heavy metals like osmium tetroxide.
Certain structures within the specimen will take up more of the stain than other parts
of the specimen, and those sections are called electron dense regions.
When electrons pass through electron dense regions they lose more energy compared to
when they pass through sparser regions.
This phenomenon is used by TEM to visualize the specimen at high levels of resolution
and magnification.
For the stained specimen to be viewed by TEM it must be dehydrated, embedded in plastic
resin, and cut into ultra-thin slices with a glass or diamond knife.
The sections must be ultra-thin so that the electrons can pass through them.
When the specimen is secured in the specimen holder, an electron beam is generated and
shot out of the electron gun.
The electron beam is accelerated down the column of the microscope after passing through
the anode.
The electron beam then passes through the specimen, once again, losing energy as electrons
pass through electron dense regions.
The electrons then pass through the objective lens, magnifying the image, before the electrons
pass through the projector, or ocular lens, which magnifies the image further and projects
it onto the fluorescent screen.
This is where we start translating the electrons into an image.
The fluorescent screen is coated with a chemical which appears as bright spots when electrons
come into contact with it.
The intensity of the bright spot depends on how much energy the electrons have when they
hit the fluorescent screen.
The camera under the fluorescent screen then captures the image made from the bright spots
which is displayed on a computer screen.
Transmission electron microscopes are used to visualize structures that are too small
to study with light microscopes such as the cell organelles, proteins, cytoskeletal filaments,
and the components of cell membranes.
But even with the unmatched resolution and magnification abilities of TEM, it still does
have its limitations.
Some of these include the fact that you cannot study living specimens or image 3D structures
because the specimens must be dehydrated and cut into ultra-thin slices before imaging.
The second type of electron microscopy is Scanning Electron Microscopy, or SEM.
With SEM, specimens are coated with vaporized gold or palladium ions, because when electrons
come into contact with this coating, they cause atoms on the surface of the specimen
to emit electrons.
These emitted electrons are called secondary electrons, and these are what SEMs use to
visualize their specimen.
After the coated specimen is secured on the stage of the SEM, the process starts out similarly
to TEM in that an electron beam is shot out and accelerated towards the specimen.
In the SEM, the electron beam then sweeps, or scans the surface of the specimen, which
causes it to discharge secondary electrons.
There are two types of detectors in the sample chamber, the secondary electron detector and
the backscatter electron detector.
The backscatter electron detector picks up backscatter electrons that come from deeper
regions of the sample that are a result of elastic interactions between the beam and
the sample.
The results from this detector are primarily used to look at crystallography, and the magnetic
field of the sample.
But for the purposes of microbiology, researchers focus on the detection of secondary electrons
which allow them to visualize the topography of the sample, or the distribution of parts
and features on the surface of the sample.
The detectors convert the electron signal into a light signal that is amplified by passing
through a photomultiplier before the final image is projected on a computer screen.
SEM imaging is primarily used to study the topography of cells and other similar structures.
With this type of microscopy, researchers are able to investigate the depth of structures,
which isn’t possible with TEM, as SEM produces a 3D image opposed to the flat image from
TEM.
Yet, just like TEM, SEM cannot visualize live specimens, and SEM has worse resolution than
TEM.
Both TEM and SEM are expensive and require a lot of training, so they are not likely
to be used apart from those pursuing a microbiology career.
We covered quite a lot in this tutorial, so let’s briefly summarize.
Electron microscopy has around 1,000 times better resolution than light microscopy because
electron microscopy uses electron beams instead of light, which have a shorter wavelength.
This allows some electron microscopes to visualize specimens at 1,000,000 times their original
size.
Transmission Electron Microscopy, or TEM, records the differing energies that electrons
possess after they pass through a specimen stained with heavy metals to visualize small,
detailed structures like proteins inside of cells, and molecules associated with viral
particles.
Scanning Electron Microscopy, or SEM, captures the secondary electrons that are emitted from
the surface of samples coated in vaporized gold or palladium ions to visualize the topography
of the specimen.
And the main drawback, as we mentioned, is that both SEM and TEM cannot be used to visualize
living specimens, and they are also quite expensive and complicated techniques to use.
And with that, we know a bit about electron microscopy.