The invention of the Electron Microscope has taken observation on a microscopic scale to a whole new level. In this blog, I explore the functioning of a Scanning Electron Microscope (SEM), one of the two most common types of electron microscopes.
Why Use an Electron Microscope At All?
The laws of physics impose a certain holy limit on the resolution of instruments we use for observation.
For instance, if we’re using light of a particular wavelength to see things, we just cannot use such light to observe things which are smaller than its wavelength.
The wavelength of the visible spectrum of light ranges from about 400 nm on the violet end to about 800 nm on the reddish end.
Thus, with visible light used to see things in an optical microscope, we just cannot observe things smaller than, say 400 nm if we’re using violet light.
It is when we want to see things smaller than that do we need to look to other methods. Basically, we would require to use some other form of wave with a smaller wavelength to see smaller things.
Now, on the level of electron, neutrons and protons – the so-called “quantum level”, an entirely different set of physical laws operate; laws which defy our intuitive sense built on a macroscopic world. One of the spooky consequences of such eerie laws is that a stream of electrons (or of any other particles of that size) behaves as a wave!
Using the laws of quantum physics, if we calculate, we find that the wavelength of such a “wave” of electrons would be only 1.23 nm. See how small that is!
Hence, we can use electrons to see things as small as 1.23 nm. With visible light, there was no way we could see things any smaller than about 400 nm. That is a huge difference, which is the reason why electron microscopes are so powerful.
They allow us to see tiny features – features tinier than the wavelength of light, which we could never have seen with an optical microscope.
What’s a Scanning Electron Microscope?
As the name implies, an SEM works with a beam of electrons that scans across the sample to be analyzed. The signals received are then analyzed, and that’s all it takes to build up an image of the sample.
Since we’re using an electron beam (and not a light beam, as we ordinarily do), some special precautions need to be taken to ensure that the sample itself does not interfere with the electron beam and therefore affect imaging.
1. Correct Size
To begin with, we need to ensure that the specimen is of the correct size, so that it can be mounted on the specimen stub.
2. Electrically Conductive
An SEM Sample is required to be electrically conductive. There is a specific reason for this.
An SEM works on the principle of analyzing both:
- The reflected electrons from its incident electron beam
- Also, the electrons emitted by the atoms of the specimen
Hence, the atoms of the sample should have enough free electrons to be emitted for the detectors on the SEM to detect them as a measurable signal and thus render the image. When a sample is electrically conductive, it means that it has a large number of free electrons. This makes its emission of electrons strong, rendering a clearer image.
3. Electrically Grounded
To prevent the buildup of electric charge on the surface of the sample – which affects imaging since electron beams are being used, the sample is required to be electrically grounded.
Preparation of the Sample
Quite naturally, before the sample is imaged, it needs to be adequately prepared to ensure that it meets these requirements.
As such, metallic objects not require much special preparation. The only procedure is the conventional cleaning of the sample, which is characteristic for optical microscopes too.
However, for the scientist operating an SEM, life isn’t so easy with non-metallic objects. Since they have no free electrons to reflect back, their surface has to be coated with an electrically conductive metal. Various metals like gold, osmium, iridium, tungsten, and graphite are used for this purpose.
However, this process may sometimes be difficult because coating certain samples, especially biological samples, is far from convenient.
The Scanning Process: Imaging the Sample
We are aware that the imaging medium for an SEM is a flow of electrons. So, the first obvious step would be to generate an electron beam.
The process used to generate the electron beam is very much similar to those big, bulky CRT monitors that were once used for your PC.
The place where the electrons are created is known as the electron gun. It consists of a tungsten filament. Tungsten is used because it has a very high boiling point and a negligible vapour pressure.
The tungsten filament is heated by passing electricity through it. This imparts extra energy to its free electrons, which help them escape from the atoms.
The particular amount of energy required for this process per electron varies from metal to metal. Once again, tungsten is well-suited for this purpose because said energy for tungsten is comparatively low.
The emitted electron beam may have an energy ranging from anything between 0.2 keV to 40 keV. The wide stream of electrons so produced has to be focused into a beam.
That is done by 2 pairs of lenses known as condenser lenses – which are basically positively charged plates that electrostatically deflect the electrons into a narrow beam.
Unfortunately, when the need arises, you can’t use an optical lens to bend an electron beam. Therefore, the “lenses” used for focusing electron beams are magnetic and electric fields, since the electron, a charged particle having its own magnetic field, interacts with both of these.
The job of the third lens, the objective lens, is to make the electron beam follow a raster scan pattern. This is done by appropriately varying the electric and magnetic fields on this lens to make the beam follow this pattern.
In a raster scan pattern, the electron beam scans the first pixel in the first row, then the second, then the third, and so on, until it is done with all the pixels in that row.
Next, it moves on to the second row, and scans it. Once done with the second row, it moves on to the next row, and so on. It repeats this sequence until it has scanned every point in the field of view.
Once focused, the electron beam converges at a point which can be 0.4 to 5.0 nanometres in size. The electron beam falls on each point, and certain electrons, along with electromagnetic radiation, is reflected back.
There are chiefly 3 types of signals that come back to the detectors:
- Backscattered High-Energy Electrons: Some of the electrons from the electron beam that had struck the sample are reflected back. They are characterised by high energy, which was characteristic of the electron beam, and a shallow reflection angle.
- Emitted Secondary Electrons: When the electron beam falls on the specimen, it imparts some energy to its free electrons. This extra energy helps them escape their respective atoms and thus get emitted. These electrons are detected as the emitted secondary electrons.
- Electromagnetic Radiation: Instead of being emitted, some of these excited electrons fall back to their initial energy state, emitting the excess energy as a pulse of electromagnetic radiation. This too is detected as a blip of X-rays.
Certain electrons are also absorbed by the specimen. These may also be taken into account while rendering an image of the specimen.
All these three kinds of reflection are analyzed by the backscatter electron detector, secondary electron detector, and the X-Ray Detector respectively. Of course, the electronic signals thus generated in these detectors is too small, and therefore, is first amplified.
The detectors are designed to detect the direction and energy of these electrons and electromagnetic radiation, and hence reconstruct its source by back-calculation. Thus, we get a distribution map of the signal for the entire sample. The image is then rendered accordingly.
An SEM can allow a magnification ranging from as basic as 10x to anything as extraordinary about 50,000x!
(False) Colour: A Means of Understanding
Electron Microscope images are rendered in grayscale, since the image is a proportional rendering of the signal present on each pixel.
However, EM images may be artificially colorized later while post-processing, due to a variety of purposes – most commonly ease of understanding/interpreting the image.
- Contour/Feature Plotting Software: Certain automated software may be used to map contours or features, and colour them according to the user’s choice.
- Special Purpose Software: Often, a colour is assigned to the image created from the three types of detectors, so that when the three inputs are combined, the colour-coding helps determine the source of each part of the image. Similar such colour characteristics may be added to various types of attributes, usually for scientific analysis later.
- Post-Processing Software: Photoshop and paint-brush tools are always there! In these cases, adding colour is all about aesthetics, and giving the image a real feel.