What is the difference in the way light microscopes and electron microscopes produce an image?

Video Transcript

The micrograph provided is of a group of human liver cells. Which type of microscope is most likely to have been used to produce this image? (A) Light microscope, (B) transmission electron microscope, or (C) scanning electron microscope.

Microscopy is a very important development in biology. Microscopes allow us to view things that are much smaller than the naked eye can see. They magnify things or make them look bigger than they really are.

There are three main kinds of microscopes: light microscopes, transmission electron microscopes, and scanning electron microscopes. Let’s look at how each one works to determine which one was most likely used to produce this image.

Here is a diagram of the path that light follows in a light microscope. A light source illuminates the specimen, the object you want to have a look at, from underneath. The light then travels through the objective lens and the eyepiece lens and into the eye of the viewer. The objective lens and the eyepiece lens both magnify the image of the specimen.

To calculate how much the specimen is magnified in total, we need to know what the magnification of the two lenses are. The total magnification can then be calculated by multiplying the two magnifications of the lenses. For example, if the eyepiece lens magnifies the specimen 10 times and the objective lens magnifies the specimen 20 times, then we calculate 10 times 20 equals 200. And we know that the specimen we see appears 200 times bigger than it actually is.

The observer who is using a light microscope sees a color image. Light microscopes are often used to visualize cells. Light microscopes have a limit of how much they can magnify an object by because of a factor called resolution. Resolution is the minimum distance between two objects at which both are still distinguishable. Since light microscopes use light as an illumination source, their resolution is limited by the wavelength of light. The wavelength of electrons used in electron microscopes is smaller. Thus, they can differentiate adjacent objects that are closer together.

Let’s now take a look at transmission and scanning electron microscopes. Transmission electron microscopes send beams of electrons through their target. This creates a black-and-white 2D image and is often used to differentiate organelles in a cell. Scanning electron microscopes bounce electrons off metal-coated specimens to look at fine details in the outer structure. It creates a 3D black-and-white image.

Now that we understand the principles involved in microscopy, let’s look at the image provided in the question again. It shows a two-dimensional image of the inside of the liver, showing some human liver cells, but doesn’t let us resolve different organelles. It is in color. Therefore, a light microscope was most likely used. So the answer is (A), light microscope.

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NOTIFICATIONS

Electron microscopes were developed in the 1930s to enable us to look more closely at objects than is possible with a light microscope. Scientists correctly predicted that a microscope that used electrons instead of visible light as the illumination source could view objects at far higher resolution than a light microscope. This is because the wavelength of visible light is what limits the resolution of light microscopes, and the wavelength of electrons is far smaller.

Over time, specialised electron microscopes have been developed that can provide information about different aspects of an object being investigated. This means that scientists can choose the microscope that is most likely to answer their questions about their sample.

Nature of science

The kind of data that scientists can collect is heavily dependent on the tools that are available to them. As microscopes have become more sophisticated, scientists have been able to view objects in greater and greater detail. In turn, they have been able to answer new kinds of questions about the objects they are studying.

What is electron microscopy?

Electron microscopes use a beam of electrons rather than visible light to illuminate the sample. They focus the electron beam using electromagnetic coils instead of glass lenses (as a light microscope does) because electrons can’t pass through glass.

Electron microscopes enable us to look in far more detail at objects than is possible with a light microscope. Some electron microscopes can detect objects that are approximately one-twentieth of a nanometre (10-9 m) in size – they can be used to visualise objects as small as viruses, molecules or even individual atoms.

Unlike light microscopes, electron microscopes can’t be used to look directly at living things because of the special preparation that samples must undergo before they are visualised. Instead, electron microscopes aim to provide a high-resolution ‘snapshot’ of a moment in time within a living tissue.

I think the electron microscope has contributed more to science than any other scientific instrument that’s ever been invented.

Allan Mitchell, Microscopy Otago

Specialised forms of electron microscopy

Several types of electron microscope have been developed to help investigate different aspects of a sample.

The transmission electron microscope (TEM) was the first electron microscope to be developed. It works by shooting a beam of electrons at a thin slice of a sample and detecting those electrons that make it through to the other side. The TEM lets us look in very high resolution at a thin section of a sample (and is therefore analogous to the compound light microscope). This makes it particularly good for learning about how components inside a cell, such as organelles, are structured.

Electron tomography is a form of TEM that lets us see a three-dimensional view of the cell or tissue being studied. Seeing structures in three dimensions can make it much easier to understand how they relate to each other. Electron tomography can also give two-dimensional images at higher resolution than conventional TEM.

The scanning electron microscope (SEM) lets us see the surface of three-dimensional objects in high resolution. It works by scanning the surface of an object with a focused beam of electrons and detecting electrons that are reflected from and knocked off the sample surface. At low magnifications, entire objects (such as insects) viewed on the SEM can be in focus at the same time. That’s why the SEM is so good at generating three-dimensional images of lice, flies, snowflakes and so on.

CryoSEM is a specialised form of SEM that’s good for looking at things that contain moisture (such as plants or food). In cryoSEM, samples are frozen in liquid nitrogen before being viewed. This avoids the need for the complex preparation steps that are done before conventional SEM (largely to remove water from the sample). Scientists often choose cryoSEM because it gives a more accurate image of what the sample looked like before it was prepared for microscopy.

Electron backscatter diffraction (EBSD) is used to look in detail at the structure of minerals (such as those in rocks). Rather than being microscopes in their own right, EBSD detectors are add-ons to SEMs. After the electron beam is fired at the rock, the EBSD detects electrons that have entered the rock and been scattered in all directions. The pattern of scattering can tell scientists a lot about the structure of the mineral and the orientation of crystals within it.

Activity ideas

In Using shadows to build 3D images, students model how scientists interpret microscope data by using shadows of an object from different angles to build up a 3D image.

In Which microscope is best?, students learn about various types of microscopes and discover which microscope is best for a specific sample type.

Useful link

Learn more about the 1986 Nobel Prize in Physics, half of which was awarded to Ernst Ruska for the design of the first electron microscope.

Plant & Food Research (PFR) have a collection of images taken using a scanning electron microscope (SEM). The images were captured within the Plant & Food microscopy laboratory and curated and hand-coloured by PFR photographer Wara Bullot.

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