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August 26, 2013

Birth of the Electron Microscope Part 3: The Scan

The creation of the transmission electron microscope (TEM) was a revolution in the field of microscopy; for the first time, it allowed humans to see things that were too small for traditional light-based microscopes to resolve, such as individual cells and large atomic molecules, by exposing samples to a beam of electrons instead of a beam of light. However, the TEM had limitations of its own; it could only resolve an image if the sample was thin enough for electrons to pass through, so biological samples had to be preserved and sliced up, destroying any potential for viewing the minute changes in a living organism and making it impossible to view a complete image of the specimen. TEM also suffered from diffraction issues, as the electron beam could only resolve to a certain magnification level before the electrons scattered too much to form a definite image.

Shortly after the TEM’s 1931 debut, a Russian scientist named Manfred von Ardenne invented a true electron-based microscope that worked on a slightly different principle, and patented the Scanning Electron Microscope (SEM) in 1937. This machine finally enabled scientists to see complete specimens in high detail, and resolve three-dimensional shapes. Instead of relying on a beam of electrons to carry the image away from the specimen, the scanning electron microscope works by scanning the beam across the specimen in a series of rectangular areas. This technique is known as raster scanning, and it is common in computer graphics; it’s how printers create images on paper, and how older CRT televisions created their images. When an SEM scans a specimen, the electron beam loses energy; this energy is converted into heat, scattered electrons, X-rays, and light emission. The SEM’s lenses can detect this energy, and it maps these signals into an image based on where the electron beam was located when it lost that particular amount of energy. By scanning in this manner, an SEM can resolve specimens as three-dimensional shapes.

The specimens in an SEM must be electrically conductive, in order to attract the electrons in the first place. While metals require very little preparation, non-conductive specimens must be coated with a very thin layer of gold, platinum, or tungsten. The SEM uses an electron gun much like the TEM, and uses a tiny cathode of tungsten at its tip. The SEM also requires the specimen to sit in a vacuum, in order to prevent interference from artifically disrupting the electron beam.

There are other types of electron microscopes, but the SEM was a major breakthrough because it allowed researchers to capture minute details of things like a house fly’s eye, a snowflake, or an ant’s head. Special environmental SEMs can observe samples that are in low-pressure environments (rather than complete vacuums) and do not require biological materials to be coated in gold. It is highly useful for seeing biological specimens, even scanning still-living insects.

May 23, 2013

Electron Microscopy – Part 1

In today’s modern era, it’s almost impossible to imagine a time when we didn’t know about microscopic particles and structures; magnification is a fundamental part of everyday life, from a pair of reading glasses to the side mirrors on a car. With the birth of the optical microscope in the 17th century, scientists were able to meticulously document, research, and discover a world of things normally invisible to the naked eye. It seemed as though the wonders of the microscopic was infinite – until the 1930s, when it became clear that optical microscopy wasn’t telling the entire story. There were forces at work that were far too small for even the most powerful lenses to see; at some point, the image simply couldn’t resolve clearly. It seemed as though optical microscopes had been pushed to their furthest possible limits. But how could something be too small for light to see, and how could you possibly examine it?

This set of problems was the catalyst for scientists to begin branching out from light-based microscopy. The theoretical framework explaining the limits of light-based magnification were already in place. The issue lay in understanding how light worked, and why it failed at certain magnifications. Visible light is electromagnetic radiation, part of a wide spectrum of rays that stretches from radio waves to X-rays, microwaves, gamma rays, and more. All EM Radiation is made of particles called photons, which travel in a wavelength; the speed and frequency of that wave will determine what sort of radiation it is, and how it interacts with various objects. X-rays, for instance, have a wavelength that is too short to bounce off of human flesh, but does reflect dense material like bone, allowing the doctor to clearly see your broken wrist. UV radiation can penetrate the skin enough to cause a sunburn, but we can’t see the rays with our eyes.

We are able to see objects because visible light bounces off of them and returns to our eyes. When that wavelength encounters an object, like a sample on a slide or the magnifying lenses of a microscope, the interference will cause the wave to spread out and weaken. As the light passes through a smaller and smaller space, it will scatter more and more. This phenomenon is called diffraction, and it is the major limit of optical microscopy: at a certain point, the photons are just too big and clunky to accurately bounce off of the sample and resolve clearly.

All of this was proven in 1873, when physicists Hermann von Helmholtz and Ernst Abbe demonstrated that optical resolution was dependent on the wavelength of the light source. They posed the crucial question: what if you could somehow use an illumination source that had a smaller wavelength than light? It was purely theoretical; the existence of electrons wasn’t proven until 1896. But the idea stayed around in various scientific circles; the theory that electrons could travel in a wave was proposed in a 1924 paper, and in 1926 German scientist Hans Busch showed that magnets could be used to direct a stream of electrons in a specific direction. The problems were in place and the theories were quickly being proven: it wouldn’t be long before scientists broke through the limits of light and found a way to see the invisible all over again.