VHA Diagnostic Electron Microscopy Program
What Is an Electron Microscope (EM) and How Does It Work?
Here we compare two basic types of microscopes - optical and electron microscopes.
The electron microscope uses a beam of electrons and their wave-like characteristics to magnify an object's image, unlike the optical microscope that uses visible light to magnify images. Conventional optical microscopes can magnify between 40 to 2000 times, but recently what are known as "super-resolution" light microscopes have been developed that can magnify living biological cells up to 20,000 times or more. However, the electron microscope can resolve features that are more than 1 million times smaller.
Electron Microscopes (EMs) function like their optical counterparts except that they use a focused beam of electrons instead of photons to "image" the specimen and gain information as to its structure and composition.
The basic steps involved in all EMs:
- A stream of high voltage electrons (usually 5-100 KeV) is formed by the Electron Source (usually a heated tungsten or field emission filament) and accelerated in a vacuum toward the specimen using a positive electrical potential.
- This stream is confined and focused using metal apertures and magnetic lenses into a thin, focused, monochromatic beam.
- This beam is focused onto the sample using a magnetic lens.
- Interactions occur inside the irradiated sample, affecting the electron beam.
- These interactions and effects are detected and transformed into an image.
At the end of the 19th Century, physicists realized that the only way to improve on the light microscope was to use radiation of a much shorter wavelength. J.J. Thompson in 1897 discovered the electron; others considered its wave-like properties. In 1924, Louis deBroglie demonstrated that a beam of electrons traveling in a vacuum behaves as a form of radiation of very short wavelength, but it was Ernst Ruska who made the leap to use these wave-like properties of electrons to construct the first EM and to improve on the light microscope.
Today there are two major types of electron microscopes used in clinical and biomedical research settings: the transmission electron microscope (TEM) and the scanning electron microscope (SEM); sometimes the TEM and SEM are combined in one instrument, the scanning transmission electron microscope (STEM):
- TEM: magnifies 50 to ~50 million times; the specimen appears flat
- SEM: magnifies 5 to ~ 500,000 times; sharp images of surface features
- STEM: magnifies 5 to ~50 million times; the specimen appears flat
In the TEM, the electrons from the electron gun pass through a condenser lens before encountering the specimen, close to the objective lens. Most of the magnification is accomplished by the objective lens system. The image is viewed through a window at the base of the column and photographed using film, or more recently a CCD camera, by raising the hinged fluorescent viewing screen.
In the SEM, electrons from the electron gun are focused to a fine point at the specimen surface by means of the lens system. This point is scanned across the specimen under the control of currents in the scan coils situated within the final lens. Low voltage secondary electrons are emitted from the specimen surface and are attracted to the detector. The detector relays signals to an electronic console, and the image appears on a computer screen.
Sometimes x-rays are detected and used to display the atomic elements within specimens. This can be very useful in analyzing the cellular or sub-cellular elemental content of tissues. TEMs and SEMs equipped with x-ray detectors are referred to as Analytical Electron Microscopes (AEMs); analyses using such instruments are described by various terms, for example electron probe x-ray microanalysis (EPMA or EPXMA) or energy dispersive x-ray analysis (EDX).
Tomographic (3-Dimensional or 3-D) images can be obtained by tilting and/or rotating the specimen while acquiring an image. Recent developments in slicing very thin sections of tissues, and imaging the face of the block of tissue, have enabled high resolution sub-cellular 3D images to be obtained.