It’s been said that “we live in the middle of infinity.” The endless boundaries of the universe are apparent to us when we hear astronomers speak about various stars and other celestial objects being “light years” away from us.
However, if we look into the smaller worlds that make up the tiny things that can be held in our hand, we can soon find out that changes in order of magnitude in the “tiny things” can be just as dramatic as they are in our understanding of the universe.
With advanced microscopes, and through the study of atomic particles we have learned that smaller, and smaller particles make up the familiar objects we live with from day to day. As we look deeper into that single plant cell, we find structures inside the cell that help it live, and within those structures we find large protein molecules, and within those protein molecules we find individual atoms, and within the atoms we find the basic atomic particles, protons, electrons, and neutrons. Atomic physicists have also discovered tinier, sub-atomic particles like quarks, and mesons, and others. It seems that every time we develop a technology that allows us to see something on a smaller level, we discover that it’s made up of even smaller building blocks. It seems like we are “living in the middle of infinity”.
It’s only through the use of instruments like magnifying glasses, optical telescopes, radio telescopes, microscopes, electron microscopes and various other devices that we’ve come to understand our universe as well as we do now.
So, what is a microscope?
A microscope (from the Greek: μικρός, mikrós, “small” and σκοπεῖν, skopeîn, “to look” or “see”) is an instrument to see objects too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope.
An early microscope was made in 1590 in Middelburg, Netherlands. Two eyeglass makers are variously given credit: Hans Lippershey (who developed an early telescope) and Hans Janssen. Giovanni Faber coined the name for Galileo Galilei’s compound microscope in 1625. (Galileo had called it the “occhiolino” or “little eye”.)
The first detailed account of the interior construction of living tissue based on the use of a microscope did not appear until 1644, in Giambattista Odierna’s L’ochio della mosca, or The Fly’s Eye.
It was not until the 1660s and 1670s that the microscope was used seriously in Italy, Holland and England. Marcelo Malpighi in Italy began the analysis of biological structures beginning with the lungs. Robert Hooke’s Micrographia had a huge impact, largely because of its impressive illustrations. The greatest contribution came from Antoni van Leeuwenhoek who discovered red blood cells and spermatozoa. On 9 October 1676, Leeuwenhoek reported the discovery of micro-organisms.
The most common type of microscope—and the first invented—is the optical microscope. This is an optical instrument containing one or more lenses producing an enlarged image of an object placed in the focal plane of the lenses.
There are several types of microscopes.
An electron microscope is a type of microscope that produces an electronically-magnified image of a specimen for detailed observation. The electron microscope (EM) uses a particle beam of electrons to illuminate the specimen and create a magnified image of it. The microscope has a greater resolving power (magnification) than a light-powered optical microscope, because it uses electrons that have wavelengths about 100,000 times shorter than visible light (photons), and can achieve magnifications of up to 1,000,000x, whereas light microscopes are limited to 1000x magnification.
The electron microscope uses electrostatic and electromagnetic “lenses” to control the electron beam and focus it to form an image. These lens are analogous to, but different from the glass lenses of an optical microscope that form a magnified image by focusing light on or through the specimen.
Electron microscopes are used to observe a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, the electron microscope is primarily used for quality control and failure analysis in semiconductor device fabrication.
Electron microscope constructed by Ernst Ruska in 1933.
In 1931, the German physicist Ernst Ruska and German electrical engineer Max Knoll constructed the prototype electron microscope, capable of four-hundred-power magnification; the apparatus was a practical application of the principles of electron microscopy. Two years later, in 1933, Ruska built an electron microscope that exceeded the resolution attainable with an optical (lens) microscope. Moreover, Reinhold Rudenberg, the scientific director of Siemens-Schuckertwerke, obtained the patent for the electron microscope in May of 1931. Family illness compelled the electrical engineer to devise an electrostatic microscope, because he wanted to make visible the poliomyelitis virus.
In 1937, the Siemens company financed the development work of Ernst Ruska and Bodo von Borries, and employed Helmut Ruska (Ernst’s brother) to develop applications for the microscope, especially with biologic specimens. Also in 1937, Manfred von Ardenne pioneered the scanning electron microscope. The first practical electron microscope was constructed in 1938, at the University of Toronto, by Eli Franklin Burton and students Cecil Hall, James Hillier, and Albert Prebus; and Siemens produced the first commercial Transmission Electron Microscope (TEM) in 1939. Although contemporary electron microscopes are capable of two million-power magnification, as scientific instruments, they remain based upon Ruska’s prototype.
Transmission electron microscope (TEM)
The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron beam to create an image. The electrons are emitted by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode typically at +100 keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the “image”) is viewed by projecting the magnified electron image onto a fluorescent viewing screen coated with a phosphor or scintillator material such as zinc sulfide.
The image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or a high-resolution phosphor may be coupled by means of a lens optical system or a fibre optic light-guide to the sensor of a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed on a monitor or computer.
Resolution of the TEM is limited primarily by spherical aberration, but a new generation of aberration correctors have been able to partially overcome spherical aberration to increase resolution. Hardware correction of spherical aberration for the High Resolution TEM (HRTEM) has allowed the production of images with resolution below 0.5 Ångström (50 picometres) at magnifications above 50 million times. The ability to determine the positions of atoms within materials has made the HRTEM an important tool for nano-technologies research and development.
Scanning electron microscope (SEM)
Unlike the TEM, where electrons of the high voltage beam carry the image of the specimen, the electron beam of the Scanning Electron Microscope (SEM)[8] does not at any time carry a complete image of the specimen. The SEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen (raster scanning). At each point on the specimen the incident electron beam loses some energy, and that lost energy is converted into other forms, such as heat, emission of low-energy secondary electrons, light emission (cathodoluminescence) or x-ray emission. The display of the SEM maps the varying intensity of any of these signals into the image in a position corresponding to the position of the beam on the specimen when the signal was generated. In the SEM image of an ant shown at right, the image was constructed from signals produced by a secondary electron detector, the normal or conventional imaging mode in most SEMs.
Generally, the image resolution of an SEM is about an order of magnitude poorer than that of a TEM. However, because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to many centimetres in size and (depending on instrument design and settings) has a great depth of field, and so can produce images that are good representations of the three-dimensional shape of the sample.
Reflection electron microscope (REM)
In the Reflection Electron Microscope (REM) as in the TEM, an electron beam is incident on a surface, but instead of using the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons is detected. This technique is typically coupled with Reflection High Energy Electron Diffraction (RHEED) and Reflection high-energy loss spectrum (RHELS). Another variation is Spin-Polarized Low-Energy Electron Microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domains.
Scanning transmission electron microscope (STEM)
The STEM rasters a focused incident probe across a specimen that (as with the TEM) has been thinned to facilitate detection of electrons scattered through the specimen. The high resolution of the TEM is thus possible in STEM. The focusing action (and aberrations) occur before the electrons hit the specimen in the STEM, but afterward in the TEM. The STEMs use of SEM-like beam rastering simplifies annular dark-field imaging, and other analytical techniques, but also means that image data is acquired in serial rather than in parallel fashion.
Low voltage electron microscope (LVEM)
The low voltage electron microscope (LVEM) is a combination of SEM, TEM and STEM in one instrument, which operates at relatively low electron accelerating voltage of 5 kV. Low voltage increases image contrast which is especially important for biological specimens. This increase in contrast significantly reduces, or even eliminates the need to stain. Sectioned samples generally need to be thinner than they would be for conventional TEM (20-65nm). Resolutions of a few nm are possible in TEM, SEM and STEM modes.
How to choose the best microscope.
Before discussing the specific varieties of microscopes on the market, there are two important features to become familiar with before going shopping—a microscope’s light source and magnification range.
Microscope Light Source: “Bargain” scopes will often have a mirror, or very small bulb, as a light source. Light is necessary for observing a specimen with a microscope. The amount of light determines the level of contrast between the object and the background. Too little light and you can’t see the specimen. Too much light and the specimen becomes washed out, and equally difficult to see. My recommendation is to stay away from cheap microscopes that use mirrors to illuminate objects. It is difficult to get sufficient light for a good image.
Microscope Magnification: Dissecting, or stereo, microscopes are designed to magnify objects that can already be seen with the naked eye, allowing the observer to discern additional detail. These are low magnification scopes with a range of approximately 10x to 40x actual size (with “x” meaning times). Microscopes with the ability to provide low levels of magnification are great for little kids who want to get a better look at things that they encounter in the world around them, such bugs, leave, flowers and other small objects.
Compound microscopes provide two sets of lenses, that together deliver high magnification, generally from 40x to 1000x, depending on the specific lenses that they come with. These high magnification scopes are great for seeing items that are not visible to the naked eye, and allow users to see essentially invisible things, such as bacteria, the details of pollen grains—tiny stuff. And high magnification scopes provide more great details.