A microscope is a device that magnifies small objects. The first microscope was developed about 1595 and was only as strong as a magnifying glass. Today, there are dozens of microscopes capable of letting us see and analyze objects as small as atoms!
Martin A. Levin
Use of Microscopes
When most people think of microscopes, biology and medicine probably come to mind. And the desire to learn about living things was most likely the main reason for the invention of the microscope. Today, however, microscopes are used in many other fields.
For example, geologists use microscopes to examine rocks and minerals and materials scientists use them to study plastics and polymers. Engineers use microscopes to study surface properties and structures of metals.
Forensic science is the study of crime scenes for the purpose of presenting evidence in courts of law. Evidence such as dust, glass, body fluids, hair, inks, and micro-organisms can be analyzed using microscopy.
Microscopes are also used in the service, manufacturing, and pharmaceutical industries to ensure the safety and quality of products. Scientists in these industries examine their products microscopically to identify any flaws or contaminants.
Common Light Microscopes
Light microscopes, also called optical microscopes, use light and lenses to magnify images. If light waves reflected off an object are passed through a lens in a certain way, they will refract, or bend. The refracted light waves are spread out and appear to be coming from a bigger object. The simplest light microscope is the magnifying glass. It is a single lens that can magnify an image up to 25 times.
The compound light microscope (also called a brightfield microscope) is the type found in most classrooms. All compound microscopes contain two magnifying lenses and work similarly. Visible light from the base passes through a condenser, through the specimen, and then into the objective lens, forming a magnified primary image. The primary image is further magnified as it passes through the ocular lens. The final image is projected onto the retina of the eye. The total magnification is calculated by multiplying the magnifying power of the objective lens by that of the ocular lens. Most microscopes give a choice of several objective lenses mounted on a rotating disc, each providing a different magnification.
A simple microscope, the stereoscopic (dissecting) microscope, is useful for viewing objects too small to be studied with the eye alone, but too large to be seen with a compound light microscope. A specimen is placed on a stage, or platform, and illuminated from above, producing an image similar to that seen with a magnifying glass. Magnifications rarely exceed 50 times.
To be viewed by a compound light microscope, a specimen must be translucent (thin enough to permit the passage of light). Microorganisms in a drop of water are thin enough to view, but what about a piece of liver? The liver must first be preserved and hardened using chemical procedures. It is then sliced very thin on a machine called a microtome, which is similar to a meat-slicing machine, and a thin slice is mounted on a glass slide. Sometimes a thin piece of glass, called a coverslip, is placed over a specimen to protect it and make it lie flat. A liver slice prepared this way is nearly colorless; therefore, the image will not be very detailed. A specimen must also have contrast to make structural details more visible.
Color must be added to provide contrast to many biological specimens. A common way to chemically add color is by using a stain, or dye. Different stains can be used to label distinct structures of the liver. Stains can be used to add color to living microorganisms as well. Some stains are toxic to living organisms, but there are a few special stains called vital dyes that add color without killing living cells.
Resolution and Magnification
Some particles, such as viruses, are too small to be seen with a compound microscope. Even if one built a light microscope capable of magnifying 1 million times, viruses would still be invisible. Why? Because viruses are smaller than the average wavelength of visible light.
Every optical instrument has a resolving power, defined as the ability to distinguish closely spaced objects and create a clear image. The resolving power depends on the wavelength of whatever produces the image (white light in the case of a light microscope) and the ability of the objective lens to capture the image-forming rays as they leave the specimen.
Resolving power limits the magnification of a microscope. If an object is too small to be seen by a particular microscope, increasing the magnification will be of no use. An object that is smaller than the wavelength of light cannot be seen with an ordinary light microscope. The maximum usable magnification for an average compound microscope is about 1,500 times. If an ocular lens with a magnification of 10 times (10X) is used in combination with a 10X, 20X, or 40X objective lens, the final magnification for each view would be 100X, 200X, and 400X, respectively. As the magnification increases, the objective lens captures fewer image-forming rays, and the magnified image becomes less distinct.
Magnifications above 400X require a different kind of objective lens to capture more of the image-forming rays. This is an oil immersion lens. A drop of oil is placed on the coverslip over the specimen, and a 100X oil immersion lens is carefully lowered into the oil. The oil acts as another lens, directing more image-forming rays into the 100X objective lens and producing a clear image (and better resolution) at 1000X.
Some light microscopes obtain higher resolutions by changing the wavelength of the light used to create the image or the ability of the objective lens to capture the image-forming rays (see the section Other Light Microscopes below). To see very small objects like viruses, drastic changes in the way the image is created are required (see the section Electron Microscopes below).
Other Light Microscopes
Some microscopes are designed to compensate for the problems of contrast and resolution that limit the function of the compound microscope. For example, the phase contrast microscope makes it easier to see living cells without staining them. This microscope processes light rays differently so that uncolored specimens appear colored. A compound microscope can be converted into a phase contrast microscope by changing the lenses and a few other parts. Phase contrast lenses are expensive and are not used in most school laboratories.
A dark field microscope can be constructed by placing an opaque disc underneath the condenser of a compound microscope to block the passage of light through the specimen. A powerful light directed at the specimen from above is reflected back from the specimen into the objective lens, producing an illuminated object on a dark background. Dark field microscopy is ideal for looking at small organisms suspended in a liquid.
The laser scanning confocal microscope was created for specialized use in research, hospital, and industrial laboratories. It uses a laser beam to increase the amount of light entering the objective lens and to scan different layers of the specimen. This produces two-dimensional, layered images that can be fed into a computer to produce a three-dimensional reconstruction of the specimen.
The fluorescence microscope uses ultraviolet light to create contrast. Specimens treated with special dyes emit visible light when exposed to ultraviolet radiation. The fluorescence microscope produces high-contrast images that are used to diagnose infections caused by many types of microorganisms.
Another light microscope is the stimulated emission depletion microscope. Through the use of lasers, this microscope allows the viewer to see cellular features one-eighteenth the size that can be observed using standard light microscopes. Although its resolving power is much less than an electron microscope, the stimulated emission depletion microscope is very useful because it can be used to view living tissue. The electron microscope is mainly limited to observing dead tissue.
German physicist Ernst Ruska knew that electrons traveled at much shorter wavelengths than light. In 1931, he built a microscope that produced an image by passing electrons through a very thin specimen. The shorter wavelength of electrons gave this transmission electron microscope (TEM) more resolving power, thereby increasing its usable magnification. Today, TEM's are capable of magnifications as high as 1 million times. Such magnification has permitted the internal structures of viruses and cells to be routinely studied in laboratories.
Just as the compound microscope passes light through a specimen to create an image, the TEM does the same with electrons. But neither microscope can observe details on the surface of an object. To view surface details, it is necessary to use a scanning electron microscope (SEM), which can show tiny surface features on biological and non-biological specimens by passing a beam of electrons across the surface of a specimen rather than through it.
The TEM and SEM are by far the most commonly used electron microscopes. However, there are other types. For example, the scanning transmission electron microscope can simultaneously provide highly detailed images and molecular information about the specimen.
In 1986, the Nobel Prize was awarded to Ernst Ruska for his design of the first electron microscope, and to Gerd Binnig and Heinrich Rohrer for the design of the scanning tunneling microscope (STM). The STM uses a very fine, pen-like electrode to scan a small area of a specimen's surface at a close distance to produce three-dimensional images of individual atoms. The atomic force microscope measures atomic-level forces between a sharp probe and electrons on the surface of a specimen. These measurements can be used to construct a highly detailed image of the surface of an object.
An atom-probe field ion microscope can simultaneously create images and provide chemical data of atoms on metal surfaces. When a sharp needle made of the metal specimen is electrically heated in a helium-filled chamber, helium atoms bouncing off the needle tip form a pattern on a television screen that can be quantified by an atom probe.
History of Light Microscopes
The invention of the microscope was preceded by the use of lenses. In the first century AD, the Roman philospher Seneca described the magnification produced by "a globe of glass filled with water." Between AD 23 and 79, Pliny the Elder, a famous Roman scholar, described the use of an emerald by Emperor Nero to better view the combat of the gladiators. However, it was the widespread use of lenses for eyeglasses in the 1400's that paved the way for the invention of the microscope.
About 1595, a young Dutchman named Zacharias Jansen and his father Hans invented the first microscope. This microscope was a long brass tube with two lenses and a maximum magnification of 9X. Within a few dozen years, there were several microscope manufacturers, including Galileo, who is more famous for his work with telescopes.
The early microscopes were good for observing insect parts, but they were too crude to view smaller objects. Then in the 1660's, Robert Hooke of England viewed smaller objects with an instrument built by Christopher Cock. While looking at thin slices of cork, Hooke used the term "cell" to describe the small air spaces he observed. In 1665, Hooke published Micrographia, one of the first books demonstrating the importance of microscopy.
Anton van Leeuwenhoek, an amateur scientist from Holland, became fascinated with Hooke's pictures of magnified textiles. He began making incredible discoveries using a tiny single-lens microscope of his own design. In 1673, van Leeuwenhoek began writing letters about his discoveries to the Royal Society of London. Throughout his life, he wrote letters describing the specimens he viewed with his microscope, which included bee mouthparts and stingers, human lice, algae, protozoa, blood cells, microscopic nematodes, bacteria, and spermatozoa.
Gradually, improvements continued to be made to the design of the microscope. Microscopes became increasingly stable and easy to use, but the images were still blurry (spherical aberration) with colorful halos around objects (chromatic aberration). An achromatic lens for the microscope was designed in 1800, and spherical aberration was fixed in 1830. Through trial and error, improvements were made to objective lenses. The relationship between resolution, wavelength, and how much of the image is collected by the objective lens was not completely understood until German physicist Ernst Abbe published his resolution formula in 1877.