INTRODUCTION TO MICROSCOPY Microscopes are instruments designed to produce magnified visual or photographic images of objects too small to be seen with the naked eye. The microscope must accomplish three tasks: produce a magnified image of the specimen, separate the details in the image, and render the details visible to the human eye or camera. This group of instruments includes not only multiple-lens (compound microscopes) designs with objectives and condensers, but also very simple single lens instruments that are often hand-held, such as a loupe or magnifying glass. The microscope that was invented by British microscopist Robert Hooke sometime in the 1660s is a simple compound microscope. This beautifully crafted microscope has an objective lens near the specimen and is focused by turning the body of the microscope to move the objective closer to or farther from the specimen.
An eyepiece lens is inserted at the top of the microscope and, in many cases, there is an internal “field lens” within the barrel to increase the size of the viewfield. The microscope is illuminated through the oil lamp and water-filled spherical reservoir. Light from the lamp is diffused when it passes through the reservoir and is then focused onto the specimen with a lens attached to the reservoir. This early microscope suffered from chromatic and spherical aberration, and all images viewed in white light contained “halos” that were either blue or red in color. More than five hundred years ago, simple glass magnifiers were developed. These were convex lenses (thicker in the center than the periphery). The specimen or object could then be focused by use of the magnifier placed between the object and the eye. These “simple microscopes” could spread the image on the retina by magnification through increasing the visual angle on the retina.
The “simple microscope” or magnifying glass reached its highest state of perfection, in the 1600’s, in the work of Anton von Leeuwenhoek who was able to see single-celled animals (which he called “animalcules”) and even some larger bacteria with a simple microscope. The image produced by such a magnifier, held close to the observer’s eye, appears as if it were on the same side of the lens as the object itself. Such an image, seen as if it were ten inches from the eye, is known as a virtual image and cannot be captured on film. Around the beginning of the 1600’s, through work attributed to the Janssen brothers in the
Compound microscopes developed during the seventeenth and eighteenth centuries were hampered by optical aberration (both chromatic and spherical), a flaw that is worsened by the use of multiple lenses. These microscopes were actually inferior to single lens microscopes of the period because of these artifacts. The images they produced were often blurred and had the colorful halos associated with chromatic aberrations that not only degrade image quality, but also hamper resolution. In the mid 1700’s lens makers discovered that by combining two lenses made of glass with different color dispersions, much of the chromatic aberration could be reduced or eliminated. This discovery was first utilized in telescopes, which have much larger lenses than microscopes. It wasn’t until the start of the 1800’s that chromatically corrected lenses became commonplace in compound microscopes. The eighteenth and nineteenth centuries witnessed a great improvement in the mechanical and optical quality of compound microscopes. Advances in machine tools allowed more sophisticated parts to be fabricated and, by the mid 1800’s, brass was the alloy of choice for the production of high-quality microscopes. A number of British and German microscope manufacturers flourished during this time period. Their microscopes varied widely in design and production quality, but the overall principles defining their optical properties remained relatively constant. The microscope illustrated in Figure 5 was manufactured by Hugh Powell and Peter Lealand about 1850. The tripod base provided a sturdy support for the microscope, which many people consider the most advanced of its period. By the end of the nineteenth century, there was a high degree of competition among microscope manufacturers and the development and production costs of microscopes became an important factor. Brass, the material of choice for microscope manufacturers, is very expensive and it was a lengthy task to machine, polish, and lacquer microscope bodies and other parts machined from this metal. To cut expenses, microscope manufacturers first started to paint the exterior portion of the microscope body and stand, as well as the stage and other non-moving parts. During the first quarter of the twentieth century, many microscope manufacturers had begun substituting cast iron for brass in microscope frames and stages. Iron was much cheaper and could be not be distinguished from brass when painted black. They also started to electroplate many of the critical brass components such as knobs, objective barrels, nosepieces, eyepieces, and mechanical stage assemblies. These early twentieth century microscopes still subscribed to a common design motif. They were monocular with a substage mirror that was used with an external lamp to illuminate the specimen. A typical microscope of the period is the Zeiss Laboratory microscope. This type of microscope is very functional and many are still in use today. Modern microscopes far exceed the design specifications of those made prior to the mid 1900’s. Glass formulations are vastly improved allowing greater correction for optical aberration than ever before, and synthetic anti-glare lens coatings are now very advanced. Integrated circuit technology has allowed manufacturers to produce “smart” microscopes that incorporate microprocessors into the microscope stand. Photomicrography in the late twentieth century is easier than ever before with auxiliary attachments that monitor light intensity, calculate exposure based on film speed, and automatically perform complicated tasks such as bracketing, multiple exposure, and time-lapse photography.