Imagining A Hidden World: The Light Microscope (1984)

[Music] [Music] The unaded eye is a powerful observational tool. But in 300 years since the invention of the microscope, we have learned of a world hidden within the very things that appear ordinary to our naked eyes. Pond water becomes under the microscope a teeming mass of microorganisms. The substance blood appears to be a uniform red liquid. It is in fact a clear fluid filled with tiny cells. All living things when enlarged show new levels of structure from leaf to cells to the tiny living particles they contain. [Music] This brass instrument made around 1900 shows all basic microscope parts. A design that hasn't changed in over 100 years. There is a mirror directing light into the microscope. A condenser lens used to focus light onto the specimen. And beneath the condenser, an iris that controls light entering the imaging system. A stage holds slides and above it most microscopes have a rotating turret with objective lenses easily replaced with modern objectives for better [Applause] imaging. Coarse and fine adjustments smoothly vary distance between objective and slide for this is how a microscope is focused. An eyepiece lens further magnifies the image beamed up from the objective and brings it to focus on the retina of a human eye. What microscope lenses do in effect is to optically move your eye right up to the slide. Of course, if you actually try this, you can't see microorganisms clearly because your eyes lens is unable to curve enough to focus at such close range. A tiny fish, by comparison, has great lens curvature. Through a minnow's eye, microorganisms look like [Music] this. The same kind of strong curvature is designed into microscope objective lenses. They focus close enough to give you a fisheye view. Compare objective lenses of different magnifications. A short 4x and a longer 20x. For a subject, a needle lit from behind. The low power 4x objective projects the needle's reversed image onto a plastic screen marked with a red circle showing the area that will be further magnified by the eyepiece. The 20X with stronger curvature focuses closer to the subject, projecting a wide cone that produces a larger but dimmer image. The part of the image sampled by the eyepiece appears highly magnified. However, with increased magnification, you must keep changing focus to see all aspects of the subject. At a quarter of a millimeter, parramium is about the smallest microorganism visible to the naked eye. Using a 40x objective and 10x eyepiece, parramium appears enlarged 400 times. With a 100x objective and a 10x eyepiece for a total magnification of 1,000, you can only see the pointed end of the parramium covered by waving cyia and nearby some rod-shaped bacteria. Light itself places a limit on any further magnification. For here we are looking at structures in the size range of the shortest wavelengths of light. For example, barely visible within the spinning bacteria are granules right at the wavelength limit. Although smaller structures are present, they are invisible, too small to affect light waves. Light may set an ultimate limit, but the condenser lens has a much more immediate effect on image quality. Before condensers were used, illumination was by mirror alone. In this kind of condenserless imaging system, leaf cells look grainy and structures blend together. Changing to a higher power objective gives no more information, just the same information enlarged. The problem here is poor resolution. The ability of an imaging system to separate or resolve details. Focusing light onto the leaf with a condenser improves resolution. However, the image now has a washed out appearance due to flare caused by light spilling in around the edges of the objective. Eliminating flare is the purpose of an iris that shapes the cone of light focused up from the condenser so that it just fills the lens. Looking in the microscope, flare vanishes as the field just begins to darken, producing a highresolution flare-free image. The plant's food making bodies resembling green jelly beans now show up clearly. When the iris is used to cut out flare contrast, how the subject stands out against its background improves. Further closing of the iris produces more contrast, but at the expense of resolution. This relationship between contrast and resolution controlled by the iris gives you some choices when viewing clear unstained specimens. A case in point, living in a lake near our laboratory are organisms that are so transparent they are virtually invisible under normal brightfield illumination. These strange creatures could only be seen by closing the iris beyond the normal amount in order to gain enough contrast to make structures like the eye and heart observable. Trading off resolution for contrast can be helpful when viewing a clear subject. However, with stained specimens, contrast is built in through color, and so the iris should be adjusted for best resolution. The use of biological stains to improve contrast and to differentiate one structure from another is largely responsible for our knowledge of the microscopic makeup of plants and animals. However, the process requires killing cells and slicing them into thin sections. An option is to study microscopic subjects live using special lighting techniques like this dramatic change in [Music] lighting. It's a technique called dark field illumination. In its simplest form, dark field is produced by blocking light from the objective lens with a black paper disc. The subject is lit by beams of light focused in from the sides. But the disc prevents light from shining directly into the objective, thus the dark background. Changing from bright field to dark field shows new features like the color of these clumps of bacteria and parramium's bluish nucleus. [Music] Many subjects are more interesting to look at in dark field. It is as though you had shrunk to their size, examining them in their own hidden world. [Music] For a variation on dark field technique, make discs from transparent colored plastic. Now subjects appear against a colored background. Again, lit by rays focused in from the sides with a colored background disc in place. Light focused onto the subject can also be colored. We tried this in order to see the faces of salt crystals. And what began as a way to get information from our crystals turned into a microscopic light show. As crystals began to dissolve and recristallize, their newly formed faces made visible in reds and greens. [Music] Heat. Heat. Another way to manipulate color is to use polarized light. These changing colors give information about the composition and thickness of growing crystals. But these effects are only visible when crystals are viewed between crossed polarizers. In this case, lenses from a pair of sunglasses. Here is the principle. Rotate the polarizing lenses until they cross, extinguishing the light. Now the crystal shows up in colors not seen in normal light. Fit your microscope with polarizers and explore a world of light and color. [Music] In living specimens, cross-polarized lighting may reveal hidden details. like the arrangement of muscle fibers in our transparent lake dwellers or crystals within a living parramium features virtually undetectable under normal brightfield illumination. These simple but powerful techniques provide a wealth of information. But to further explore cells and tissues, biologists require special microscopes. The two main kinds are the phase contrast microscope and the differential interference microscope. Both use complicated manipulations of light to produce their revealing images. In phase contrast lighting, clear structures stand out without sacrificing resolution. This kind of revealing image showing living processes in action is what makes phase contrast such a valuable imaging tool. The other and newer lighting method, differential interference, uses a polarization process to produce a different kind of image. Here there is a feeling of looking at an optical slice cut through a living cell. A researcher may need to use all of these forms of lighting to interpret the hidden world. For example, consider Uglina, a primitive one-sellled organism. Under normal bright field lighting, Uglina's green chloroplasts and red eye spots show up. Dark field emphasizes Uglina's cell wall. Phase contrast shows the whip-like fleellum, an important structure often difficult to see in normal bright field. Differential interference gives a different impression of the fleellum and shows further details of the cell. These techniques complement each other. No one method of lighting tells the whole story. Or as Robert Hook, one of the first biologists to explore the hidden world, wrote over 300 years ago. I can never describe the specimen accurately without first observing it in several lights in different positions to these lights. Only in this way have I been able to discover its true form. Using a microscope can be a mindstretching adventure. a way to discover through observing. But the light microscope is not only a valuable scientific tool. Used with skill, it becomes a window into an amazing hidden world. [Music] [Applause] [Music]