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Because many organisms are quite small and even the largest organisms are made up of a multitude of very small tissues, cells and organelles, biologists frequently use microscopes to study them. The first microscopes were invented in 1590 by two Dutch spectacle makers, Zacharias and Francis Janssen, who combined two convex lenses in a tube to magnify objects. In the 17th century another Dutchman, Anton van Leeuwenhoek made hundreds of microscopes and published many detailed observations of a variety of microscopic organisms and structures. Because of this he is often credited with perfecting the first microscopes. The inability of the human eye to see microscopic objects is a consequence of the arrangement of receptor cells at the rear of the eye. Two objects must be at least 100 µm apart for their reflected light to fall on different receptor cells. If they are closer together than this their light falls on the same receptor cell and therefore they are perceived as one object. Resolution is the minimum distance that two points can be separated and still be distinguished as two separate points. At less than 100 µm apart the human eye can not resolve two points or objects. Resolution may be increased by increasing magnification, i.e. making objects appear larger. For most everyday objects this is quite simple, just move closer to the object or bring it closer to you. However, our eyes can not focus closer than about 25 cm. But putting a convex lens, such as a magnifying glass or microscope lens, between the eye and object provides a clear image at a much closer range. Because the object is closer, it projects a larger image on the back of the eye, and it appears larger. Since this larger image is spread over more receptor cells the resolving ability of the eye is improved, making it easier to see smaller objects. Microscopes which use two or more magnifying lenses are called compound because the lenses work in tandem to magnify the image which ultimately appears on the back of the eye. The objective lens closest to the specimen magnifies and projects the image into the body tube of the microscope where it is further enlarged and projected to the eye via the ocular lens. Thus total magnification is a product of the magnifying power of the objective times that of the ocular. Magnifications of light microscopes are limited to about 1,500 X because you can not resolve objects smaller than the wavelength of light being used. Consequently increased magnifications under visible light, no matter how large, will not improve resolution. The wavelength of the electrons used in electron microscopy have much smaller wavelengths than visible light, therefore they can resolve much smaller objects. This is why electron microscopes are used to provide higher magnifications. When the parallel rays of a light source strike a convex lens the rays will concentrate or focus at a specific point, the focal point. If you have ever started a fire with a magnifying glass, you are acutely aware of the concentration or focusing effect of a lens. The distance between the center of the lens and the focal point is called the focal length. Focal length is related to lens power (magnifying ability). A powerful lens will have a short focal length, thus requiring that it be placed very close to the object being examined. This means that as magnification increases, the working distance between the objective lens and object decreases. Students often either forget or ignore this fact and broken slides and/or scratched objectives may result. Never use the coarse focusing knob on anything but low power. Focal plane (depth of focus) is another important concept you should become familiar with. Hold a page of text up at normal reading distance and focus on a few words. Note that background and peripheral objects, although noticeable, are out of focus. Now focus on a background object and note that the writing on the paper is no longer in focus. All lenses, including those in your eyes, have limited distances or ranges in which two objects on different planes (focal planes) may be simultaneously in focus. For purposes of microscopy it is important to note that as magnification increases, focal plane (depth of field) decreases. In fact at high magnifications, the focal plane is usually less than the thickness of the object being examined. Therefore to see all of an object it is necessary to focus through it, i.e. constantly focus up and down with the fine focus knob of the microscope. The quality of a microscope image is greatly affected by contrast, the differential absorption of light by different parts of the specimen. Many cellular components are translucent and therefore there is little contrast between them. Dyes and stains which absorb at different wavelengths are used to increase contrast. USE OF THE MICROSCOPE Microscopes are expensive, precision instruments with parts that easily get out of alignment. Even the slightest misalignment will greatly affect image quality and detract from your lab experience. Handle them with care!
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