Microscopes magnify images so that we can see things that otherwise would be invisible with the naked eye. The most common type, light microscope, uses glass lenses to focus light and create a high resolution image. Some light microscopes collect light reflected from the surface of solid objects. Using stereo (twin) eyepieces, they produce a three-dimensional view of surfaces but usually don't reveal any internal structure. This type of instrument usually is limited to enlargements between 10 and a few hundred times. Large plant or animal cells are generally visible in at this magnification but smaller structures are mostly indistinguishable.
The microscopes most commonly used in biology laboratories transmit light through a thin specimen held on a glass slide. A condenser lens between the light source and the specimen increases illumination brightness while one or more objective lenses collects light passing through the specimen and forms an image. The most common magnification ranges for these instruments is 4x, 10x, 40x, and 100x. The image usually is further magnified 10 or 15 times by an eyepiece. The objective lens most important in determining both ultimate magnification and resolution of the instrument. With a very good objective lens, the highest useful magnification for a light microscope is about 1,000 diameters, meaning that smallest space between objects that can be resolved is about 0.2 micrometers.
The most important limit to resolution in any optical system is caused by diffraction as light rays pass through the lenses. Late in the nineteenth century, the German physicist Ernst Abbe showed both theoretically and experimentally that best possible resolution in a light microscope is equal to about 0.6 times the wavelength of the illuminating light divided by a factor called the numerical aperture that combines the refractive index of the medium through which light passes and the diameter of the lens. The best possible numerical aperture of an oil immersion lens (one in which the specimen and the lens are immersed in oil) is about 1.4. The wave length of visible light is approximately 0.5 micrometer and thus the limit to resolution is about 0.2 micrometers.
Some very small biological specimens such as living bacterial or protozoan cells can simply be suspended in a drop of water and observed directly with a light microscope. For most tissues from higher plants or animals, however, elaborate preparation techniques are required to preserve specimens, stain them to make internal structures visible, and to make it possible to cut slices as thin as about 1 micrometer that are relatively transparent to light. The thinner the section is, the crisper the resulting image. Chemical fixatives such as formaldehyde are used to preserve tissue. Organic solvents such as alcohol are used to extract water and to allow an embedding medium such as paraffin wax to infiltrate the specimen. The most common stains in light microscopy are organic dyes such as hematoxlyn and eosin, which bind to proteins and nucleic acids to create distinctive colors that help identify cellular components.
A recent development that provides high resolution images with thick sections is called confocal light microscopy. In these instruments, a very narrow beam of light produced by a laser shining through tiny pin-hole aperture focuses at a very specific spot in the specimen. Fluorescent light re-emitted from special stains in the specimen at a wavelength different than that in the incident illumination is collected by a mirror and passed through a pin-hole that is confocal (that is at exactly the same focal length) with the laser aperture. The fluorescent light is collected by an extremely sensitive photo-detector, amplified, and displayed on a video terminal. Because computer controls the position of the confocal apertures very exactly, the image is as clear as if it had been made with an extremely thin section. Digital enhancement of the electronic signal from the photodetector gives very good images of structures that would otherwise be invisible in a light microscope.
The resolution limits imposed by light optics are overcome by electron microscopes, which use a beam of very high energy electrons rather than light to create an image. Although roughly a thousand times larger and more expensive than an ordinary light microscope, these instruments also have approximately one thousand times better resolution. This means that magnifications of a million times or more are possible and that objects as small as a fraction of a nanometer can be resolved. Large molecules are generally visualized easily in an electron microscope and in special cases even individual atoms can be seen.
Preparation of biological tissues in electron microscopy is difficult because the specimen must be able to withstand both very high vacuums and the intense bombardment of the electron beam. Biological samples are usually fixed (preserved) with glutaraldehyde or some other chemical that retains fine structure. They are then dehydrated and embedded in a very hard epoxy resin that can be sliced into sections about 20 nanometers thick by an extremely sharp diamond knife. The only stains useful in a TEM are heavy metals such as lead, tungsten, uranium, or gold. In some cases these stains can be coupled to antibodies or unique enzymatic reactions to give useful information about the location of specific cellular components. An alternative technique for sample preparation for the TEM involves freezing biological tissues in liquid nitrogen, fracturing them in a high vacuum system, and then making a very thin carbon-platinum replica of the fractured surface. This freeze-fracture technique has been useful for studying membrane structure.
In spite of the expense of confocal and electron microscopes, and the difficulties in preparing tissues for observation, a good deal of what we know about the internal organization of cells has come about since the introduction of these instruments in the past few decades. An explosion of information in cell biology brought about in recent years by higher resolution instruments and better preparation techniques brought us a wealth of information about cellular structure and how living organisms function.
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