William R. Bowen Microscopy Center
Learning About Microscopy
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THE LIGHT MICROSCOPE
Probably the most important instrument for many biologists is the microscope. The most commonly used is a light microscope. In order to understand the principles of the Scanning Electron Microscope it is expected that you first gain a basic understanding of the principle and function of standard type light microscopes. While, it is a delicate and expensive piece of equipment, the light microscope is relatively easy to use and care for compared to many other laboratory instruments. It may be for these reasons that the microscope is often treated so casually and, therefore, abused, resulting in expensive repairs. The purpose of this section is for the student to gain working knowledge of the parts, purpose, and proper use and care of the microscope so that he or she will feel comfortable and competent when using this instrument. It also provides a brief description of some of the laboratory techniques used in light microscopy and comparisons will be made for sample preparation and theories of focus, when discussing the Scanning Electron.
The Compound Microscope
The microscope most commonly in use in the microbiology laboratory is the compound microscope. A compound microscope is one that uses a combination of lens systems: the objective lens system and the ocular or eyepiece lens system. The objective lenses are the lenses nearest the specimen or object while the ocular or eyepiece lenses are the ones nearest the eye. Other important parts of a compound, bright-field microscope include an illuminator system, a substage condenser system, an iris diaphragm, Nicol prisms, a tubular barrel and a mechanical state. Light from the illuminator system passes through a filter (usually a blue filter) and is collected into the substage condenser. The substage condenser then focuses the light on the specimen while the iris diaphragm regulates the amount f light. Light, as it passes through the specimen, requires an image that is magnified by the objective lens system. The objective lens acts as a small projection lens by forming an enlarged, inverted image of the specimen about 11 mm from the top of the microscope tube; this image is referred to as a primary or aerial image. Since the image is optically too close to the eye to be observed clearly, the ocular or eyepiece lens acts as a magnifier to help the eye to focus on the primary image. The final image formed in the retina appears to come from a point below the specimen, about 10 inches from the eye, and is referred to as the virtual image. The final magnification of the specimen is a product of the magnifying power of both the objective and ocular lens systems.
Objective Lens System
The most important factor in determining the quality of an image produced by a microscope is the objective lens system. The system consists of three to four objectives of varying magnifying powers attached to a revolving nosepiece. The degree of initial magnification by a particular objective is usually etched on a barrel of the lens and refers to an increase in the diameter of the specimen. Also, the various lenses often have color coded bands around the barrel for easy reference. The most common objectives used in a microbiology laboratory are the: 10x low power lens; the 45X high, dry power lens; and the 100x box oil immersion lens. Most of today's compound microscopes are parfocal meaning it only takes a minor fine adjustment to bring the viewing field into focus when switching from one objective to another. One must keep in mind that as one increases magnification, the total area of the specimen seen through the microscope decreases. Therefore, only the center portion of the viewing field will be in view when going from a lower to a higher power objective. In the microbiology laboratory, the objective most often employed is the oil immersion objective. Light, when it goes from a medium of a high refractive index to a medium of a lower refractive index is bent toward the medium having the higher refractive index. Therefore, light passing through a glass slide (refractive index of 1.52) into air (refractive index of 1.0) is reflected toward the slide instead of through the objective lens. This results in a fuzzy image and a loss of light. Immersion oil, with a refractive index comparable to glass, when placed between the slide and the oil immersion lens will correct this problem. The objective lens will help prevent the light from being deflected from the aperture of the objective. The oil generally used is a specially prepared oil that is a non-drying, reflectively stable aqueous liquid with a refractive index of 1.51. ** NOTE: It is very important to use the oil only on the oil immersion objective lens and to clean the lens completely when you are through with the microscope.
Mechanical Lens System Deflection of light through air and oil
The ocular or eyepiece lens system is relatively simple in comparison to the objective lens system. Whereas the objectives act as small projection lenses, the oculars act as magnifiers of the aerial image produced by the objectives. The magnifying power to most eyepieces used in a microbiology laboratory is 10X. Even though the eyepiece lens can easily be changed, one would not necessarily benefit from using an ocular of higher power. Using an ocular lens that is too strong would only make the object appear blurry and actually result in a loss of resolution.
Many of today's more expensive compound microscopes used a matched pair of eyepieces or oculars binocular) instead of just one ocular (monocular). The binocular microscope uses a system of mirrors and prisms located in the body tube to split the incident beam of light from the objective into two identical images that an be seen at the same time by both eyes. This helps prevent eyestrain and fatigue for personnel, involved in prolonged microscopy. Therefore, it is important for the student of microscopy to learn how to use the binocular microscope properly.
Substage Condenser and Iris Diaphragm
When using lenses of high magnifying power, it is essential that the aperture of the objective be filled with optimal light. This is accomplished by using a brightfield substage condenser. The substage condenser is actually a third lens system positioned between the light source and the specimen. It concentrates light beams from the light source into a thin cone shaped line. Thereby, regulating the height of the condenser focuses this cone on the specimen and up through the aperture of the objective.
The amount of light allowed to concentrate on the specimen is regulated by the iris diaphragm. When the iris diaphragm is opened too much, the intense light will cause glare, making the microorganisms virtually impossible to see. On the other hand, closing the diaphragm too much will not allow sufficient light to strike the specimen and the numerical aperture of the objective will be reduced.
Illumination system
Most of today's better microscopes have an Illuminating system built into the base of the microscope. The advantage of this type of substage Illuminator is that it can be permanently aligned and is easy to use. The lamp houses a high intensity tungsten filament lamp bulb that can range from a 10-15-watt refrigerator bulb to a tungsten halogen lamp of 15 - 100 watts. Many have a rheostat (dimmer switch) attached that can be used to control power and, thus, brightness.
To be satisfactory, a microscope must provide adequate magnifying power, possess high resolution and produce an image of good definition.
The degree of magnification possible with a compound microscope is practically limitless, but there is a limit to its usefulness. Useful magnification is based on the resolution of resolving power of a microscope. The resolving power can be defined as the ability to separate and distinguish the fine details of the object under view. The limit of resolution can be measured in mathematical terms as the minimal distance with which one can discriminate between two closely spaced objects. (Think of it as the analogy of looking at two of your fingers from arms length away and slowly bringing them closer and closer to your nose, eventually you get to the point where you lose the ability to see both fingers and only see one.)
The resolving power is primarily dependent upon the design of the objectives, particularly the numerical aperture of the objective lenses. The numerical aperture determines the size of the cone of light transmitted through the lens--the larger the cone of light, the greater the resolving power. Resolving power is also dependent upon the numerical aperture of the condenser and the wavelength of the light source used. Once the limit of resolution is met, an increase in magnification only results in a blurred and indistinct image. This is referred to as "empty magnification".
The maximum magnification that still retains acceptable resolution is approximately l,000 times the numerical aperture of the objective lens. Therefore, when using an oil immersion lens with a N.A. of 1.30, the highest magnification that would still give good detail is about 1,300 diameters.
The numerical aperture is etched on the condenser and objective. With most modern microscopes, when the objective and condenser lenses are properly focused, the numerical aperture of each should be almost equal. It is definition that gives the image contrast, brilliance, sharpness, and clarity. A high-quality lens will help eliminate spherical and chromatic aberrations, curvature of the field, distortion and astigmatism of the image.
Laboratory Lecture
The brightfield, compound microscope is probably the most important instrument used regularly by the typical biologist. It is expensive, delicate, and probably the most abused instrument in the laboratory. To get satisfactory results and avoid expensive repairs, strict adherence to the guidelines regarding the use and care of the microscope is necessary.
The objective lens system is the single most important part of the microscope. It consists of at least three objectives of varying magnifying powers, usually l0x low power; 45X high dry power; and 100x oil immersion lenses. The ocular lens most often used is a l0x eyepiece lens. The total magnification of the specimen is a product of the magnifying powers of the objective lens used and the ocular lens.
The lens system nearest the eye. It magnifies the aerial image produced by objective lens The part of the microscope through which light and the image formed by the objective lens passes and is directed toward the eyepiece raises or lowers the objectives rapidly to bring the specimen into approximate focus. The iris diaphragm lever raises or lowers the condenser controls the amount of light reaching the specimen opens and closes the iris diaphragm and moves the objective lens slowly to bring the specimen into sharp focus.
The part of the microscope to which the objective lenses is attached. Can be rotated to change from one objective to another, which magnifies the specimen and projects the image to form the primary or aerial image. Usually 3 different lenses--10x low power, 45X high dry power, and 100X oil immersion lenses condenses the light from the illumination system into a pencil shaped cone and focuses it on the specimen.
The light source that can either be built into the microscope base or are separate and focused on the condenser by mirrors. Some allow control of light intensity supports the slide and the mechanical stage allows the slide to be moved. The mechanical stage allows the slide to be moved back and forth and side to side
Care of the Microscope
Always use two hands when carrying the microscope. Grasp the arm of the microscope with one hand and place the other hand under the base. Never tilt the microscope so the oculars could fall out. Place the microscope far enough from the edge of the workbench that it cannot be knocked off.
The microscope should always be kept clean and clear of dust. It is a good idea to clean the lens systems (eyepiece, objectives, and condenser) before using the microscope. It is absolutely mandatory that the lenses be cleaned after each use. Use special lens-paper when cleaning the lenses. Clean the oil immersion lens, last so as not to transfer any oil from the lens paper to another lens. A small amount of xylene on a clean piece of lens paper can be used to remove any excess immersion oil. Wipe any remaining xylene away with another clean piece of lens paper.
When changing objective lens be careful not to allow the 45x high, dry lens to come into contact with any immersion oil on the slide. The working position of the high power lens is low enough that it could touch the oil which, is damaging to the high power lens.
Do not remove any parts of the microscope or change a light bulb without consulting the instructor. Never touch lenses with fingers When finished using the microscope:
a. Clean lenses.
b. Place the low power objective in the working position and rack down.
c. Turn light control to lowest setting, then turn light off and unplug if removing the microscope from the workbench.
d. Replace dust cover.
The Microscope Figure 1
Use of the Microscope
Materials: Microscope, Immersion oil, Lens-paper, 1/unit
Microscope slides, Microscope cover-slips, Plastic pipette
Broth culture of Saccharomyces cerveisiae with mixed bacteria
Wright's stained slide of human blood cells
Throughout this manual, the amount of materials needed. Is given per working unit, which may be individuals, pairs, or groups at the instructor's discretion. This may be modified according to the availability of supplies and the number of students per laboratory.
Procedure:
1. Using both hands to carry the microscope, place it on the workbench with the arm of the microscope toward you.
2. Remove the dust cover, clean lenses, plug in light source.
3. Using,The MICROSCOPE Figure 1 locate the various parts of your microscope.
4. Place one of the prepared, stained slides into the slide holder of the mechanical stage and, using the mechanical stage controls, position the portion of the slide with the specimen directly under the objective lens.
5. Bring the low power lOX objective into working position by turning the revolving nosepiece until you hear a click. Using the coarse adjustment knob, bring the objective down to its lowest position while striking the slide does not damage watching from the side of the microscope to make sure the lens. (Never use the coarse adjustment to lower any objective while looking through the eyepiece).
6. Using the condenser adjustment knob, raise the condenser to its highest position without striking the underside of a slide.
7. Turn on the light source. Some microscopes come with a rheostat to adjust light intensity.
8. Adjust the light by using the iris diaphragm lever to open and close the diaphragm. Proper lighting is essential in any microscopy work and more light is not necessarily better. One wants to provide contrast without destroying resolution. Even though it might be necessary to adjust the condenser some-what, it is best to leave it in an upward position and adjust the light by using the iris diaphragm.
9. With the lOX objective in a lowered position, slowly use the coarse adjustment knob to raise the objective until the specimen is in approximate focus. Use the fine adjustment knob to bring the specimen into sharp focus.
10. With the specimen in focus under low power, you can then easily increase magnification. Center the portion of the field that you wish to observe and rotate the revolving nosepiece until the high power 45X objective clicks into place. If you’re microscope is parfocal, only fine adjustment should be necessary for focusing
11. The stained slides of bacteria and blood cells require the oil immersion lens for viewing. Swing the high power objective away from the slide, place a small drop of immersion oil on the slide, and bring the lOOX oil immersion objective into position. When doing this, take special care not to run the high power objective through the oil because it can damage the lens. If this does happen, use lens paper to remove the oil immediately. After the oil immersion lens has been positioned, adjust the focus with the fine adjustment knob. Since the focal distance of the oil immersion objective is short, take special precautions with any downward movement of the objective so it will not hit the slide.
12. When reviewing the specimen, it is best to do so in an orderly rather than a random fashion so to cover a representative area without repeating areas. Using the mechanical stage knobs, go across the width of the slide, then move the slide over one viewing field and then back across the width and continue in like fashion until you feel a representative area has been searched. Make drawings of your observations on both stained slides. Note size of differences with various magnifications. (See Sketch Below).
Figure 2: Method for observing a slide. Note that the organized manner allows you to view the entire slide without going over the same area several times.
Scanning Electron Microscopy:
Scanning Electron Microscopy (SEM) in concept is very similar to the information you just reviewed on Light microscopy. In the case of SEM electrons are produced by applying voltage to a specific type of filament (tungsten) which then travels down a column to strike the specimen. The electrons "bounce" off the specimen and stub and produce several kinds of energy products (backscattered electrons, x-rays, 2ed electrons…). Dependent on the type of detector and it's orientation to the specimen you can convert this information into an image. The trick is that you have a strong electron beam which must be controlled by magnets in the column and can damage a specimen. This caused us to have to use several different methods of preparing the samples for observation. The samples are typically observed after coating them with a very thin layer of gold/palladium. You should now go to JEOL's web site that has "A guide to Scanning Microscope Observation" for a review of the basics.
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