Parts and Specifications
Historians credit the invention of the compound microscope to the Dutch spectacle maker, Zacharias Janssen, around the year 1590. The compound microscope uses lenses and light to enlarge the image and is also called an optical or light microscope (vs./ an electron microscope). The simplest optical microscope is the magnifying glass and is good to about ten times (10X) magnification. The compound microscopehas two systems of lenses for greater magnification, 1) the ocular, or eyepiece lens that one looks into and 2) the objective lens, or the lens closest to the object. Before purchasing or using a microscope, it is important to know the functions of each part.
Eyepiece Lens: the lens at the top that you look through. They are usually 10X or 15X power.
Tube: Connects the eyepiece to the objective lenses
Arm: Supports the tube and connects it to the base
Base: The bottom of the microscope, used for support
Illuminator: A steady light source (110 volts) used in place of a mirror. If your microscope has a mirror, it is used to reflect light from an external light source up through the bottom of the stage.
Stage: The flat platform where you place your slides. Stage clips hold the slides in place. If your microscope has a mechanical stage, you will be able to move the slide around by turning two knobs. One moves it left and right, the other moves it up and down.
Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can be rotated to easily change power.
Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. To have good resolution at 1000X, you will need a relatively sophisticated microscope with an Abbe condenser. The shortest lens is the lowest power, the longest one is the lens with the greatest power. Lenses are color coded and if built to DIN standards are interchangeable between microscopes. The high power objective lenses are retractable (i.e. 40XR). This means that if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide. All quality microscopes have achromatic, parcentered, parfocal lenses.
Rack Stop: This is an adjustment that determines how close the objective lens can get to the slide. It is set at the factory and keeps students from cranking the high power objective lens down into the slide and breaking things. You would only need to adjust this if you were using very thin slides and you weren't able to focus on the specimen at high power. (Tip: If you are using thin slides and can't focus, rather than adjust the rack stop, place a clear glass slide under the original slide to raise it a bit higher)
Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen. Condenser lenses are most useful at the highest powers (400X and above). Microscopes with in stage condenser lenses render a sharper image than those with no lens (at 400X). If your microscope has a maximum power of 400X, you will get the maximum benefit by using a condenser lenses rated at 0.65 NA or greater. 0.65 NA condenser lenses may be mounted in the stage and work quite well. A big advantage to a stage mounted lens is that there is one less focusing item to deal with. If you go to 1000X then you should have a focusable condenser lens with an N.A. of 1.25 or greater. Most 1000X microscopes use 1.25 Abbe condenser lens systems. The Abbe condenser lens can be moved up and down. It is set very close to the slide at 1000X and moved further away at the lower powers.
Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide. There is no set rule regarding which setting to use for a particular power. Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire and the particular objective lens in use.
How to Focus Your Microscope: The proper way to focus a microscope is to start with the lowest power objective lens first and while looking from the side, crank the lens down as close to the specimen as possible without touching it. Now, look through the eyepiece lens and focus upward only until the image is sharp. If you can't get it in focus, repeat the process again. Once the image is sharp with the low power lens, you should be able to simply click in the next power lens and do minor adjustments with the focus knob. If your microscope has a fine focus adjustment, turning it a bit should be all that's necessary. Continue with subsequent objective lenses and fine focus each time.
What to look for when purchasing a microscope.
If you want a real microscope that provides sharp crisp images then stay away from the toy stores and the plastic instruments that claim to go up to 600X or more. There are many high quality student grade microscopes on the market today. They have a metal body and all glass lenses. One of the most important considerations is to purchase your instrument from a reputable source. Although a dealer may give you a great price, they may not be around next year to help you with a problem. One dealer that we can highly recommend is Microscope World. They offer a wide variety of instruments at very competitive prices.
(This passage was adapted from Microbiology: A Laboratory Manual,5th edition, Cappuccino, J.S. and Sherman, N., Benjamin/CummingsScience Publishing.)
1.To become familiar with the history and diversity of microscopeinstruments.
2.To understand the components, use, and care of the compoundbrightfield microscope.
3.To learn the correct use of the microscope for observation andmeasurement of microorganisms.
Microbiology, the branch of science that has so vastly extendedand expanded our knowledge of the living world, owes its existence toAntony van Leeuwenhoek. In 1673, with the aid of a crude microscopeconsisting of a biconcave lens enclosed in two metal plates,Leeuwenhoek introduced the world to the existence of microbial formsof life. Over the years, microscopes have evolved from the simple,single-lens instrument of Leeuwenhoek, with a magnification of 300,to the present-day electron microscopes capable of magnificationsgreater than 250,000. Microscopes are designated as either lightmicroscopes or electron microscopes. The former use visible light orultraviolet rays to illuminate specimens. They include brightfield,darkfield, phase-contrast, and fluorescent instruments. Fluorescentmicro-scopes use ultraviolet radiations whose wavelengths are shorterthan those of visible light and are not directly perceptible to thehuman eye. Electron microscopes use elec-tron beams instead of lightrays, and magnets instead of lenses to observe submicro-scopicparticles.
Essential Features of Various Microscopes
This instrument contains two lens systems for magnifyingspecimens: the ocular lens in the eyepiece and the objective lenslocated in the nose-piece. The specimen is illuminated by a beam oftungsten light focused on it by a sub-stage lens called a condenser,and the result is that the specimen appears dark against a brightbackground. A major limitation of this system is the absence ofcontrast between the specimen and the surrounding medium, which makesit difficult to observe living cells. Therefore, most brightfieldobservations are performed on nonviable, stained preparations.
This is similar to the ordinary light microscope; however, thecondenser system is modified so that the specimen is not illuminateddirectly. The con-denser directs the light obliquely so that thelight is deflected or scattered from the spec-imen, which thenappears bright against a dark background. Living specimens may beobserved more readily with darkfield than with brightfieldmicroscopy.
Observation of microorganisms in an unstained state is possiblewith this microscope. Its optics include special objectives and acondenser that make visible cellular components that differ onlyslightly in their refractive indexes. As light is transmitted througha specimen with a refractive index different from that of thesurrounding medium, a portion of the light is refracted (bent) due toslight varia-tions in density and thickness of the cellularcomponents. The special optics convert the difference betweentransmitted light and refracted rays, resulting in a significantvari-ation in the intensity of light and thereby producing adiscernible image of the struc-ture under study. The image appearsdark against a light background.
This microscope is used most frequently to visualize speci-mensthat are chemically tagged with a fluorescent dye. The source ofillumination is an ultraviolet (UV) light obtained from ahigh-pressure mercury lamp or hydrogen quartz lamp. The ocular lensis fitted with a filter that permits the longer ultravioletwavelengths to pass, while the shorter wavelengths are blocked oreliminated. Ultraviolet radiations are absorbed by the fluorescentlabel and the energy is re-emitted in the form of a differentwavelength in the visible light range. The fluorescent dyes absorb atwavelengths between 230 and 350 nanometers (nm) and emit orange,yellow, or greenish light. This microscope is used primarily for thedetection of antigen-antibody reactions. Antibodies are conjugatedwith a fluorescent dye that becomes excited in the presence ofultraviolet light, and the fluorescent portion of the dye becomesvisible against a black background.
This instrument provides a revolutionary method of microscopy,with magnifications up to one million. This permits visualization ofsubmicroscopic cel-lular particles as well as viral agents. In theelectron microscope, the specimen is illu-minated by a beam ofelectrons rather than light, and the focusing is carried out byelec-tromagnets instead of a set of optics. These components aresealed in a tube in which a complete vacuum is established.Transmission electron microscopes require speci-mens that are thinlyprepared, fixed, and dehydrated for the electron beam to pass freelythrough them. As the electrons pass through the specimen, images areformed by direct-ing the electrons onto photographic film, thusmaking internal cellular structures visi-ble. Scanning electronmicroscopes are used for visualizing surface characteristics ratherthan intracellular structures A narrow beam of electrons scans backand forth, producing a three-dimensional image as the electrons arereflected off the specimen's surface.
While scientists have a variety of optical instruments with whichto perform routine laboratory procedures and sophisticated research,the compound brightfield micro-scope is the "workhorse" and iscommonly found in all biological laboratories. Although you should befamiliar with the basic principles of microscopy, you probably havenot been exposed to this diverse array of complex and expensiveequipment. Therefore, only the compound brightfield microscope willbe discussed in depth and used to examine specimens.
USE OF THE MICROSCOPE
To become familiar with the:
1.Theoretical principles of brightfield microscopy.
2.Component parts of the compound micro-scope.
3.Use and care of the compound microscope.
4.Practical use of the compound microscope for visualization ofcellular morphology from stained slide preparations.
Microbiology is a science that studies living organisms that aretoo small to be seen with the naked eye. Needless to say, such astudy must involve the use of a good compound microscope. Althoughthere are many types and variations, they all fundamentally consistof a two-lens system, a variable but controllable light source, andmechanical adjustable parts for determining focal length between thelenses and specimen.
Components of the Microscope
A fixed platform with an opening in the center allows for thepassage of light from an illu-minating source below to the lenssystem above the stage. This platform provides a surface for theplacement of a slide with its specimen over the central opening. Inaddition to the fixed stage, most microscopes have a mechanical stagethat can be moved vertically or horizontally by means of adjustmentcontrols. Less sophisticated micro-scopes have clips on the fixedstage, and the slide must be positioned manually over the centralopening.
The light source is positioned in the base of the instrument.Some microscopes are equipped with a built-in light source topro-vide direct illumination. Others are provided with a mirror; oneside flat and the other concave.
An external light source, such as a lamp, is placed in front ofthe mirror to direct the light upward into the lens system. The flatside of the mirror is used for artificial light, and the concave sidefor sunlight.
This component is found directly under the stage and contains twosets of lenses that collect and concentrate light passing upward fromthe light source into the lens sys-tems. The condenser is equippedwith an iris diaphragm, a shutter controlled by a lever that is usedto regulate the amount of light entering the lens system.
Above the stage and attached to the arm of the microscope is thebody tube. This structure houses the lens system that magnifies thespecimen. The upper end of the tube contains the ocular or eyepiecelens. The lower portion consists of a movable nosepiece containingthe objective lenses. Rotation of the nosepiece posi-tions objectivesabove the stage opening. The body tube may be raised or lowered withthe aid of coarse-adjustment and fine-adjustment knobs that arelocated above or below the stage, depending on the type and make ofthe instrument.
Theoretical Principles of Microscopy
To use the microscope efficiently and with minimal frustration,you should understand the basic principles of microscopy:magnification, resolution, numerical aperture, illumination, andfocusing.
Enlargement or magnification of a specimen is the function of atwo-lens system; the ocular lens is found in the eyepiece, and theobjective lens is situated in a revolving nose-piece. These lensesare separated by the body tube. The objective lens is nearer thespecimen and magnifies it, producing the real image that is projectedup into the focal plane and then magnified by the ocular lens toproduce the final image.
The most commonly used microscopes are equipped with a revolvingnosepiece containing four objective lenses possessing differentdegrees of magnification. When these are combined with themagnification of the ocular lens, the total or overall linearmagnification of the specimen is obtained.
Resolving Power or Resolution
Although magnification is important, you must be aware thatunlimited enlargement is not possible by merely increasing themagnifying power of the lenses or by using additional lenses, becauselenses are limited by a property called resolving power. Bydefinition, resolving power is the ability of a lens to show twoadjacent objects as discrete entities. When a lens cannotdiscriminate, that is, when the two objects appear as one, it haslost resolu-tion. Increased magnification will not rectify the loss,and will, in fact, blur the object. The resolv-ing power of a lens isdependent on the wave-length of light used and the numericalaperture, which is a characteristic of each lens and imprinted oneach objective. The numerical aper-ture is defined as a function ofthe diameter of the objective lens in relation to its focal length.It is doubled by use of the substage condenser; which illuminates theobject with rays of light that pass through the specimen obliquely aswell as directly. Thus, resolving power is expressed mathematically,as follows:
Resolving power = Wavelength of Light .
2 (Numerical Aperture)
Based on this formula, the shorter the wave-length, the greaterthe resolving power of the lens. Thus, short wavelengths of theelectromag-netic spectrum are better suited than longer wavelengthsin terms of the numerical aperture.
However; as with magnification, resolving power also has limits.You might rationalize that merely decreasing the wavelength willautomati-cally increase the resolving power of a lens. Such is notthe case, because the visible portion of the electromagnetic spectrumis very narrow and borders on the very short wavelengths found in theultraviolet portion of the spectrum.
The relationship between wavelength and numerical aperture isvalid only for increased resolving power when light rays areparallel. Therefore, the resolving power is dependent on anotherfactor, the refractive index. This is the bending power of lightpassing through air from the glass slide to the objective lens. Therefractive index of air is lower than that of glass, and as lightrays pass from the glass slide into the air, they are bent orrefracted so that they do not pass into the objective lens. Thiswould cause a loss of light, which would reduce the numericalaperture and diminish the resolving power of the objective lens. Lossof refracted light can be compensated for by interposing mineral oil,which has the same refractive index as glass, between the slide andthe objective lens. In this way, decreased light refraction occursand more light rays enter directly into the objective lens, producinga vivid image with high resolution.
Effective illumination is required for efficient magnification andresolving power. Since the intensity of daylight is an uncontrolledvariable, artificial light from a tungsten lamp is the most commonlyused light source in microscopy. The light is passed through thecon-denser located beneath the stage. The condenser contains twolenses that are necessary to produce a maximum numerical aperture.The height of the condenser can be adjusted with the con-denser knob.Always keep the condenser close to the stage, especially when usingthe oil-immersion objective.
Between the light source and the condenser is the iris diaphragm,which can be opened and closed by means of a lever; therebyregulating the amount of light entering the condenser. Excessiveillumination may actually obscure the specimen because of lack ofcontrast. The amount of light entering the microscope differs witheach objec-tive lens used. A rule of thumb is that as themag-nification of the lens increases, the distance between theobjective lens and slide, called working distance, decreases, whereasthe numerical aperture of the objective lens increases.
Use and Care of the Microscope
You will be responsible for the proper care and use ofmicroscopes. Since microscopes are expensive, you must observe thefollowing regu-lations and procedures.
The instruments are housed in special cabinets and must be movedby users to their laboratory benches. The correct and only acceptableway to do this is to grip the microscope arm firmly with the righthand and the base with the left hand, and lift the instrument fromthe cabinet shelf. Carry it close to the body and gently place it onthe laboratory bench. This will prevent collision with furniture orco-workers and will protect the instrument against damage.
Once the microscope is placed on the laboratory bench, observe thefollowing rules:
1.Remove all unnecessary materials such as books, papers, purses, and hats from the laboratory bench.
2.Uncoil the microscope's electric wire and plug it into an electrical outlet.
3.Clean all lens svstems; the smallest bit of dust, oil, lint, or eyelash will decrease the efficiency ot the microscope. The ocular; scan-ning, low-power, and high-power lenses may be cleaned by wiping several times with acceptable lens tissue. Never use paper tow-eling or cloth on a lens surface. If the oil-immersion lens is gummy or tacky, a piece of lens paper moistened with methanol is used to wipe it clean. If the lens is very dirty it may be cleaned with xylol however the xylol cleansing procedure should be performed only by the instructor, and only if necessary. Consistent use of xylol may loosen the lens.
The following routine procedures must be followed to ensurecorrect and efficient use of the microscope while focusing.
1. Place the microscope slide with the specimen within the stage clips on the fixed stage. Move the slide to center the specimen over the opening in the stage directly over the light source.
2. Rotate the scanning lens or the low power lens into position. While watching from the side to insure that the lens doesn't touch the specimen, turn the coarse focus knob to move the stage as close as it can get to the lens without touching the lens. (Always watch from the side whenever you move a specimen towards any objective lens to make sure the lens doesn't crash through the specimen and get damaged!)
3. Now, while looking through the ocular lens, turn the coarse focus knob carefully, and slowly move the stage away from the lens until the specimen comes into vague focus. Then, use the fine focus knob to bring the specimen into sharp focus.
4. If this is the first specimen of the day, you should Kohler your microscope at this point (while it is in focus). Otherwise, if your microscope has already been Kohlered you won't need to do it again
5. Routinely adjust the light source by means of the light source transformer setting, and/or the iris diaphragm, for optimum illumination for each new slide and for each change in magnification.
6. Our microscopes are parfocal, which means that when one lens is in focus, other lenses will also have the same focal length and can be rotated into position without further major adjustment. In practice, however; usually a half-turn of the fine-adjustment knob in either direction is necessary for sharp focus.
7. Once you have brought the specimen into sharp focus with a low-powered lens, preparation may be made for visualizing the spec-imen under oil immersion. Place a drop of oil on the slide directly over the area to be viewed. Rotate the nosepiece until the oil-immersion objective locks into position. Care should be taken not to allow the high-power objective to touch the drop of oil.The slide is observed from the side as the objective is rotated slowly into position. This will ensure that the objective will be properly immersed in the oil. The fine-adjustment knob is readjusted to bring the image into sharp focus.
8. During microscopic examination of microbial organisms, it is always necessary to observe several areas of the preparation. This is accomplished by scanning the slide with-out the application of additional immersion oil. This will require continuous, very fine adjustments by the slow, back-and-forth rotation of the fine adjustment knob only.
On completion of the laboratory exercise, return the microscope toits cabinet in its original condition. The following steps arerecommended:
1.Clean all lenses with dry, clean lens paper. If you need to, you can use a drop or two of methanol to help clean the lens. Use xylol to remove oil from the stage only.
2. Place the low-power objective in position and bring the stage and objectives close together.
3.Center the mechanical stage.
4.Coil the electric wire around the body tube and the stage.
5.Carry the microscope to its position in its cabinet in the manner previously described.