Tuatara: Volume 23, Issue 1, July 1977
How to Use Your Microscope
How to Use Your Microscope
Optical Apparatus Technician, Department of Zoology, Victoria University of Wellington
These practical notes on the microscope are intended for the biologist, who uses a microscope as an analytical tool in his research work. It is not necessary to become a full-time professional research microscopist in order to get the best out of a microscope. For this reason these notes do not go into the laws of optics as applied to microscopy.
The ‘biological microscope’ discussed here includes all types in general use in teaching laboratories that employ bright field illumination. The parts of a typical instrument are shown in Fig. 1. Low-power dissecting microscopes, i.e. stereo microscopes, have been omitted as they are easily set up and hence less likely to be used incorrectly.
The optical information which can be obtained from a biological microscope (also known as a compound microscope) is sufficient for most biological demands. However, it has its limitations as a bright field observation microscope. Its utilisation can be enhanced by adaptation for dark field, phase contrast, polarised light, differential interference-contrast and U.V. fluorescence microscopy. It should be noted that only modern high quality microscopes can be converted from one phase of observation to another. In fact each one then becomes a specialist instrument. In order not to confuse the beginner this article will deal only with the basic compound microscope. The higher facets of microscopy are nevertheless built on these foundations.
The Microscope Image
Magnification in the microscope is collectively produced by two independent optical systems — objective and eyepiece. The image formed is inverted and reversed, i.e. it is seen upside down, and left to right is also reversed. The objective projects an image which is located below the eyepiece at a point which falls in the front focal plane of the eyepiece (known as the aerial image). The eyepiece, because it is acting as magnifier, enlarges the already greatly magnified image coming from the objective to produce at the exit pupil of the eyepiece an even wider visual angle and thus higher magnification.
The objective and eyepiece have been designed to work together to give best results at a particular tube length. 160 mm is one of the standardised tube lengths. This length is the distance from the flanged shoulder of the objective to the upper end of the eyepiece tube.
A Generalised Bright-FIELD Microscope
(1) A, eyepiece; B, diopta ring; C, eyepiece interpupillary slide. (2) Binocular inclined tube. (3) Revolving objective nosepiece. (4) Mechanical stage. (5) Sub-stage condenser assembly: A, swing-out top lens; B, centering screw (one each side of condenser carrier); C, aperture iris; D, filter carrier; E, auxiliary condenser lens. (6) Field stop diaphragm. (7) Lamp mount. (8) Base plate. (9) Coarse focus adjustment. (10) Fine focus adjustment. (11) Sub-stage condenser drive. (12) Operating knob for mechanical stage. (13) Stand with tube carrier.
It should be noted that a limiting factor that is generally termed ‘useful magnification’, namely the limit of resolving power, is also to be found at a specific point. Beyond this limit further resolution of fine detail is no longer accomplished even when magnification is further increased by the use of a higher-powered eyepiece. To do so would only increase the initial magnification without resolving any new detail. We can calculate the limits of ‘useful magnification’ by knowing the numerical aperture of a particular objective.
Numerical Aperture — (N.A. Value)
The useful magnification of an objective is found by multiplying the N.A. by a minimum of 500 and a maximum of 1000. An eyepiece must then be selected to produce the appropriate magnification up to, or within, these two limits. The numerical aperture is engraved on most objectives. For example, we may find ‘40/0.65′ engraved on a typical ‘medium power’ objective. This indicates that 40x is the objective's magnification and 0.65 is the maximum numerical aperture of its front lens. Multiplying 0.65 by 500 and 1000 gives a useful magnification range from 325x - 650x. As the magnification of the objective is 40x in our example and as total magnification of a microscope is found by multiplying objective and eyepiece magnifications, we can calculate that to remain within the limits of useful magnification an eyepiece should be selected which has a magnification within the range of 325/40 to 650/40— that is, between about 8x and 16x magnification. Eyepiece magnification is normally engraved on top of the eyepiece. To exceed (as many beginner microscopists do) the useful magnification range of an objective by using an eyepiece of too high a magnification only distracts from the excellence of the objective.
We have seen that resolving power is the measurement of the degree to which an optical system can create separate images of two points closely set together. This ability to distinguish fine detail is thus not governed by magnification alone but rather by the N.A. value. The higher the N.A. the greater the, resolving power.
To help understand the theory behind the formula which determines the N.A. value, one must firstly comprehend that any light entering the optical system of a microscope manifests itself in the form of waves (the fundamental restricting element in all optical microscopes). These light waves have a natural spreading tendency. Furthermore, as they traverse through the specimen and cover glass, they become refracted from the optical axis, i.e. the cone of rays originating at the object. The outermost rays thus fall in intensity as their angle of refraction is increased. Conversely the central area of light is concentrated because of the smaller refracted angle.
In practical terms it is impossible to achieve a figure greater than 0.95 for dry systems. This is due to the failure of the aperture angle within the objective to attain a half-angle of 90°, a limiting factor governed by optical theory. If a medium which has a greater refractive power than air is used between the front lens and cover glass (e.g. immersion oil, N.A. = 1.515) a greater angle of acceptance is achieved. Consequently this type of objective, known as an ‘immersion system’, has a superior ability to resolve through the gain in numerical aperture. Immersion objectives are therefore assigned higher N.A. values than those designated to dry systems — and are used to obtain higher magnifications. At this point it is important to stress the influence of illumination and the sub-stage condenser on the microscope's total resolving capability.
There are various kinds of illumination on biological microscopes.
|(a)||The most basic type employs the use of daylight via a collecting mirror which has both plane and concave sides. The use of daylight is somewhat inconvenient, because light intensity is so changeable. It is therefore not recommended for prolonged observation.|
An improvement on daylight illumination is to use a desk lamp with an opalescent bulb. Place it about 25cm from the plane side of the mirror.
Note: Generally use the plane mirror in (a) and (b). However, when the microscope has no sub-stage condenser or you wish to remove unwanted images cast by the plane mirror, use the concave side.
|(c)||An improvement on (b) is to fit a sub-stage illuminator in place of the mirror. This can take the form of a simple lamp-holder and bulb with a frosted glass screen. More basic models are plugged directly into a 240-volt power supply. Others work with a low-voltage bulb through a transformer with intensity steps. This gives some degree of control over the illumination.|
|(d)||A type of illumination preferable to (c) also uses low-voltage illumination. However, it incorporates two superior features —- a lamp condenser and an iris diaphragm (known as a field stop diaphragm) at the light source. This provides Kohler illumination. This system of exacting control of the light rays will usually be found only on the higher quality microscopes. Nonetheless some manufacturers do make free-standing illuminators of this pattern which can be utilised for microscopes with mirrors.|
This assembly has a very important role to play in the total balance of microscope illumination. Its task is to collect as much page 15 light as possible from the light source and to concentrate that light on to the specimen. A good quality condenser ensures that the numerical aperture of the higher-powered objectives is fully utilised in providing maximum resolving power.
A basic bright-field condenser can be vertically racked up and down. It consists of a fixed lens and an iris diaphragm. More sophisticated condensers incorporate a swing-out top lens or one which can be screwed off and interchanged. Most microscopes with this type of unit also have the extra facility whereby the condsenser can be centred in the horizontal plane. This is a prerequisite for Kohler illumination unless the light source itself can be centred.
Operation and Setting up of the Microscope
Basic preparatory points are often neglected by the student microscopist.
Firstly, it is important to avoid the use of mismatched components on your microscope. Do not use different makes of objectives and eyepieces. They may work and produce a somewhat satisfactory image. However, they are usually not ‘parfocal’ with the other objectives. (Each objective must have the same focal length to the mechanical tube length.) It is important that when the nosepiece is revolved to a different objective, the focal plane does not deviate from the last objective used — a significant factor when changing from low power to an oil immersion system when damage to the objective and specimen can occur if parfocalisation is not maintained. When revolving the nosepiece make sure that it positions itself at the click stop. Failure to do this will cause the objective to be out of alignment with the optical axis. Check, too, that the objectives are fully screwed in. A dirty or finger-printed front lens will give an upsharp image lacking in contrast. When the lamp collector, condenser lens or eyepiece are dirty, this will usually be indicated as blurred spots or flecks which remain in the field of view as the preparation is moved. Their location can be easily found by rotating either the eyepiece, objective or by moving the sub-stage condenser. When using a microscope with a monocular body tube, it is recommended that you keep the unused eye open to lessen eye fatigue. In order not to harm the muscles by this form of ‘disconnection’, the eyes should be used alternately. This problem does not apply to binocular microscopes. However, it is important to adjust the eyepieces for individual interpupillary distance. This is accomplished by lateral movement of the eyepieces until only a single image is seen in union with both eyes. Also the eyepiece must be adjusted to suit the operator's individual eyesight. This is done by focusing sharply on to the specimen while observing only through the fixed eyepiece (the other eye remaining closed). Now open the closed eye and bring into focus the image seen in this eyepiece, by adjustment page 16 of the diopta ring. Some binocular microscopes have diopter rings on both eyepieces, with a small engraved disc set between the eyepieces. The indicated value shown must be transferred to both diopta rings (this compensates for any change in tube length which might have occurred while adjusting for interpupillary distance). If the observer has a visual defect, focus the microscope with the strongest eye only, then adjust the diopta ring of the weaker eye.
Often the novice does not correctly relax his eyes while focusing the instrument. The image should be imagined to be set at infinity. Invariably this is mistakenly viewed as if it were closely set inside the eyepiece. This form of accommodation by the eye will be found to be very tiresome. If one is working in a laboratory that has a window, a good practice is to periodically glance at some distant object. You should be able to look from the window back to the specimen image without refocusing. Another good practice is to constantly alternate the focus by means of the fine motion adjustment. This will prevent your eyes from accommodating a fixed point, thus alleviating eye strain.
As stated earlier, the most basic illumination device found on microscopes consists of a mirror, either utilising daylight or some form of artificial illumination. For the sake of convenience this has generally been substituted by a fixed light source mounted on the base plate. For this reason we will describe the adjustments required for the latter system. The manner of operation will, of course, also apply to other bright field observation instruments, irrespective of what system is used.
Setting up for Critical Illumination
Place the microscope in a convenient working position. Switch on the light source, then check that all optical components are clean and that the rest of the instrument is functioning correctly. Before placing a specimen slide on the stage, the following important points must be observed:
Never try to set a slide under the objective without firstly either lowering the stage assembly so that there is some distance between slide and objective (on some microscopes the stage is fixed and the body tube is the moving part) or, alternatively, if you do not wish to lose your focused setting, you may turn the objective away from the optical path. e.g. slanting it halfway between click stops. However, this is not recommended when using oil immersion objectives, i.e. unless they are of the spring-mounted type (pushed up and locked).
The above prictice, if adopted, will safeguard the objective's front lens from being inadvertently scratched by the sharp edge of the slide. The importance of this exercise cannot be overstressed and should in time become second nature to the operator.page 17
|2.||Select a low power objective (10x) and locate it in the optical path. While viewing from the side of the microscope, place a suitable slide on the stage in the aforementioned manner. Carefully lower the objective by the coarse motion to within a short distance of the cover glass. Look through the eyepiece and focus the image sharply with the fine adjustment knob.|
|3.||Fully open the sub-stage condenser aperture iris. If a detached light source is used via a collecting mirror make sure that the plane side of the mirror is facing the light source and that the field of view is uniformly illuminated. This can be checked by removing the eyepiece and manipulating the light so that the back lens of the objective is completely filled with light.|
|4.||Rack the condenser to its upper limit. If it embodies a swing-out top lens, place it into operation. Now bring the whole assembly down till the granular diffuser of the light source becomes visible through the specimen. In order to facilitate the location of this image it is suggested you either close down the condenser iris or rotate the illuminator back and forth on its axis. Having thus found the focal plane, slowly move the condenser upwards until the frosted image has just disappeared. If the above adjustments have been carried out correctly, the top lens of the condenser should be seated slightly down from the underside of the stage.|
Finally, rotate the nosepiece to the required objective that is to be used for observation. Remove the eyepiece and look down the tube at the objective. Take care when doing this so that dust does not enter the tube or eyepiece. By using the aperture iris, mask the visible field by approximately one third. It is important to remember that each time a different objective is used the aperture must be correspondingly adjusted.
A point of caution here. — Never control the light intensity by using the iris diaphragm on the condenser as the resolving power will be adversely affected. Should the image be too bright, regulate the lamp voltage or insert a filter (e.g. neutral density or blue) between condenser and light source.
Setting up for Kohler Illumination
Observe and carry out steps 1 and 2 as previously described under Critical Illumination'.
|A.||Open fully the lamp field stop (diaphragm installed in the microscope base). Rack the condenser with the top lens in place to the upper end of its travel, i.e. to the underside of the stage. If your microscope has an auxiliary bottom condenser lens, put it into operation.|
|B.||Having focused on to the specimen, almost fully close down the field stop. Lower the condenser slowly until the image of the diaphragm edge is focused sharply within the specimen. This circle page 18 of light, also called the radiant field stop, must be correctly centred so that it is set in the middle of the field of view. To align it use the centering screws located on the condenser carrier. Microscopes fitted with a fixed condenser, i.e. without centering screws, are adjusted by moving the lamp within its socket mount until the field of view appears centralised. This type of instrument will usually have a ‘sliding insert’ which is located above the lamp assembly. Use this to make the final centering correction. If neither of the above centering descriptions seem to apply, consult the operating instructions supplied with your microscope.|
|C.||Open the radiant field stop until the edge of the diaphragm has just moved beyond view. Finally, check that the whole field is uniformly illuminated. Should it require adjustment, reposition the lamp housing as outlined above.|
Then carry out step 5 as described under ‘Critical Illumination’.
|(i)||When using scanning objectives, (i.e. objectives with initial magnifications between 2.5 and 4x), swing out the condenser front lens and open the condenser iris.|
|(ii)||Before setting a microscope up for Kohler illumination check if it has a filter between the sub-stage condenser and the radiant field stop. If it has, make sure that it is of the clear type and not frosted glass. The matt surface will obstruct the image of the field stop iris and will thus make it impossible to achieve Kohler illumination.|
|(iii)||Double immersion is the means by which maximum aperture utilisation of a given lens system is obtained. When the condenser's front lens has a high N.A. value, e.g. 1.25 or greater, immersion oil is applied between the underside of the specimen slide and condenser front lens. This will ensure the highest concentration of light rays falling within the objective's front lens aperture.|
|(iv)||If unstained specimens are to be used, close down the condenser aperture till approximately half of the objective's field is masked. This will improve the contrast of the specimen. However, care must be taken not to close down too much, as this will then be at the expense of losing resolution. Unstained specimens are best examined under phase contrast or interference phase contrast. (Methods used for preparations poor in contrast.)|
|(v)||A brief note on synthetic immersion oils. Recent research has shown that most modern immersion oils contain harmful and toxic chemicals, in the form of PCB compounds (polychlorinated biphenyls). When using immersion oil strict care should be taken not to transmit any part of it to the mucous membranes, i.e. lips, tongue, nostrils and eyelids. After use it must be page 19 washed immediately from the hands as it can be absorbed through the skin. PCB-free immersion oil can be obtained, however, from one known manufacturer (Carl Zeiss, West Germany).|
Hints on Maintenance of the Microscope
Remove dust and dirt from the microscope body, i.e. painted surfaces, with a damp cloth (never use harsh or abrasive solvents on any part of the microscope) and if required use a small amount of hand soap. Dry and polish with a soft linen cloth. When the above exercise is carried out always remove the objectives and eyepieces (replace with dust plugs).
The optical components of the microscope such as eyepieces, objectives and condenser should never be attempted to be dismantled. Clean only the outer surfaces in the following manner:
Remove loose dust with a degreased (in ether) camel-hair brush or a soft ‘brush-blower’. However, check that the inside of the ‘blower’ is of a type that is chalk-free. Rigid and encrusted dirt should be removed by firstly breathing on to the lens surface and then cleaning with a clean, lint-free cloth. Avoid using circular motions that produce harmful ‘sandpaper’ effects. Lateral movement is far better as it minimises the risk of scratching the lens surface. Should a closer inspection (e.g. with a magnifier) reveal still some residual dirt, try using a cotton wool applicator moistened with a little xylol. (Never use alcohol as this may dissolve the cement between lens elements.) Note of caution here is to remember that xylol is carcinogenic so that inhalation or contact with the skin must be avoided. This also applies to benzene which is frequently used as a cleaner. If possible safer alternative solvents should be used, e.g. diethyl ether. It goes without saying that all chemicals should be used sparingly and with care.
Never attempt to oil any part of the microscope. Special lubricants have been used in its manufacture. If it requires servicing due to a stiff focusing mechanism, condenser drive or a tight mechanical stage, take the microscope to an authorised workshop.
Finally, protect the instrument against dust by covering it when not in use, e.g. plastic cover or placed in a cabinet.
In conclusion, remember that the microscope is only as good as the operator and its performance will be dictated by the user's own technique.