The light microscope is called so because it uses visible light to detect small objects. It is the most well-known and commonly used research tool in biology, as well as in routine clinical labs for diagnostic purposes. Because the light microscope is a delicate and expensive instrument, special care must be taken while using, cleaning and storing it. In this article, we will discuss the various parts, usage, handling, and care of a light microscope.
Light microscopes are used in many clinical lab departments to magnify structures or cells too small to be seen with the naked eye. They are used to evaluate stained blood smears and tissue sections, perform cell counts, examine urine sediment, observe cellular reactions and observe and interpret smears containing micro-organisms.
Magnification, however, is not the most important issue in microscopy. Magnification alone without added detail and clarity is not useful. It would be similar to enlarging a small photograph which will not reveal any more detail but will only increase the blurring. The main function of any microscope is to produce a better resolution than the eye. Resolution is the ability to differentiate between two objects as separate entities, and not seeing them as a blurred image or as a single smudge. There is a limit to the degree of magnification that can be obtained with the microscope and still yield a clear image.
Electron microscope in comparison with a light microscope is used by scientists and in research labs to get detailed knowledge of even the smallest microorganisms or cell organelles. The light microscope uses light (approx wavelength 400-700 nm) as the source of illumination while the electron microscope uses a beam of electrons as the source of illumination. The resolving power of the electron microscope (0.001µm) is about 250 times higher as compared to a light microscope (0.25µm to 0.3µm) Light microscope has a magnification of 40 X to 1500X while electron microscope has a magnification of about 100,000X to 300,000X.
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Parts of Light Microscope
The compound bright-field microscope is the type used in most clinical labs.
A compound microscope has 2 lens systems. The lens system nearest the eyes is the ocular (eyepiece) lens. The other lens system is the objective that is nearest to the object being viewed.
A microscope may be monocular or binocular.
Monocular microscopes have only one eyepiece. They are inexpensive but cause eyestrain.
Binocular microscopes have 2 eyepieces to allow viewing with both eyes, which results in less eyestrain.
The oculars or eyepieces located at the top of the microscope are attached to a barrel or tube connected to the microscope arm. The oculars through which object is viewed contain lenses that magnify objects.
The underside of the arm contains a revolving nose piece to which the objectives are attached. Most microscopes have at least 3 objectives or magnifying lenses
Low power objective which magnifies 10 times.
High power objective which magnifies 40 times.
Oil immersion objective which magnifies 100 times.
Each objective is marked with color-coded bands and the power of magnification.
To determine the degree of magnification, multiply the magnification listed on ocular (usually 10 x) by the magnification listed on the objective being used.
Eg. An object viewed with a 10 X ocular and 40 X objective would be magnified 40 times.
While an object viewed with a 10 X ocular and 100 X objective would be magnified 1000 times.
Light Source, Condensor and Diaphragm
The arm of the microscope connects the objectives and eyepieces to the microscope base which supports the microscope. The base also contains the light or mirror which supplies light to the object viewed. The light or mirror has a movable condenser and iris diaphragm located above it. The condenser focuses or directs the available light into the objective as it is raised or lowered. The iris diaphragm located in the condenser unit regulates the amount of light that strikes the object being viewed. The iris diaphragm may be adjusted by a movable lever.
Coarse and fine adjustments
The two focusing knobs may also be located just above the base. The coarse adjustment is used to focus with the low power objective only. The fine adjustment is used to give a sharper image after the object is brought into view with the coarse adjustment.
Working distance is the distance between the objective and the slide when the object is in sharp focus. The higher the magnification of the objective, the shorter the working distance will be.
The stage of the microscope is supported by the arm and is located between the nose piece and the light source. It serves as the support for the object being viewed i.e., the slide and has a stage clip to keep the slide stationary. The stage can be moved by using knobs located just below the stage. These move the stage left and right or backward and forward.
Using the Light Microscope
Adjusting oculars on binocular microscopes
These must be adjusted for each individual eye. The ocular (interpupillary distance) should be adjusted so that one image is seen. The object is then brought into sharp focus with the coarse and fine adjustments while looking through the right ocular with the right eye. The right eye is then closed and the knurled collar on the left ocular is used to bring the object into sharp focus while viewing the object through the left ocular with the left eye. This is called dioptic adjustment.
Using the objectives
Low power objective
It is used for initially locating objects and for viewing large objects. The slide is secured, specimen side up on the stage with the clips. Low power objective is rotated into position and microscope light is turned on. The coarse adjustment is used to bring the objective and the slide as close together as possible. Then while looking through the ocular, the coarse adjustment is used to move the objective and slide apart until the object on the slide comes into focus. A clearer image is then achieved by focusing with the fine adjustment.
High power objective
It is used when greater magnification is needed. After initial focusing, with the low power objective, the high power objective is carefully rotated into position. The fine adjustment is used to bring the object into sharp focus.
Most microscopes are parfocal and therefore require only slight changes in the fine adjustment.
Use only the fine adjustment when using high power objectives.
Oil immersion objective
It gives the highest magnification of all bright field objectives. After initial focusing with low power, the objective is slightly rotated to the side. A drop of immersion oil is placed on the slide directly over the condenser. The oil immersion objective is then carefully rotated into the drop of oil taking care not to allow any other objective to come in contact with the oil. Fine adjustment is used to focus the object. Coarse adjustment should not be used when the oil immersion objective is in position. Immersion oil should never be used on any objective other than the one marked oil immersion.
When the slide has been examined, the low power objective is rotated into position and the slide is removed from the stage. All oil should be cleaned from the objective with lens paper.
Adjusting the light
Condenser and diaphragm must be adjusted according to the objective being used and the type of specimen being observed.
When viewing objects using the low power objective, the condenser may need to be lowered somewhat to reduce the brightness of the light.
The condenser should also be lowered and the iris diaphragm adjusted when viewing unstained specimens such as urine sediments or cell dilutions to be counted with high power objectives. This provides more contrast between the constituents being viewed and the background.
The condenser should be raised and the diaphragm opened when viewing most stained preparations with the high power objective. When viewing specimens such as stained blood smears with the oil immersion objective, the condenser should be raised until it is almost touching the bottom of the slide. The diaphragm should be completely open to provide maximum light.
If the illumination is built-in, make sure that the lamp voltage is turned down before switching on the microscope, then turn up the lamp until it is around 70 % of maximum power.
Care of Light Microscope
- The microscope should be left in a permanent position on a firm surface so that it does not vibrate.
- While transporting, it should be held securely with one hand supporting the base and the other holding the arm.
- The underside of a glass slide should be completely dry before it is placed on to the stage.
- It is always advisable to start viewing with the low power objective (10 X).
- The specimen should be viewed with or without oil depending on the type of objective employed.
- Microscope lenses should be cleaned with lens paper before and after each use. A little xylene may be used if dirt is difficult to remove. Never use alcohol because it can dissolve the cement used to keep the lenses together.
- Never leave the lenses with oil on them because the oil will soften the cement (glue) that holds the lens in the objective.
- When the microscope is not being used, it should be left with the low power objective in position and the nosepiece in the lowest position. The stage should be centered so that it does not project from either side of the microscope.
- The microscope should be stored under a protective cover.
- Never force the controls. If the movement of the focusing screws or mechanical stage becomes difficult, lubricate them with a small drop of machine oil.