In this article we will discuss about:- 1. Definition of Microscope 2. Objectives of Microscopic Components 3. Types 4. Components 5. Unit 6. Steps 7. Cure.
Definition of Microscope:
A microscope is a tool or machine with the ability to increase the visual size of all the objects making it easier to see. All types of microscopes must perform two important functions – they must magnify (enlarge) the specimen to see a size that can be seen by the human eye, and they must provide a clear image that will enable the microscopist to distinguish the component parts of the specimen—a feature known as resolution.
These may be accomplished by using visible (white) light, ultraviolet light or electron beams. Various forms of energy share certain qualities. One is that they behave as if they were waves. The wave like form of energy such as radio-waves, X-rays, and light make up the electron magnetic spectrum. A wave consists of high point (crest), followed by a low point (trought), and again rises to high point.
The distance from crest of one wave to the crest of next wave is one wavelength. In the electron magnetic spectrum, wavelength is usually measured in nanometers (nm). A nanometer is one billionth meter (10-9m) long.
In order to see the components of an object, light rays coming from the objects must be separated. If very two small objects move closer and closer, there will be a point where two will be seen one. The smallest distance between two objects at which they may be seen as separate objects is the resolving power or resolution of the lens system.
A human eye has resolving power of about 0.1 mm or 100 micrometers. This means that the person can tell by looking that there are two separate objects if they are separated by a distance of at least 0.1 millimeter. If they were any closer together, they would appear as a single object.
The resolving power of the lens system depends on the ability of light to pass between the objects being viewed. The shorter wavelengths of light are able to pass between the pair of close objects much easier than the longer wavelengths.
This means that the components of the specimen will be seen more clearly than the wavelength of the light being used. Thus we see that the shorter the wavelength of blue light provides the most clear images when viewed with a light microscope.
Skill in the use of the microscope must be acquired by practice, but if simple rules are followed, anyone can learn to use it without difficulty and with enjoyment in a short time. If each of the following steps are carried out carefully, in the order given, the beginner will find most of the difficulties solved.
Objectives of Microscopic Components:
These microscope components are considered to be the most important of the optical parts, primarily because they affect the quality of the image seen by the observer. The major objective types are in us –achromatic, apochromatic and fluorite. The first of these objective types is the least expensive and simplest in construction.
The latter two are more expensive and are used in more critical type of work as they are corrected for most defects commonly encountered. Most laboratory instruments are equipped with three objectives that have different magnifying powers – the low power, high power (or high-dry), and the oil immersion lenses.
Several microscope manufacturers differentiate the individual objectives by sets of differently coloured rings. For example, green is used for the low-power, yellow for the high-dry and red or black for oil immersion objectives. If not colour-coded, the individual objectives commonly can be distinguished from one another on the basis of their respective lengths (low power is the shortest, while the oil immersion lens is the longest).
The primary functions of the objective lenses include:
1. Gathering or concentrating the light ray coming from the specimen being viewed,
2. Forming the image of the specimen and
3. Magnifying the image and several important properties of microscope are directly associated with the objectives. One of these is resolving power (RP) or resolution, which is defined as the ability to distinguish clearly two points that are close together within the structure of a particular object. This feature is largely determined by the wavelength of light source (with a shorter wavelength providing finer detail) and the angular aperture of lens system being used. The resolution is also affected by refractive index of the medium through which light passes before entering the microscope objective.
The relationship of these factors is expressed in the combined formula:
Numerical Aperture (NA) = ƞ sinθ
where ƞ represents the refractive index of the medium through which the light passes before entering the objective lens; and sinθ is the trignometric sign of half the angle formed by the light rays (in the shape of the cone) coming from the condenser and passing through the specimen. Light in this form frequently is referred to as ‘a pencil of rays’.
The formula clearly depicts the explanation of numerical aperture. Values for NA are engraved on the barrel of objectives and are used to determine the maximum resolution obtainable.
Another important dimension of modern-day instruments is the property of par focal. Stated simply, this means the changing of objectives without major focusing adjustments. Thus, if a higher magnification is needed during the course of examining a specimen, one would just rotate the desired higher objective into place and make some minor focusing adjustment to bring the specimen into view.
The highest magnification microscope objective utilized in general courses is the oil-immersion on objective. It can magnify specimens 1000 times. In order to obtain best possible results, the objective is immersed in a medium which has approximately the same index of refraction as glass, specifically, 1.6. One medium commonly employed for this purpose is cedar wood oil. Other materials containing mineral oils also are in common use.
Oils have the advantage of not drying out when exposed to air for long periods of time. Further, oil provides an ‘optically homogenous path’ for light rays to pass from the specimen, through the oil, and into the front lens of the oil-immersion objective. An entirely different pitcher results if air is present between the specimen and the objective.
In this case, as some light is lost due to the air, the image observed usually is fuzzy, and the finer details cannot be seen. The resolving power of the oil-immersion objective definitely is enhanced by the oil medium. Individuals using this objective for the first time have a tendency to use large quantities of oil. One good drop of the medium usually is sufficient.
The oil-immersion objective is used in various phases of microbiology. To insure the effectiveness of the microscope component, care should be exercised to remove the oil from the objective and all other parts after use. Lens paper is usually employed for this purpose.
An adjustable mirror can be used with microscopes to reflect light up into the instrument’s condenser, thus aiding in the illumination of a specimen. Today, however, many instruments do not utilize a mirror.
Low Power Objective:
This objective is useful for the examination of protozoa and other larger micro-organisms and it may be used for study of colonies of growing organisms, but individual bacteria can scarcely be made out with this lens. The low power objective is usually much shorter than the other two, and it is certain to have much larger lens at its end than either of the others. Different manufacturers use various systems for marking objectives. The low power is often marked “3”, or “2/3”, (meaning 2 inch) or “16 mm”.
High Power Objective:
This objective is used in microbiology for the examination of living micro-organisms suspended in drops of water or other fluid. In most microscopes, the high power objective is longer and more slender than the low power objective, and the visible lens at its end is smaller than that of the low power, though still larger than that of the oil-immersion objective. It may be marked “6” or “1/6” (meaning 1/6 inch) or “4 mm”.
Oil Immersion Objective:
This objective is indispensable to the bacteriologists. It is always used for examination of stained smears of bacteria. The objective may be long or short, but it will always have a very small lens visible at its end. It is usually marked “oil immer” or “homogimmer” (meaning homogeneous immersion). Also the figure “1/12” (inch) “1.9” m.m or “1.8 m.m.” often are engraved upon it.
The figures 16 mm, 4 mm, etc., on the objectives refer to what is called the equivalent focal length. They give an idea as to the distance there will be between the end of the objective and the object when in focus. Thus, the low power objective will be in focus about 16 mm, above the object on the microscopic slide, but the oil immersion lens will be focused at less than 2 mm. above it.
Types of Microscopes:
i. The Dark Field Microscope:
In a dark cell as the dust in the air is practically invisible, but if a streak of sunlight crosses the room, the dust immediately becomes obvious as bright particles in the beam of light. Thus dust particles become visible because they refract the strong light, sending some rays in the direction of the observer. By this indirect method of illumination, very small objects, invisible by ordinary light, are made visible.
Specimens examined with this technique usually are seen as bright objects against a black or dark background. This is the effect opposite to the one obtained with bright field microscopy, where specimens usually appear darker against light backgrounds. The dark field procedure commonly is performed by fitting an Abbe condenser with an opaque disc or “dark field stop”.
In the dark field microscope, a special condenser fits into the sub-stage in place of the ordinary condenser. The center of the top lens of this special condenser is opaque, so that none of the central rays of light can pass through it, and the object is illuminated only with very oblique rays.
None of the light goes directly up the objective as in the ordinary way, but instead the light rays pass through the object almost at right angles to the objective and nearly parallel to the stage. Through the microscope, the field appears dark, but micro-organisms or other objects in the preparation stand out sharply as very bright retractile bodies just as the dust particles appear in the beam of light across the cellar.
The dark field microscope is used for the examination of unstained micro-organisms or other objects suspended in fluids. It is specially useful for the study of very small and delicate organisms, such as spirochetes, which are invisible or nearly invisible when viewed in the ordinary way. Extremely tiny particles, much smaller than the ordinary bacteria, may be seen in a dark-field preparation.
ii. Fluorescence Microscopy:
This type of microscopy provides a means of studying structural details and other properties of a wide variety of specimens. Such materials differ in ‘fluorescing power’ from their surroundings. This property of fluorescence is noted when such substances become luminous upon their exposure to ultraviolet rays (UVL).
When certain substances, including some dyes, fat, oil droplets and uranium ores are exposed to this form of radiation, they absorb the energy of the invisible ultraviolet light waves and emit it in the form of visible light waves.
The fact that particular dyes fluorescence is utilized in studies is concerned with some tissues, cells and bacteria. Dyes of this nature include acridine orange R, auramine O, primulin and thiazo yellow G. These substances apparently also possess a particular selective action for micro-organisms and their components. For example, the fluorescent dye auramine O is used in a detection procedure for Mycobacterium tuberculosis.
The dye, which glows yellow when exposed to UV, has a strong selective action for the wax like substances which in part comprise this organism. Auramine is applied to a smear of a sputum specimen suspected of containing M. tuberculosis. Excess dye is removed by washing. Then the stained preparation is examined in the dark with the aid of the so-called “fluorescence microscope”.
The presence of the Tubercle bacilli is indicated by the bright yellow organisms against a dark background (although the effect produced is similar to that observed with a dark-field microscope, the principals involved differ significantly).
Instruments used in this type of microscopic application do not differ optically or mechanically from the conventional microscope, but do require special filter systems. Many manufacturers provide complete units which in addition to these filters, include objectives, condensers and a suitable source of illumination. A special non- fluorescent type of immersion oil or glycerine is recommended for fluorescence microscope procedures.
iii. Differential Interference Contrast (DIC) Microscopy:
Differential interference contrast (DIC) microscopy is an optical method that makes use of the wavelike properties of light and the way in which those properties change when traveling through a specimen. As with phase-contrast microscopy, DIC microscopy depends on changes in the phase of the light wave as it passes through the specimen.
In this case, however, gradients in the refractive index in the sample (between the specimen and the surrounding medium, for example) are detected. In fact, the DIC image is really a map of the differences in phase along the direction, achieved by comparing the phase change of two nearby paths of light.
Because the biggest phase changes usually occur at cell edges (the refractive index is more constant within the cell), the outline of the cell typically gives a strong signal. The image appears three-dimensional as a result of a shadow-casting illusion that arises because differences in phase are positive on one side of the cell but negative on the opposite side of the cell.
The optical components required for DIC microscopy consists of a polarizer, an analyzer, and a pair of Wollaston prisms. The polarizer and the first Wollaston prism split a beam of light, creating two beams that are separated by a small distance along the direction. After traveling through the specimen, the beams are recombined by the second Wollaston prism.
DIC imaging provides a sensitive means for optical sectioning because this interference only occurs in the focal plane of the objective lens. This technique is especially useful for studying living, unstained specimens. By combining this technique with video microscopy, the dynamics of cellular morphogenesis cell division can be studied.
iv. Video Microscopy and Electronic Imaging:
The advent of solid-state light detectors has in many circumstances made it possible to replace photographic film with an electronic equivalent-that is, with a video camera or a digital-imaging camera. These developments have given rise to the techniques of video microscopy and electronic imaging. By placing a video camera in the image plane formed by the ocular lens, one can record and store the microscope images.
Video images, which are in analogue form, can be stored either on videotape or in a computer in digital format. However, images stored on videotape are of low fidelity, so most modern video microscopy is stored digitally, and an increasing number of such images are now produced digitally as well.
Digital storage allows images not only to be viewed in higher fidelity, but also to be enhanced with computer-based image processors, increasing the contrast and eliminating distracting defects of the optical system. These techniques have been particularly successful in applications to DIC and bright field microscopy for the study of cell motility and cell division.
v. Confocal Microscopy:
This technique is designed to improve the effective resolution along the optical axis of the microscope—that is, to provide depth selection in the specimen. In this way, structures in the middle of a cell may be distinguished from those on the top or bottom. Likewise, a cell in the middle of a piece of tissue can be distinguished from cells above or below it.
To understand this type of microscopy, it is first necessary to consider the paths of light taken through a simple lens. To understand what your eye would see, imagine placing a piece of photographic film in the plane of focus. Now ask how the images of other point of light placed further away or closer to the lens contribute to the original image.
As you might guess, there is a precise relationship between o, the distance of the object from the lens i, the distance from the lens to the image of that object brought into focus, and f, the focal length of the lens. This relationship is given by the equation
1/f = 1/0 + 1/i
The light arising from the points that are not focus covers a greater surface area on the film because the rays are still either converging or diverging. Thus, the image on the film now has the original point source that is in focus, with a superimposed halo of light from the out-of-focus objects.
If we were only interested in seeing the original point source, we could mask out the extraneous light by placing an aperture, or pinhole, in the same plane as the film. This principle is used in a confocal microscope to discriminate against out-of-focus rays. In a real specimen, of course, we do not have just a single extraneous source of light on each side of the object we wish to see, but a continuum of points.
vi. Phase Contrast Microscopy:
The phase contrast microscope is useful for visualizing cells or organisms because it permits viewing of cellular structures without the necessity of staining. Light passing through a cell of higher refractive index (one that has a greater ability to change the direction of a ray of light) than the surrounding medium is slowed down relative to the light that passes directly through the less dense background medium.
The greater the refractive index of the cell or cellular structure, the greater the retardation of light wave. Thus, when light passes through a cell, there is a slight alteration in the phase stage of the cyclic movement of the light wave.
The phase contrast microscope is designed to take the advantage of the difference in refractive indexes between structure of a cell, translating differences in the phase of light into changes in light intensity that are visible to the eye.
Even difficult to stain structures often are conspicuous under a phase contrast microscope because small phase changes give rise to high contrast images. Thus, with the phase contrast microscope, living organisms can be clearly observed in great detail without staining them, permitting the study of their movements in the medium in which they are growing.
Components of Compound Microscope:
The mechanical parts of the microscope are concerned with the support and adjustment of the optical parts, whose function is to make an enlarged image of the object which we see. The student should have a microscope at hand while the following description is being studied.
i. Base, Pillar and Inclination Joint:
The base and the pillar serve to support the entire instrument. They are made heavy in order to minimize vibration as much as possible. The inclination joint and the pillar permit the microscope to be tipped back to any degree desired by the observer.
ii. Arm and Body Tube:
The arm supports the body tube to which the principal lenses are attached. The newer microscopes are so made that the instrument may be carried by the arm, but this may be harmful to some older makes.
iii. Draw Tube, Resolving Nose-Piece; Tube Length:
Fitting inside the upper end of the body tube, which may be drawn upward within it fits the ocular. The purpose of draw tube is to adjust the tube length, that is, the distance between the top lens of the ocular above and the attachment of the objective into the resolving nose-piece below. The system of lenses in objectives and oculars is made to function best when ocular and objective are a definite distance apart, that is, at a definite tube length.
In Bausch and Lomb, Spencer and Zeiss microscopes, the proper tube length is 160 millimeters; in Leitz microscopes it is 170 millimeters. The draw tube is usually marked with a millimeter scale. The thickness of the nose piece (usually 5 millimeters) must be subtracted from the scale on the draw tube in order to adjust it to the right tube-length.
That is, instead of pulling out the tube to the mark 160 (or 170) on the scale, it should be placed at the mark 145 (or 155). The proper tube length in many microscopes is indicated by a line running completely around the draw tube. Some microscopes have no draw tube, and the proper tube length is fixed when the instrument is made.
When the draw tube is pulled out beyond the point which gives the best tube length, the image is larger, but not quite so distinct in outline.
iv. Coarse and Fine Adjustment (for Focusing):
The entire body tubes with its attached lenses are moved up and down by means of the rack and pinion of the coarse adjustment. The tube is likewise raised and lowered by very slight degrees by means of the fine adjustment. The purpose of these adjustments is to bring the object into focus, so that its outlines are sharp and clear.
Both the coarse and fine adjustment should be manipulated carefully, and especially the latter, for it is a very delicate machine. The range of fine adjustment screw is limited. At one end of its range, it comes to a stop, and at the other, it goes beyond the limit of movement and has no effect. It should always be kept near the mid-point.
v. Stage:
This is the part of the microscope on which the object to be examined is placed. In most bacteriological work, the object is a transparent smear or other preparation on a glass slide.
vi. Mirror:
The mirror collects and reflects light up into the microscope. One side of the mirror is a plane mirror, the other is a concave mirror. Since in most bacteriological work, a large amount of light is needed, the concave mirror is most useful, for it helps to concentrate the light. When a bright artificial light is used, the plane mirror may suffice.
vii. Sub-Stage Condenser and Diaphragm:
Before the light reaches the object on the stage, it is condensed and focused by passage through the large condensing lens, commonly called the Abbe Condenser, in the sub stage. The result is that the maximum amount of light is directed upon the object, a necessity when the higher powers of the microscope are used.
Often, however, an object is too brilliantly illuminated if all the light from the mirror passes to the condenser. For this there is placed beneath the condenser an iris diaphragm. The size of the opening in the diaphragm may be reduced to that of a pin head or any intermediate size by moving a hand lever, so that by this means, the amount of light admitted to the condenser can be very accurately controlled. If a diffuse light is required, the entire sub-stage may be lowered.
Unit of Microscopic Objects:
The unit of microscopic measurement is one thousandth part of a millimeter i.e. 0.001 mm, and it is called one micron and written as 1µ. The measurement is most easily carried out by the use of a micrometer placed in the eye piece, the ocular micrometer consists of a disc of glass with a scale engraved on it.
Unscrew the top lens of the ocular and place the disc (so that the numbers on it can be read) on the stop or diaphragm present in the eye piece, moving the stop up or down a little if necessary until the scale is in focus – screw on the top and place the ocular in the tube.
Special micrometer eye pieces are procurable, fitted with a scale and an arrangement by which the scale can be focused accurately. When the disc is in proper position, focus the divisions and numbers on it can be easily read on looking down the tube of the microscope.
We have to find out the actual size of one division of this ocular micrometer scale – this is accomplished by the use, with it, of a scale called the scale micrometer which is mounted on a slide, and has divisions of a definite value in microns engraved on it. The scale on the stage micrometer consists usually of 1 mm divided into a hundredth part of a millimeter, i.e. 0.01 mm = 10µ.
Calibration of the ocular micrometer with the aid of the stage micrometer is carried out as follows – the ocular micrometer having been placed in the selected eye piece, the stage micrometer slide is put on the microscope stage, and the scale on it is focused. There are now two scales in focus, that of the ocular and that of the stage micrometer.
Arrange them super-imposed one on the other, so that one of the lines of the ocular micrometer scale coincides exactly with a line on the stage micrometer scale, then look along the super imposed scales until a point is reached where another pair of lines is found, as far away from the first pair as possible, which also exactly coincide.
Count the divisions of the ocular scale and the stage scale in the interval between these two points, and calculate from these numbers, the value of one division of the ocular micrometer. For example, if 20 divisions of ocular scale are equal to 16 divisions of stage scale, each of which latter divisions we know is equal to 10µ, then the value in microns of the ocular scale is 16 x 10µ = 160µ, i.e., 20 ocular divisions 160µ; therefore one division of the ocular scale equals 8µ.
If a micrometer eye piece is not available, it is recommended that one ocular be kept, with this scale in it, for making measurements, and that the lower one, the X6, be used; a spare X6 is a convenience for this purpose. All that is now necessary is that the scale in this ocular shall be calibrated by means of stage micrometer, for the X10, division of the ocular scale has been found for each objective, the stage micrometer is removed, cleaned with xylol and put away.
Any microscopic object can now be substituted for the stage micrometer and measured directly with the ocular scale alone. All measurements must be made with the tube of the microscope in the same position as when the calibration is done, i.e., in this case with the tube down, and with the same ocular, otherwise the measurements will not be correct. It saves time and repetition of calculation.
If the actual values for 1, 2, 3……. and so on, divisions of the ocular micrometer scale, used in combination with each of the objectives, are set down on paper in tabular form and the table kept with the microscope. It is then possible to read off directly by reference to this table the size of objects, being the value in µ for the appropriate number of divisions of the ocular micrometer with the objective in use.
For examples, such a table might read as follows:
Minute Objects:
Very small objects below 10µ in size must be measured under the oil immersion objective if gross errors are to be avoided. Large objects, such as eggs of helminths, may be measured under the X40 objective.
Units of Measure:
In microbiology, as well as in other branches of the biological sciences, several common metric units are used to express the dimensions of microscopic objects under study. Until recently the micron, is symbolized by the Greek letter µ, with the basic unit of measurement. It measured one-thousandth (0.001 or 10-3) of a millimeter (mm).
The micron was further subdivided into one thousand units, each of which was called a millimicron (mµ). One mµ in turn made up 10 Angstrome units (Å). The relationships of these various units to one another, as well as to other components of the metric system and equivalent values of the English system, are given in the table.
Recently new prefixes to express lengths, volumes and weights have been adopted by the scientific community. In the case of length, nanometer (nm) and micrometer (µm) are used instead of the units.
Steps to be Followed While Using a Microscope:
1. Clean the microscope
2. Place the microscope towards a source of light and take a comfortable position at the instrument.
3. Place the object on the stage, swing into place the objective you wish to use, and while watching from the side, lower it to a point just under the position it will have when in focus.
4. Secure the proper amount of light by manipulating the mirror, sub-stage, condenser and diaphragm.
5. Focus, first with the coarse, then with the fine adjustment; always focus up.
6. Maintain the focus by continual manipulation of the fine adjustment.
1. Cleaning:
Obviously, dirty lenses will not give good images. The mirror, ocular, objectives, and condenser should be whipped free of dirt with a piece of clean lens paper or soft cotton. Do not grind the dirt into the lenses, but wipe off the dust lightly, the polish afterward. Do not attempt to take the ocular or objectives apart; clean the outside lenses only. Never touch the fingers to any of the lenses, as this will spoil them.
The high power and the oil immersion objectives must be perfectly clean. Immediately after the use of oil immersion objective, the excess oil must be wiped off with clean lens paper or cotton. If oil has dried on the lens, it may be removed by wiping the lens paper or cotton moistured with a little xylol.
2. Position at the Microscope:
Place the microscope on a table of convenient height, towards a source of light, and sit at ease before the instrument. With most preparations, the microscope may be tilted back slightly at the inclination joint, so that it is possible to look down the tube without straining the neck of course, when wet preparations are being examined, it will be necessary to have microscope erect.
Throughout microscopic examination, keep both eyes open. Look down the tube with either eye. Keep the eye very close to the ocular. Never strain to see; remember that the microscope is meant to be adjusted to suit your eyes. Try to keep your eyes at rest, just as if you were looking at some distant pleasant landscape.
If you do not see the object clearly, adjust the light by manipulation of the mirror and diaphragm, or correct the focus, until you do not see well. The more at ease you are, the more you will see, and the less fatigued you will be.
3. Placing Objective in Position:
Revolve the nose piece until the objective to be used snaps into place. All dry stained smears should be examined by the oil immersion object and no other. Wet preparations are examined by the high power objective when the oil immersion lens is to be used, place a drop of immersion oil direct upon the smear. Use plenty of fresh oil. Never use oil with other objectives.
Lower the body tube with the coarse adjustment, while watching from the side until the objective is approximately in its focal position and just below it. This means in case of oil immersion lens, here the objective must be lowered into the oil over the smear until it almost but not quite touches the slide. The high power objective should also be lowered as close to the slide as possible without actually touching the preparation. The low power objective should be lowered to about one quarter inch above the slide.
4. Securing Proper Illumination of the Object:
This is the most important step, though most often neglected by the beginner. If the illumination is correct, focusing and studying the object will be found to be a simple matter.
Before any attempt is made to focus, look into the microscope, and by manipulation of the mirror, secure an even bright light over the field. The concave side of the mirror will give the best light in most cases. The iris diaphragm should be wide open while the mirror is being adjusted, then, if the field is too brilliantly lighted, the light may be reduced by partially closing the diaphragm.
Use no more light than necessary, in order to spare the eye. When uncoloured microscopic objects in fluids such as living bacteria are to be examined with the low or high power objective, the light must be reduced to the level of dullness.
5. Finding the Object; Focusing:
Having secured good illumination, with the objective in the approximate focal position, the next step is to bring it into true focus. It is necessary to hold the object slide firmly with the fingers or by means of clips. Focusing must be done by raising the body tube very slowly with the coarse adjustment until the object is seen. The exact focus is then found with fine adjustment.
The body tube should never be lowered with the eye at the ocular, because the objective may be driven down so far as to break the slide or injure the lens. To focus up means, in most microscopes, it is important to turn the coarse adjustment screws towards the observer.
This must be done very slowly and carefully while the eye watches for the glimpse of the object and often the object can be more quickly located if the slide is moved slightly back and forth with one hand. As soon as the object can be seen, however dimly, the fine adjustment is brought into use and the exact focus found.
6. Keeping the Focus with the Fine Adjustment:
The exact focus is a very fine point. The slightest movement will throw the lenses out of position. In order to keep the object in focus, one hand must be continually on the fine adjustment. This adjustment should be moved a little, first one way, then the other, so as to reveal different levels of the object.
Although an object is focused for your eyes, it may not be for someone else, and when another person looks into your microscope, it will be necessary for him to move the fine adjustment until he sees the object clearly. A person accustomed to the use of the microscope always keeps one hand engaged in delicate manipulation of the fine adjustment.
Care of Microscopes:
The microscope should be kept in its box when not in use. If it is in fairly constant service, it must be protected during intervals of works, from dust and also from strong light, specially in the tropics. This can be done by wrapping around it a clean duster large enough to protect the condenser and mirror.
After use, the oil should be wiped off the immersion lens which is cleaned with a drop of xylol, and dried with lens tissue, a silk cloth or soft handkerchief kept for the purpose.
In cleaning oculars, objectives or the surface of the sub stage condenser, it is advisable not to rub them in a circular manner but wipe across them – it is essential that the cloth should be clean and free from particles of grit. Alcohol must not be used for cleaning lenses as it dissolves the cement in which they are set.
Dirt on Lenses, Condensers and Objectives:
If, on looking down the microscope, dust is seen, locate it as follows:
(a) Move the slide by means of the mechanical stage; if the dirt moves, it is on the object or slide.
(b) If the dirt does not move, rotate the eye piece while looking steadily down the microscope – if the dirt now moves, it is either on or in the eye piece.
(c) If neither of these tests moves the dirt, then it is either on the condenser or on the objective, wipe the condenser and objective clean; if this fails, unscrew the objective from the nose piece and wipe the upper surface of the lens gently with a soft handkerchief; breathing into the objective will, as a rule, only make the dust particles stick more firmly and should be avoided. The component parts of the objective lenses should be unscrewed except as a last resort, and only in cases where sending it to a maker is impossible. In order to prevent dust passing into the tube, an ocular should always be kept in it.
Oiling:
The friction surface of the moving parts of the microscope and mechanical stage should be oiled very sparingly with a little thin machine oil from time to time.