MICROSCOPY
Modern medical training as well as clinical diagnosis utilises the facility of microscope (Fig. 1.1) to such an extent that today it has become an indispensable tool for a practitioner, student, teacher, research worker. Fine structural organisation could only be appreciated after the advent of microscope. Today, a range of different microscopes from simple magnifying lens to electron microscope are utilised for various purposes in the medical, biological and geological studies. Selection of the individual type of microscope depends upon the purpose of the investigator.
4How microscopy has evolved and reached to its present minitext as since 1695 when Robert Hook made first compound microscope, the microscopy had undergone tremendous improvement to reach the present day perfection. However, certain types of use in medical and biological field are discussed here.
Before various types of the microscopes are described a word about microscopy is essential. Microscopy is a technique by which a small object is magnified and observed without compromising with the resolving power of the eye and the colour of the object. Microscopy works on the basic principles of optics. In electron microscopy, the same principles are transpolated by the use of a series of electromagnets used for converging or diverging the beam of electrons. Thus, broadly microscopy is of two types viz. Light Microscopy and Electron Microscopy. Light microscopy involves the use of either artificial composite light or monochromatic light or natural sunlight while electron microscopy involves the use of a beam of electrons which, by virtue of refraction/diffraction or absorbance through a series of converging/diverging electrostatic plate lenses or magnets, renders the view of the object as enlarged one. The efficiency of the instrument or system depends upon resolving power of the observer and the instrument and non-splitting of incident light into a diffuse hand of coloured light, which may occur on account of prismatic effect of the lenses. In this chapter only light microscopes are introduced. There are following types of light microscopes.
SIMPLE MICROSCOPES
Simple microscopes are of various types ranging from simple biconvex magnifying lens to a combination of two to three lenses. These work on the principle of simple biconvex lens. The object lies within the focus of single or composite lens and eye also remains close to the lens. The image formed is virtual, erect and at 10″ away from the eye. Physically we come across many a type, e.g., simple examination loop, simple magnifier, dissecting microscopes and binocular magnifier. A good simple microscope would have optics from chromatic aberration and spherical aberrations. The magnification of the simple microscope may range from 2× to 20×. In 20× magnification, simple microscope has to be too near the object and thus 5renders dissection impossible. Further, these simple microscopes do not give a three dimensional appreciation of the object in a way similar to most of the compound microscopes to be described later. They are of immense use in preliminary screening examination of the object and also in the event where dissection of, or effect of manocuring with the object has to be viewed.
COMPOUND MICROSCOPES
These are combinations of a double sets of lenses. One set of lens lies in the proximity of the object while the other is near the eye. Accordingly, the two sets are called objective and eye- piece. There are various types of compound microscopes to meet the requirements of diversified field of study.
- Monocular compound microscope with vertical tube
- Inclined monocular compound microscope
- Binocular compound microscope
- Trinocular compound microscope
- Phase contrast microscope
- Polarising microscope
- Ultraviolet microscope
- Ultra microscope
- Fluorescence microscope
- Interference microscope.
These various types differ either in their light sources or in their lenticular system, but they all have eye pieces and objectives in common. These days it is a fashion to refer to even electron microscopic structures either as ultrastructure or ultramicroscopic view of the object. In fact, ultramicroscope is quite different from an electron microscope. The former one is a light microscope while the later utilizes a beam of electrons. The ultra microscope is basically utilised for detecting the matter of particulate size and works on the principle of Tyndall effect. It incorporates double objective and a ray of right perpendicular to the optical axis in jet dark field. It is used to detect the size of the colloid particles.
Parts of a Compound Microscope
In case of negative eyepiece The focus is inside the lenticular system with diaphragm in the ocular. This cannot be utilised for simple microscopy, because the image is formed from objective at its diaphragm.
In case of positive eyepiece The focus is outside the lens and primary image is formed within the diaphragm. Accordingly, later one can be utilised for simple microscopy. Former one is also known as Hygenian eyepiece invented by Christian Hygens while later is also known as Ramsden eyepiece. Routinely, microscopes are supplied with eyepieces of the magnifications 6×, 10×, and 15×. In some microscopes, an additional eyepiece of 20× may also be provided. For Binocular microscope, these eyepieces are supplied in pairs.
Objectives
These are functionally of three types:
- In those which have not been corrected either for colour aberrations or spherical aberrations
- Achromatic objective which have been corrected for one colour
- Apochromatic objectives, those which have been corrected for two colours. From the use point of view there are two types: dry objectives and wet or immersion objectives. Magnifications of these objectives range from 3× to 100×. Dry lenses usually have magnification of the order of up to 45× while objectives with magnification above 60× are usually wet or immersion type. Wet or oil immersion types of objectives are better if have a double barrel, i.e., one sliding in the other on a spring mount, such construction adds to the safety of the slide and the objective itself.
Body
It consists of several parts, viz., body tube, stage, mechanical vernier, stage body proper with rank and pinion adjustment for coarse and fine adjustment and the stand. Body tube houses on the top, the draw tube while on the lower side it houses the nosepiece carrying a battery of objectives. Stage is a mechanical platform having a central hole for light to pass. The stage may be circular capable of rotatory motions coupled with vernier graduation in degrees of the movement or may be of fixed 7square type where movement of the entire stage is not needed. Body proper hinges along a joint on the tripod stand. The hinge joint enables the observer to tilt the microscope to adjust the proper illumination as well as observe angle. An angle of 40 degree is adequate enough to enable the observer to observe without any strain for prolonged periods. The stage has on its lower side a condenser with iris diaphragm attached with the body proper through another rack and pinion adjustment screw by which the condenser can be brought to desired heights below the stage. Such movements are necessary to adjust the amount of light needed for different objectives of varied magnifications. The body as such may be of two types, in one the stage is fixed and the body tube can be moved up or down while in other, the body tube is fixed and the stage is moved up or down. In any case these movements are controlled by rack and pinion operated fine and coarse adjustments.
Condensers
The purpose of the condensers is to bring the light rays from source of illumination to be focussed at the plan of the specimen. The concave mirror attached at the bottom of the instrument serves as a condenser for objective of low power. For high power objectives, this condenser is inadequate and a special substage condenser is required with which concave mirror should not be used. Plain mirror is used for directing the light beam to condenser. The substage condenser may be of many types. The two lens type condenser is uncorrected for spherical and chromatic aberrations while four lens condenser may be corrected one either for spherical or chromatic aberration or for both. There are several type of condensers meeting the specialised requirements, but for routine work the Abbe condenser with numerical aperture 1.20 or 1.25 gives satisfactory performance with objective of nearly all available power. To name a few others are Watson holoscopic condenser, Cardiod condenser, Dark field condenser and Rheinberg differential colour illumination condenser. As they are also not routinely used, they are discussed in brief in this chapter.
Vernier Stage
This is a detachable device of immense importance for scanning or examining desired and several fields of the object without 8repetition. It is fixed on the stage and has a spring finger which fixes the slide as if in a claw and the entire set can be moved in two directions over the stage perpendicular to each other on the horizontal plane of the stage. The slide can be moved with the help of two screws 25 mm forward and 75 mm sidewards on the stage proper. In medical microscopes, the vernier mechanical stage is attached at right working hand side and requires special care for operation, because it does not sustain the untender and hard use.
Coarse and Fine Adjustments
These are provided on either side of the ordinary fixed stage compound microscope or fixed body tube microscope. The coarse adjustment is identified by bigger size of the knob while fine adjustment has a smaller knob. With practice objects can be focussed very clearly with the aid of coarse adjustment only under ordinary high power objective while under oil immersion objective, the object could be brought to near focus. Fine adjustment in some microscopes is provided with either a vernier scale or the entire knob or drum divided into 100 or 50 equal divisions. By this graduation movements of body tube or the stage in the optical axis can be monitored in the tune of 0.5 to 2.0 microns. This device helps to measure/caliberate the thickness of the slide, coverslip, object (section thickness) or even the refractive index of the liquids.
Illumination
Routinely, day light illumination is used. When artificial light is to be used then the illumination is achieved with the help of microscopical lamps which may give adequate illumination. For better illumination special illuminators based on Nelson's principle of critical illumination of Kohler method of illumination are employed. On the later principle there is available “Pancratic Condenser” manufactured by Zeiss which has a built-in illuminating device for all powers of objectives.
ELECTRON MICROSCOPE
By substituting a beam of electrons instead of light rays, a deface of resolution of much greater deface, could be obtained. 9Since at the subjection of 50,000 volts; electrons exhibit a wavelength of 0.001 µ only, a resolving power of 0.00005 µ could be applied enabling the molecules to be seen thereby. The use of special hard planters, special value microtomes, and steel knives cannot be used to cut these sections. Glass or diamond knives could be used instead.
Terminologies used in Microscopy
- Resolving power of the eye It indicates the capacity of eye to differentiate in between two points kept close to each other. Resolving power of the eye is highest for monochromatic yellow light (575 millimicron) next in order are green, red and blue. It also depends on the quantity of light. Optimum light intensity for maximum resolving power is light of 10 to 20 foot candles. Under best illumination eye can distinctly separate 1/2000th part of an inch.
- Magnification of the microscope Usually it is calculated by multiplying the magnifying power of the eyepiece with the magnifying power of the objective. But taking into consideration the resolving power of the eye only the magnification capable of separating 1/100th of an inch would be useful. Further magnification shall cause strain over the eye.
- Optical index It is an index of the usefulness of the objective and is calculated as follows OI = NA × 1000/objective magnification. OI = Optical index, NA = Numerical aperture. High optical index indicates high resolving power.
- Numerical aperture It is the product of the refractive index of the medium in front of the lens and sine of the half of the angular aperture. NA = n sine u, n = refractive index, u=half of the angular aperture. High numerical aperture indicates high resolving power of the lens system.
- Angular aperture The angle of the light rays taken in the objective from a point on the subject.
- Units Micron=0.001 mm, 1 millimicron=0.000001 mm (10−6 mm) etc.1 Angstrom (A) = 0.1 millimicron or 0.0000001 mm (10−7 mm)
Cover glass thickness = 0.17 = 0.18 mm
Additional magnifications due to:
a. Binocular head | = | 1.5 times. |
b. Camera | = | 1.5 times usually. |
Precautions in Handling Microscope
- Always clean all the lenses with fine linen cloth or tissue lens paper and if necessary with little xylene. Avoid excess use of xylene as it may dissolve the lens cement.
- Always focus first under low power then gradually increase power. When using oil immersion objective only cedar wood oil is to be used because it has same refractive index as that of glass and least dispersion of light rays occurs. If the oil has gone too thick it should be discarded. It should not be used after thinning it with xylene.
- Primary adjustments are to be made with coarse adjustment and then high power, and then if need be then oil immersion lens is to be used.
- When examination is done under low power the mirror could be concave and condenser slightly lower than the stage. But a low situation of condenser with plain mirror is preferred. When high power or oil immersion lens is used the condenser is kept on top situation and plain mirror is used.
- When oil immersion is used a drop of oil cover condenser is also desired but should only be used when being directed to do so.
- If the objectives and condensers are not par focal (same optical axis) then centering is achieved through the help of a mechanic.
- Each microscope should be thoroughly checked by mechanic at least once a year to ensure long trouble-free service.