CHAPTER II
COMPOUND MICROSCOPES
The =compound microscope= has undergone wonderful changes since 1667, the days of Robert Hooke. When we consider the crude construction and the limitations of Robert Hooke’s microscope, we marvel at the structural perfection and the unlimited possibilities of the modern instrument. The advancement made in most sciences has followed the gradual perfection of this instrument.
The illustration of Robert Hooke’s microscope (Fig. 7) will convey to the mind more eloquently than words the crudeness of the early microscopes, especially when it is compared with the present-day microscopes.
STRUCTURE OF THE COMPOUND MICROSCOPE
The parts of the compound microscope (Fig. 8) may be grouped into--first, the mechanical, and, secondly, into the optical parts.
THE MECHANICAL PARTS
1. The =foot= is the basal part, the part which supports all the other mechanical and optical parts. The foot should be heavy enough to balance the other parts when they are inclined. Most modern instruments have a three-parted or tripod-shaped base.
2. The =pillar= is the vertical part of the microscope attached to the base. The pillar is joined to the limb by a hinged joint. The hinges make it possible to incline the microscope at any angle, thus lowering its height. In this way, short, medium, and tall persons can use the microscope with facility. The part of the pillar above the hinge is called the _limb_. The limb may be either straight or curved. The curved form is preferable, since it offers a more suitable surface to grasp in transferring from box or shelf to the desk, and _vice versa_.
[Illustration: FIG. 7.--Compound Microscope of Robert Hooke]
3. The =stage= is either stationary or movable, round or square, and is attached to the limb just above the hinge. The upper surface is made of a composition which is not easily attacked by moisture and reagents. The centre of the stage is perforated by a circular opening.
4. The =sub-stage= is attached below the stage and is for the purpose of holding the iris diaphragm and Abbé condenser. The raising and lowering of the sub-stage are accomplished by a rack and pinion.
5. The =iris diaphragm=, which is held in the sub-stage below the Abbé condenser, consists of a series of metal plates, so arranged that the light entering the microscope may be cut off completely or its amount regulated by moving a control pin.
6. The =fine adjustment= is located either at the side or at the top of the limb. It consists of a fine rack and pinion, and is used in focusing an object when the low-power objective is in position, or in finding and focusing the object when the high-power objective is in position.
7. The =coarse adjustment= is a rack and pinion used in raising and lowering the body-tube and in finding the approximate focus when either the high- or low-power objective is in position.
8. The =body-tube= is the path traveled by the rays of light entering the objectives and leaving by the eye-piece. To the lower part of the tube is attached the nose-piece, and resting in its upper part is the draw-tube, which holds the eye-piece. On the outer surface of the draw-tube there is a scale which indicates the distance it is drawn from the body-tube.
9. The =nose-piece= may be simple, double, or triple, and it is protected from dust by a circular piece of metal. Double and triple nose-pieces may be revolved, and like the simple nose-piece they hold the objectives in position.
THE OPTICAL PARTS
1. The =mirror= is a sub-stage attachment one surface of which is plain and the other concave. The plain surface is used with an Abbé condenser when the source of light is distant, while the concave surface is used with instruments without an Abbé condenser when the source of light is near at hand.
[Illustration: FIG. 8.--Compound Microscope
Eyepiece Draw Tube Body Tube Coarse Adjustment Revolving Nosepiece for three Objectives Fine Adjustment Stage Objectives Limb Abbi Condenser Iris Diaphragm Hinge for Inclining Substage Attachment Mirror Pillar Foot]
2. The =Abbé condenser= (Fig. 9) is a combination of two or more lenses, arranged so as to concentrate the light on the specimen placed on the stage. The condenser is located in the opening of the stage, and its uppermost surface is circular and flat.
[Illustration: FIG. 9--Abbé Condenser]
3. =Objectives= (Figs. 10, 11, and 12). There are low, medium, and high-power objectives. The low-power objectives have fewer and larger lenses, and they magnify least, but they show more of the object than do the high-power objectives. There are three chief types of objectives: First, dry objectives; second, wet objectives, of which there are the water-immersion objectives; and third, the oil-immersion objectives. The dry objectives are used for most histological and pharmacognostical work. For studying smaller objects the water objective is sometimes desirable, but in bacteriological work the oil-immersion objective is almost exclusively used. The globule of water or oil, as the case may be, increases the amount of light entering the objective, because the oil and water bend many rays into the objective which would otherwise escape.
[Illustration: FIG. 10.]
[Illustration: FIG. 11.]
[Illustration: FIG. 12. Objectives.]
4. =Eye-pieces= (Figs. 13, 14, and 15) are of variable length, but structurally they are somewhat similar. The eye-piece consists of a metal tube with a blackened inner tube. In the centre of this tube there is a small diaphragm for holding the ocular micrometer. In the lower end of the tube a lens is fastened by means of a screw. This, the field lens, is the larger lens of the ocular. The upper, smaller lens is fastened in the tube by a screw, but there is a projecting collar which rests, when in position, on the draw-tube.
[Illustration: FIG. 13.]
[Illustration: FIG. 14.]
[Illustration: FIG. 15. Eye-Pieces.]
The longer the tube the lower the magnification. For instance, a two-inch ocular magnifies less than an inch and a half, a one-inch less than a three-fourths of an inch, etc.
The greater the curvature of the lenses of the ocular the higher will be the magnification and the shorter the tube-length.
FORMS OF COMPOUND MICROSCOPES
The following descriptions refer to three different models of compound microscopes: one which is used chiefly as a pharmacognostic microscope, one as a research microscope stand, while the third type represents a research microscope stand of highest order, which is used at the same time for taking microphotographs.
[Illustration: FIG. 16.--Pharmacognostic Microscope]
PHARMACOGNOSTIC MICROSCOPE
The =pharmacognostic microscope= (Fig. 16) is an instrument which embodies only those parts which are most essential for the examination of powdered drugs, bacteria, and urinary sediments. This microscope is provided with a stage of the dimensions 105 × 105 mm. This factor and the distance of 80 mm. from the optical centre to the handle arm render it available for the examination of even very large objects and preparations, or preparations suspended in glass dishes. The stand is furnished with a side micrometer, a fine adjustment having knobs on both sides, thereby permitting the manipulation of the micrometer screw either by left or right hand. The illuminating apparatus consists of the Abbé condenser of numerical aperture of 1.20, to which is attached an iris diaphragm for the proper adjustment of the light. A worm screw, mounted in connection with the condenser, serves for the raising and lowering of the condenser, so that the cone of illuminating pencils can be arranged in accordance to the objective employed and to the preparation under observation. The objectives necessary are those of the achromatic type, possessing a focal length of 16.2 mm. and 3 mm. Oculars which render the best results in regard to magnification in connection with the two objectives mentioned are the Huyghenian eye-pieces II and IV so that magnifications are obtained varying from 62 to 625. It is advisable, however, to have the microscope equipped with a triple revolving nose-piece for the objectives, so that provision is made for the addition of an oil-immersion objective at any time later should the microscope become available for bacteriological investigations.
THE RESEARCH MICROSCOPE
The =research microscope= used in research work (Fig. 17) must be equipped more elaborately than the microscope especially designed for the use of the pharmacognosist. While the simple form of microscope is supplied with the small type of Abbé condenser, the research microscope is furnished with a large illuminating apparatus of which the iris diaphragm is mounted on a rack and pinion, allowing displacement obliquely to the optical centre, also to increase resolving power in the objectives when observing those objects which cannot be revealed to the best advantage with central illumination. Another iris is furnished above the condenser; this iris becomes available the instant an object is to be observed without the aid of the condenser, in which case the upper iris diaphragm allows proper adjustment of the light. The mirror, one side plane, the other concave, is mounted on a movable bar, along which it can be slid--another convenience for the adjustment of the light. The microscope stage of this stand is of the round, rotating and centring pattern, which permits a limited motion to the object slide: The rotation of the microscope stage furnishes another convenience in the examination of objects in polarized light, allowing the preparation to be rotated in order to distinguish the polarization properties of the objects under observation.
[Illustration: FIG. 17.--Research Microscope]
[Illustration: FIG. 18.--Special Research Microscope]
SPECIAL RESEARCH MICROSCOPE
A =special research microscope= of the highest order (Fig. 18) is supplied with an extra large body tube, which renders it of special advantage for micro-photography. Otherwise in its mechanical equipment it resembles very closely the medium-sized research microscope stand, with the exception that the stand is larger in its design, therefore offering universal application. In regard to the illuminating apparatus, it is advisable to mention that the one in the large research microscope stand is furnished with a three-lens condenser of a numerical aperture of 1.40, while the medium-sized research stand is provided with a two-lens condenser of a numerical aperture of 1.20. The stage of the microscope is provided with a cross motion--the backward and forward motion of the preparation is secured by rack and pinion, while the side motion is controlled by a micrometric worm screw. In cases where large preparations are to be photographed, the draw-tube with ocular and the slider in which the draw-tubes glide are removed to allow the full aperture of wide-angle objectives to be made use of.
[Illustration: FIG. 19.--Greenough Binocular Microscope]
BINOCULAR MICROSCOPE
The =Greenough binocular microscope=, as shown in Fig. 19, consists of a microscope stage with two tubes mounted side by side and moving on the same rack and pinion for the focusing adjustment. Either tube can be used without the other. The oculars are capable of more or less separation to suit the eyes of different observers. In each of the drub-like mountings, near the point where the oculars are introduced, porro-prisms have been placed, which erect the image. This microscope gives most perfect stereoscopic images, which are erect instead of inverted, as in the monocular compound microscopes. The Greenough binocular microscope is especially adapted for dissection and for studying objects of considerable thickness.
POLARIZATION MICROSCOPE
The =polarization microscope= (Fig. 20) is used chiefly for the examination of crystals and mineral sections as well as for the observation of organic bodies in polarized light. It can, however, also be used for the examination of regular biological preparations.
[Illustration: FIG. 20.--Polarization Microscope]
If compared with the regular biological microscope, the polarization microscope is found characteristic of the following points: it is supplied with a polarization arrangement. The latter consists of a polarizer and analyzer. The polarizer is situated in a rotating mount beneath the condensing system. The microscope, of which the diagram is shown, possesses a triple “Ahrens” prism of calcite. The entering light is divided into two polarized parts, situated perpendicularly to each other. The so-called “ordinary” rays are reflected to one side by total reflection, which takes place on the inner cemented surface of the triple prism, allowing the so-called “extraordinary” rays to pass through the condenser. If the prism is adjusted to its focal point, it is so situated that the vibration plane of the extra-ordinary rays are in the same position as shown in the diagram of the illustration.
The analyzer is mounted within the microscope-tube above the objective. Situated on a sliding plate, it can be shifted into the optical axis whenever necessary. The analyzer consists of a polarization prism after Glan-Thompson. The polarization plane of the
## active extraordinary rays is situated perpendicularly to the plane as
shown in the diagram. The polarization prisms are ordinarily crossed. In this position the field of the microscope is darkened as long as no substance of a double refractive index has been introduced between the analyzer and polarizer. In rotating the polarizer up to the mark 90, the polarization prisms are mounted parallel and the field of the microscope is lighted again. Immediately above the analyzer and attached to the mounting of the analyzer a lens of a comparatively long focal length has been placed in order to overcome the difference in focus created by the introduction of the analyzer into the optical rays.
The condensing system is mounted on a slider, and, furthermore, can be raised and lowered along the optical centre by means of a rack-and-pinion adjustment. If lowered sufficiently, the condensing system can be thrown to the side to be removed from the optical rays. The condenser consists of three lenses. The two upper lenses are separately mounted to an arm, which permits them to be tilted to one side in order to be removed from the optical rays. The complete condenser is used only in connection with high-power objectives. As far as low-power objectives are concerned, the lower condensing lens alone is made use of, and the latter is found mounted to the polarizer sleeve. Below the polarizer and above the lower condensing lens an iris diaphragm is found.
The microscope table is graduated on its periphery, and, furthermore, carries a vernier for more exact reading.
The polarization microscope is not furnished with an objective nose-piece. Every objective, however, is supplied with an individual centring head, which permits the objective to be attached to an objective clutch-changer, situated at the lower end of the microscope-tube. The centring head permits the objectives to be perfectly centred and to remain centred even if another objective is introduced into the objective clutch-changer.
At an angle of 45 degrees to the polarization plane of polarizer and analyzer, a slot has been provided, which serves for the introduction of compensators.
Between analyzer and ocular, another slot is found which permits the Amici-Bertrand lens to be introduced into the optical axis. The slider for the Bertrand lens is supplied with two centring screws whereby this lens can be perfectly and easily centred. The Bertrand lens serves the purpose of observing the back focal plane of the microscope objective. In order to allow the Bertrand lens to be focused, the tube can be raised and lowered for this purpose. An iris diaphragm is mounted above the Bertrand lens.
If the Bertrand lens is shifted out of the optical axis, one can observe the preparation placed upon the microscope stage and, depending on its thickness or its double refraction, the interference color of the specimen. This interference figure is called the orthoscopic image and, accordingly, one speaks of the microscope as being used as an “orthoscope.”
After the Bertrand lens has been introduced into the optical axis, the interference figure is visible in the back focal plane of the objective. Each point of this interference figure corresponds to a certain direction of the rays of the preparation itself. This arrangement permits observation of the change of the reflection of light taking place in the preparation, this in accordance with the change of the direction of the rays. This interference figure is called the conoscopic image, and, accordingly, the microscope is used as a “conoscope.”
Many types of polarization microscopes have been constructed; those of a more elaborate form are used for research investigations; others of smaller design for routine investigations.
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