There are many methods for determining the focal length of the
objective. The objective to be examined is placed on the stage, and
in the manner just shown, the distance of the focal plane from the
edge of the fittings or to the surface plane of the front lens is determined.
Any plane object a few yards distant can be used. If the
object can be seen by using the mirror, the plane mirror must be
used; then the actual size of the object and of the image produced by
the objective is measured (of the image by a micrometer ocular).
The distance of the object from the nearer focus of the objective
is next determined. This distance is composed of the distance of the
object from the centre of the plane mirror, and of the distance of
the focus of the objective on the stage plate from the centre of the
plane mirror. Let the size of the object be y, the size of the image y ′
the distance of the object from the focus x, then y/y ′＝x/f1 from
which f1 can be calculated (see Lens). The same method can be
used to determine the focal length of the eyepiece. These are the
dimensions necessary for determining the magnification of the microscope,
viz. the optical length of the tube Δ, the focal lengths of the
objective f1′, and of the eyepiece f2.
The focal length of an objective can be more simply determined by placing an objective micrometer on the stage and reproducing on a screen some yards away by the objective which is to be examined. If the size of the image of a known interval of the objective micrometer is determined by an ordinary scale, and the distance of the image from the focal plane of the objective belonging to it is measured, then the focal length can be calculated from the ratio y/y ′＝f1′/x1′, in which y is the size of the object, y ′ that of the image, and x1′ the distance of the image from the focal plane belonging to it.
Besides this indirect method of determining the magnification there is also a direct one, in which it is not necessary to first measure f1, f2 or Δ. If a drawing prism is used above the eyepiece, and an objective micrometer is inserted, then if a scale is laid on the drawing board which is 25 cm. distant from the exit pupil, one or more intervals of the objective micrometer can be seen projected on the scale lying on the board. The comparison of the two scales gives directly the magnification. The course of the light within the drawing prism must be taken into account when determining the distance of the scale from the exit pupil. Although this method does not give very accurate results, it is more convenient and simple than the indirect method.
Bibliography.—E. J. Spitta, Microscopy (2nd ed., 1909); Sir A. E. Wright, Principles of Microscopy (1906); W. B. Carpenter, The Microscope and its Revelations (8th ed. by W. H. Dallinger, 1901); J. Hogg, The Microscope (15th ed., 1898); H. van Heurck, The Microscope (Eng. trans. by W. E. Baxter, 1893). W. Kaiser, Technik des modernen Mikroskopes (Vienna, 1906), deals with the practical aspects, whilst the theory is treated in M. von Rohr (Die Theorie der optischen Instrumente, Berlin, 1904) and in S. Czapski (Grundzüge der Theorie der optischen Instrumente; ed. by O. Eppenstein, Leipzig, 1904). (O. Hr.)
MICROTOMY (Gr. τόμη; τέμνειν, to cut), the term applied to
the preparation of minute sections of organic tissue for the
microscope. In 1875 the methods were yet in their infancy;
their development has enabled observers to achieve the most
exact study of minute anatomy, in the case of small objects,
which without these methods could only be investigated by the
unsatisfactory process of focusing with the microscope through
the solid object.
It is not necessary here to detail at length the wet method of preparing sections. Briefly, the tissue is soaked in a solution of gum, or of gum and syrup, and after being frozen by ether spray, or by a mixture of ice and salt, is cut into sections either by the Rutherford, Cathcart or some similar section-cutter, or by apparatus which can be fitted to the more modern types of microtome referred to below. This method, which is to-day used mainly by pathologists, has two main disadvantages: the prolonged action of watery fluids on the tissues, and the impossibility of getting ribbons, each section having to be picked up separately.
The general processes of the dry method employed in zoological and botanical microtomy are, up to a certain point, practically identified with those used for the preservation of animals and their tissues for other branches of microscopic work. In the first place the tissues must be killed; in the second, they must be fixed, i.e. the protoplasm must be set or coagulated as far as possible in the condition in which it appears in life; and in the third, they must be hardened, i.e. in most cases dehydrated. Killing may be effected by asphyxiation or narcotization (nicotine, cocaine, chloral hydrate, &c.) in special cases, but is generally achieved by fixing reagents, of which corrosive sublimate and other chlorides, picric, acetic, osmic and chromic acids, alone or in combination, chromates and strong alcohol are the most usual. These serve to a great extent also as hardening agents, but alcohol, used after them, completes this process effectively, and when not too strong (70%) is the best storage fluid. The second set of processes relates to the staining, without which transparent sections are almost invisible. The stains are divisible into general stains, which dye the tissue practically uniformly and indifferently; and selective stains, which have affinity for special tissues or cell elements. Of the latter group some fasten on nuclei, others only on the chromatin of the nuclei; some on connective tissues, others on muscle fibres and so on. It is probable that the action of all these selective stains is produced by definite chemical combination with compounds originally present in, generated in, or introduced into the tissue selected. The most generally useful stains for ordinary work belong either to the cochineal series (borax-carmine, carmalum, &c.), or to the logwood series (haematoxylin, haemalum, iron haematoxylin, &c.); in both of these great improvements have been introduced of late years by Dr Paul Mayer. The activity of these stains apparently depends upon the presence of alumina or of some similar base. For more special researches, such as cytology, neuropathology, neurohistology, and so forth, greater dependence is placed on the coal-tar colours, the name of which is legion. Some of these, such as safranine or gentian violet, are regressive stains; that is to say, the tissues are overstained uniformly, and the superfluous colouring matter washed out either by alcohol or by weak hydrochloric acid from the unselected parts. Others, such as methyl green, are progressive—that is, the colour is brought up to the pitch required and the reaction promptly stopped. The coal-tar stains can be used singly, or in combinations of two or three. Some of the best, unfortunately, are not permanent. A third group of stains is furnished by such reagents as silver nitrate, gold chloride, and the like (impregnation stains), which can be made not only to stain, but also to deposit a fine metallic precipitate on certain structures. In the case of small and delicate objects, the staining is done in the mass before any further preparation for sections, but with larger animals, or large pieces of resistant tissue, the stain is applied to the sections only. The processes so far mentioned are applicable to many branches of microscopic work.
When preparing tissues for sections the first step is complete dehydration, generally effected by bringing the object into absolute alcohol. It is then transferred to one of a group of reagents, which are miscible with absolute alcohol, but would form an emulsion with water, and are solvents of the embedding medium. The embedding mass in most general use is paraffin wax, melting at a temperature of 54° to 60° C., according to the character of the object and the thickness of section required. The object is transferred from absolute alcohol to benzol, chloroform, cedar oil, or similar fluid to the melted paraffin; the fluid diffuses and evaporates, leaving the tissues to be completely permeated by the paraffin. This process can be greatly hastened by the use of a partial vacuum. When impregnation is complete the paraffin is cooled rapidly, so as to assume a homogeneous non-crystalline condition, and the tissue thus comes to form part of a block of soft but tenacious material, which protects it from damage by air or damp, and can be readily cut by a razor. The block is then trimmed to the form of a triangle or rectangle, and fixed by a clamp or by local melting in the holder of the microtome.
The first automatic microtome suitable for cutting a block of tissue into a continuous series of sections was made in 1883 in the university Workshops of Cambridge, from a design by W. H. Caldwell and R. Threlfall. Only a single machine was made, but in 1884 twelve machines were made by the Cambridge Scientific Instrument Company from a design by Caldwell. Since then numerous excellent and simpler forms of microtome have been evolved. Some of these have distinct advantages over others, but with microtomes as with other tools—the success of the results depends very largely on the manipulator, for every one works best with his accustomed instrument. In one type of microtome the razor is attached at one end only to a heavy
