Mountants for specimens

Definition, particularly of colourless structures, is increased by choice of a mountant of refractive index different from that of the object. A mountant of lower refractive index is to be preferred so that the outline shadow is on the side away from the object. The ratio refractive index of object to refractive index of mountant should be of the order of 1.06. The value of this relative refractive index for cellulose (cotton) to water is 1.17, to chloral hydrate solution (5:2) 1.08 and to glycerin 1.06. This serves to emphasize the value of chloral hydrate and glycerin as mountants for plant structures.

Magnification and field of view

For work in pharmacognosy, microscopes are usually fitted with two objectives, 16 mm and 4 mm, two or three eyepieces and a condenser. The procedure for making microscopical measurements is described below. Different combinations of eyepiece and objective give different magnifications and fields of view, as indicated in the table below.

When using the microscope, it is useful to know the size of the fields of view. For instance, if we know that using a 4 mm objective and a ×6 eyepiece our field of view is approximately 0.5 mm, or 500 μm, the size of objects such as the Arachnoidiscus diatom in agar (100–300 μm) or the large rosette crystals of calcium oxalate in rhubarb (up to 200 μm) may be roughly estimated. For accurate measurement, however, an eyepiece micrometer or camera lucida is used.

Apparatus for making microscopical measurements and drawings to scale

Microscopical measurements can be made using a stage micrometer in conjunction with an eyepiece micrometer, camera lucida or microprojector.

Micrometers

Two scales are required, known, respectively, as a stage micrometer and an eyepiece micrometer. The stage micrometer is a glass slide 7.6 × 2.5 cm (3 × 1 inch) with a scale engraved on it. The scale is usually 1 or 1.1 mm long and is divided into 0.1 and 0.01 parts of a millimetre. The eyepiece micrometer may be a linear scale (Fig. 43.1A and the scale 0–10 in Fig. 43.1B) or it may be ruled in squares. The value of one eyepiece division is determined for every optical combination to be used, a note being made in each case of the objective eyepiece and length of draw-tube.

Fig. 43.1 A, Eyepiece micrometer; B, eyepiece micrometer superimposed on portion of stage micrometer scale.

To do this, unscrew the upper lens of the eyepiece, place the eyepiece micrometer on the ridge inside, and replace the lens. Put the stage micrometer on the stage and focus it in the ordinary way. The two micrometer scales now appear as in Fig. 43.1B, when the 4 mm objective is in use. In the example figured, it will be seen that when the 7 line of the stage micrometer coincides with the 0 of the eyepiece, the 10 of the stage coincides with 7.7 of the eyepiece. As the distance between 7 and 10 on the stage scale is 0.3 mm, 77 of the small eyepiece divisions equal 0.3 mm or 300 μm; therefore, 1 eyepiece division equals 300/77 or 3.9 μm.

Camera lucida

Various forms of apparatus have been designed so that a magnified image of the object under the microscope may be traced on paper. The Swift-Ives camera lucida and the Abbé drawing apparatus are examples. The former fits over the eyepiece, and when in use light from the object passes direct to the observer’s eye through an opening in the silvered surface of a prism. At the same time, light from the drawing paper and pencil is reflected by a second prism and by the silvered surface, so that the pencil appears superimposed on the object, which may thus be traced.

When the instrument is in use, the illumination of both object and paper must be suitably adjusted and the paper must be tilted at the correct angle to avoid distortion. The Abbé drawing apparatus utilizes, instead of the adjustable prism, a plane mirror carried on a side-arm; with the mirror at 45° to the bench surface, no inclined board is necessary.

To make measurements or scale drawings, the divisions of a stage micrometer are first traced on paper and then, using the same objective, eyepiece and length of draw-tube, the object to be recorded is traced.

The above camera lucidas which have served pharmacognosists well in the past do not now appear to be available commercially. This seems apparent from some of the line illustrations now submitted for publication!

Microprojection

With a suitable apparatus objects beneath a microscope can be transmitted to a screen or on to paper. Suitable particles can then be traced at the desired magnification.

Photomicrography

Modern research microscopes have built-in facilities for photo- micrography; with less expensive or with older microscopes it is necessary to fit a suitable camera to the equipment. The photographic record may seem, at first sight, to dispense with the older tracing techniques discussed above. However, although the photographic record is suitable for thin sections of plant material, rarely is it completely so for powdered drugs. A photograph contains everything visible in a particular field of view and much of this is usually uninformative; rarely does a single field of view show all the diagnostic features of the powdered drug to best advantage, and also, owing to limitations on depth of focus, structures such as large fibres, vessels, trichomes, etc. are seldom seen with all details completely in focus. Fragments of disintegrated material adhering to, or partly covering, the element photographed are also confusing to the inexperienced observer.

Polarization

The apparatus consists of a polarizer, or Nicol prism, fitting below the microscope stage, and a similar prism forming the analyser fitting above the objective. As in the case of the polarimeter, with which students will be familiar, one Nicol is kept stationary while the other is rotated. With geological microscopes, polarizers are usually permanently fitted, but for botanical work the analyser is usually fitted when required, either between the objective and nosepiece or over the eyepiece. Less cumbersome are discs cut from polaroid sheets; one of these can be of a size to fit into the filter holder of the microscope and the other to rest on the eyepiece.

When both the polarizer and analyser have their diagonal surfaces parallel, the ray of plane polarized light is transmitted by the analyser. If now the polarizer is revolved, the light diminishes in intensity until at a position 90° from the first it is entirely extinguished, the polarized light being now totally reflected by the analyser. This position, when the diagonal surfaces of the two Nicols are at right angles, is termed ‘crossed Nicols’.

Isotropic substances are characterized by having the same physical properties in all directions (e.g. gases, liquids and isometric crystals). Such substances are monorefringent (i.e. they have only one refractive index). Isotropic substances are not visible, however they be oriented, when examined between crossed Nicols. They in no way affect the polarized light passing through them from the polarizer.

Anisotropic substances exhibit different physical properties according to the direction along which they are examined. Such substances show more than one refractive index. The great majority of crystalline materials show birefringence. When a uniaxial crystal is placed with its optical axis horizontal to the stage and examined between crossed Nicols, then as the stage is rotated it will alternately shine bright (or coloured) and disappear. Through the 360° it becomes invisible (i.e. shows extinction) four times. The examination of crystals between crossed Nicols enables us to determine their crystal system (see ‘Calcium oxalate’ Chapter 42). The crystal is placed with its axis parallel to the longer diagonal of the polarizing Nicol. If the crystal belongs to the tetragonal system, the polarized light passes unchanged and on reaching the analyser is completely absorbed, the field appearing dark (i.e. extinction takes place). Conversely, monoclinic crystals show extinction only when the vertical axis makes an angle with the diagonal of the Nicol known as the extinction angle.

Many crystalline substances show brilliant colours when examined in polarized light (e.g. asbestos, sucrose, cinnamic acid). Starch grains often show a black cross, a phenomenon due to the crystalline refraction of the material. Polarized light is useful for the detection of calcium oxalate, especially when only small quantities are present in the tissues under examination. It appears bright on a black background.

Phase-contrast microscopy

This has proved particularly useful for the examination of living cells, the constituents of which normally show little differentiation. Monochromatic light is employed; light directly transmitted through the sample is reduced in intensity and the deflected light is brought half a wavelength out of phase with the transmitted light. Strong contrasts in the material under examination are thereby obtained without reduction in the resolving power of the microscope.

Ultraviolet microscopy

The limit of resolution of any microscope is governed by the wavelength of the beam employed; the shorter the wavelength, the smaller the object which can be resolved. The ultraviolet microscope with lenses of fused quartz will transmit radiation down to the wavelength of 240 nm. The images produced are recorded photographically. The instrument has been valuable in the study of cell division and differentiation.

Electron microscopy

Just as a beam of light can be focused by an optical lens, so a stream of electrons can be focused by an electromagnet acting as a lens. Objects placed in the path of the electrons produce an image which can be recorded either on a fluorescent screen or on a photographic plate. Both the focal length and the magnification can be varied by regulation of the field strength, which is controlled by the current passing through the lens. Good stabilization of the lens current is essential for the best lens performance. Because gas molecules will cause a scattering of electrons, electron images are formed only in a high vacuum (less than 10⁻⁴ mmHg). Although commercial electron microscopes were available in 1939, it was not until the 1950s that their potential could be fully exploited for biological work. The breakthrough in this field centred on the preparation of ultra-thin sections of biological tissue by the use of glass knives and on the development of suitable staining, fixation and embedding materials. To prevent complete scattering of the electrons by the tissue, sections of the order of 20–200 nm are used and a buffered solution of osmium tetroxide is commonly employed for fixation and staining. Unstained cell components of a tissue have a fairly uniform electron scattering power, similar to that of the embedding medium, so that little contrast of the image is obtainable. However, the incorporation of electron-dense atoms (osmium) into the cell organelles enables a good degree of contrast to be obtained on the electron micrographs of the sections. There is, at present, no objective way of determining how much the fine structure of cells is altered by the fixation methods employed, but indirect correlation of the results obtained with those from other techniques is reassuring.

The light microscope gives magnifications of the order of ×1000 with a resolution, set by the wavelength of the light employed, down to about 0.2 μm for visible light; no further magnification of the image can increase the detail. The theoretical limit of resolution of the electron microscope is similarly governed by the wavelength of the electrons (about 0.003 nm) and in practice electron microscopes give resolutions to about 0.4 nm. Magnifications of ×10 000 to ×24000 are commonly employed and to show all the available detail on high-quality electron micrographs, prints at magnifications of around ×500 000 may be required.

Much knowledge of the detailed structure of the living cell has only been made possible by the advent of the electron microscope. For the routine examination of vegetable drugs the light microscope with polarizing attachment is generally fully adequate, but scanning electron micrographs at a much lower magnification than the above can be extremely useful for depicting structural details not obvious with the light microscope, for example, maize starch and digitalis (Fig. 23.17).

Drawings for publication and thesis work

For the detailed steps involved in the preparation of these line drawings, see earlier editions of this book.

Distribution of tissues

A general idea of the distribution of tissues can be obtained by the examination of transverse and radial and tangential longitudinal sections. Such sections should first be mounted in water or dilute glycerin. Subsequently sections should be cleared by means of chloral hydrate or other clearing agents (see below) and some stained as follows.

Clearing, defatting and bleaching

Structures are frequently obscured by the abundance of cell contents, the presence of colouring matters and the shrinkage or collapse of the cell walls. Therefore, reagents are used for the removal of cell contents, for bleaching and for restoring as far as possible the original shape of the cell wall. If the microscopical examination is to be made from the section mounted in the clearing agent, the refractive index of the latter is important. It may be advisable to wash the section and mount in a different medium. The commonly used mountants glycerin, alcohol, carbolic acid, lactophenol, clove oil and Canada balsam all have some clearing effect. The following clearing and bleaching agents are particularly useful.