The microscope; a practical handbook

Front matter

VtlCROSCOl'l;

O

AMU

Contents

PAGE

Inthoduction by the Reviser . . ... 7

CHAPTER

Introduction

This handbook, written many years ago by the late Mr. Lewis Wright, has proved so extremely useful to both amateurs and students that it has been decided by the publishers to have it rewritten and brought up to date. During recent years microscopic science has made remarkable progress, and in consequence thereof a very large portion of the work had become quite out of date.

As far as has been possible in re-writing the work I have endeavoured to follow the arrangement of Mr. Wright. In the chapters on apparatus I have adhered to the original plan, viz. to show the reader exactly what he can obtain for a given expenditure. It must be borne in mind, however, that since the war microscopes have increased in price very greatly, and that although recently there has been a tendency. for prices to fall it seems unlikely that English instruments will ever be as cheap as formerly. Every stand referred to is known personally to me, and I have mentioned no lenses that I have not examined many times, so that the list given will be found perfectly trustworthy.

With regard to the selection of instruments, I have assumed that the reader has a real desire to study and learn something of the marvellous world revealed by the microscope. With this object in view I have endeavoured

8 INTRODUCTION

to guide the student to select an instrument with which real work may be done, and which he will not need to discard at a later date owing to its shortcomings. In describing manipulation, I have endeavoured to show the student in as simple language as possible how to use his microscope to the best advantage.

The later chapters, dealing with some of the revelations of the microscope, have undergone considerable change from the original form. I have purposely devoted more space in this section to the Protozoa and allied forms, as there is much useful work to be done here by the amateur, and work moreover which apart from the microscope does not need any costly appliances. With the view to assisting the student as far as possible to identify his specimens, several plates have been introduced in which the various organisms have been figured by means of line drawings.

Considerable space has been given to the consideration of the best methods of obtaining critical images, as this subject is still far from being understood either by Professors of histology or their students. It cannot be too strongly insisted that the microscope is not a mere magnifying glass, and can only be used as such, with an immense loss of resolution. There is probably no other scientific instrument which is put into the hands of the student without a detailed explanation as to its use ; yet how often is instruction regarding the microscope given the students by their teachers ! Indeed the Professors are in many cases as ignorant of the proper use of the instrument as are their students. Only recently, at a

INTRODUCTION 9

meeting where many microscopic objects were being shown, the writer heard one teacher of pathology remark, "I cannot understand all this talk of critical images," and yet this man was engaged in teaching students subjects requiring the constant use of the microscope.

The chapter on section cutting and mounting has been considerably enlarged, so as to enable the student to gain some knowledge in the preparation of his own specimens.

It has been my aim in rewriting this work to extend its usefulness, and to provide an elementary text-book, which will prove of assistance, not only to the amateur, but to the medical and biological student, in seeking to learn the proper use of the microscope, which is essentially an instrument of the utmost precision.

I have to thank Messrs. Baker, Beck, Leitz, Ogilvy, Watson, Swift and Zeiss for the loan of numerous blocks, and also Dr. J. A. Murray and Mr. W. Pilgrim for kindly making a number of drawings.

A. H. DREW.

Mttngo Ponton, del

Fig. 80a.— Fossil Radiol&.ria (Polycystina) x 100

THE MICROSCOPE A PRACTICAL HANDBOOK

CHAPTBE I

EAYS, IMAGES, LENSES

Foe the proper selection and intelligent use of a microscope and its accessories, some comprehension of the fundamental principles upon which the formation of images is based is essential.

Images and Magnification. — The physical act of ' seeing ' results from an ' image ' or picture of an object being thrown upon the retina or sensitive screen of nerve endings lying behind , the crystalline lens of the eye. The apparent size of an object depends upon this image which is formed on the retina, and to magnify an image is to increase the visual angle formed on the retina (fig. 1).

The eye itself is similar in construction to a camera, having a lens in front (crystalline lens and cornea) and a sensitive receiving screen (retina) behind.

Images are formed by the lens in front of the eye focussing the waves of light proceeding from the object on

Fig. 1. — Visual Angle to the retina. The angle formed by the extreme rays of the object causes us mentally to form an impression of the size and distance of the object. Eor visual purposes of seeing an object closely the unit of distance employed is 10 inches. In magnification the eye mentally refers back all objects to this distance, and thus if an object is examined with a lens which permits it to be seen clearly at a distance of 1 inch, the angle so formed is ten times as great. The brain mentally refers back light along this angle to a distance of 10 inches, and the object appears correspondingly magnified.

Images formed by Rays.— In the last paragraph reference has been made to images formed by rays of light being focussed by the lens of the eye as in a camera. To appreciate this it must be understood that each ray of light is capable of forming an image, and this function is utilised by lenses. A single experiment will prove this. Light a candle in a dark room and support a sheet of card or white paper two feet away. Take also a large blackened card and prick a pinhole in the middle, holding this midway between candle and white card. By this means rays will be separated from the source of light, and it will be seen they form a real ' image or picture of the

light source upon the card.

Inversion and size of Image. — The image formed in this roanner is inverted, as is evident from fig. 2. Eays proceed from each point on the source of light o through the pinhole a to the image i, so that the ray from the top proceeds to the bottom, and vice versa. It will also be observed that the size of the image must be in proportion to its distance from the pinhole and the position ,

Fjg. '1, — Inverted Image

J? AYS, IMAGES, LENSES 13

of the light source. Move the white card further away or the black card nearer the candle, and the image becomes both larger and of necessity fainter.

This simple fact about rays applies to all optical systems. No matter for the moment what the rays are, they travel in straight lines so long as they traverse undisturbed the same medium (air or glass, etc.), and objects are visible by rays from each point which diverge or proceed from that point in all directions (where not stopped by something or the object itself) into any transparent medium.

Refraction of Rays. — The rays can readily be reflected into some other direction, as by means of a mirror (image reflection) or by other objects (which we then see by this scattered reflection when not self-luminous). What, however, is of more importance is that they can be bent aside or refracted by the surface and density of any fresh transparent medium through which they then proceed in some other straight line. In such cases it must be remembered that the image is apparently seen in the final direction from which the rays enter the eye. Thus in the looking glass we see an apparent image, and a stick plunged into water appears bent, the lower end appearing higher up than it actually is.

The Law of Refraction. — For simplicity in diagrams rays of light are always represented as proceeding in a straight line from the object or light source, but actually this is not the case. Light proceeds in concentric ring waves from any source. As a simple illustration consider a stone thrown into a sheet of calm water. The displacement causes a series of waves spreading out in all directions horizontllay and vertically from the centre.

This is applicable to light, and any point on the diagrammatic line showing its direction -may be considered as a wave front and can be treated as a source of light.

These points are essential to the consideration of refraction. Let the lines w Wi inig. 3 represent a block of glass with air above and below. The line n is normal or at right angles to the surface, and light travelling in this line is

retarded, but its direction remains unchanged.

On the margin of the first wave reaching the denser medium there is a distinct retardation (as, for example, with a man walking in water).

This retardation is progressive and consequently alters the whole direction of the light wave. On again reaching air there is a reverse action, the light proceeding at a greater speed and thus attaining a path parallel to its original direction.

Thus we say that light entering a denser medium is refracted towards the normal, but on entering its original medium is refracted back to its original path and emerges parallel to the incident line.

As a simple experiment take a block of glass and place on the one side two pins at an angle to the surface but in a straight line with each other, then looking through the glass at the pins insert two other pins separated by 4 to 6 inches, so that all four are in an apparently continuous line. Eemove

Fig. 3. — Illustrating the Law of Refraction

JiAVS, IMAGES, LENSES the glass and join the pinholes with a pencil line, and it will appear as the diagram above.

Total Reflection : Critical Ang^le. — Eefraction at a surface takes, place up to a spot where the line between two media becomes opaque to the ray. Assume that the air ray 1 grazes the surface of the glass. This surface then becomes the sine. It niust be borne in mind that whilst the ratio of reflections remains the same in any one medium whatever the angle of incidence, there is an increasing amount of inclination the greater the angle the incident ray makes from the perpendicular, until arriving at what is known as the ' critical angle,' when the refracted i-ay cannot emerge from the denser medium to which its refraction is due. This is as a result totally reflected back at the same angle, and is readily demonstrated by holding a glass of water above the eye. The surface looked at from below is of mirrorlike brightness and reflections can easily be distinguished in it.

The law of this refraction is of interest and very important. Let us take water and air, and let w w^ (fig. 4) represent the surface of the water, and draw a perpendicular, p f, through some point, c, where every ray is to strike the water, and also describe a circle round c. Any ray, p c, striking the surface perpendicularly, goes straight on to p. Take some other ray, A c , striking the water at an angle a c p with the perpendicular.i Part of it will be reflected at an equal angle, as if the surface were a mirror ;

' Optical angles are measured from the perpeodicptar, called 'quvfital,' and not with the surface.

■P

-Law of Eefraction i6 THE MICROSCOPE

but the part that enters the water is refracted at c to a, down towards the perpendicular, because the water is denser than the air.i That is the first law. In the denser medium the rays are refracted towards the perpendicular. Conversely, on entering a rarer medium, they are refracted from the perpendicular, so that a ray from a to c in the water would be refracted to a. This involves another important fact, that if we trace a ray the other way, its path is exactly the same, or it is reversible.

But the measure of the refraction is most important to us. Draw the line a s from where the ray cuts the circle, at right angles to the perpendicular p c ; then a s is the geometrical sine of the angle a c p. Divide c w and c w^ each into six parts numbered, and let the ray be such that a perpendicular from a meets c w^ at the fourth division : this gives the measure of as on c w^ as on a scale. Treat the sine a s of the refracted ray c a; in the same way ', we shall find a s (in water) measures three divisions, a s and as are in the proportion of 4:3. Now take some other inclined ray b o — for convenience we take it here, so that the sine b s of its angle is exactly half a s, or two divisions. Then the refracted ray c h will be such that its sine h s will also measure half of a s. And so with any other rays ; the sines of the incident and of the refracted rays are always in. the same proportion of 4:3.

It is the same with all transparent media, including glass. The proportion differs in each, according to its density, so that in crown glass the ratio is about 3:2. But the ratio is the same for all rays, and that enables us to put the ratio into a simpler number compared with air as 1, and call that number the ' refractive index ' of the fluid or glass. Thus the index of ordinary optical crown glass is

' The optical density is meant. As a rule heavier fluids or glasses refract more, but not always. Thus oil of turpentine floats on water, but has more optical refractive power.

RAYS, IMAGES, LENSES i;

1-516. And when we know the index, it is easy to calculate the direction in which a ray. entering at any angle will go.

The diagram demonstrates, among other things, how fast refraction increases with the angle, and this fact brings us to a curious limit. Let the air ray just graze the surface w c^, then this surface itself is the sine, or 6 divisions. Conversely, the ray b c will just escape from the water in the path o w^. If, then, we take some ray in the water of greater angle than k c ^, what is to happen to that, since it cannot get out of the water ? It does not do so, but is totally reflected back at the same angle, with a brilliance no mirror can equal ; as can be s§en by holding high a tumbler of water, and observing the bright reflection from under the surface. This reflection at the surface of a rarer medium is the only case of total reflection of rays in nature; but the very important point to us is that rays cannot get out of a denser medium, beyond what is called the 'critical angle,' ecr. This angle is plainly less, the denser the medium. In water the critical angle is 97|°, in crown glass 82J°.

Action of a Leas. — The reason that an object is not displaced when looking at it through a sheet of glass, such as a window, will be readily understood from the foregoing description of the law of Eefraction, as the rays which on passing through the glass are bent towards the perpendicular, return parallel to their original direction on re-entering the air. If, however, the glass has an inchned surface and unequal thickness, as is the case with a prism, the rays on emerging follow a different path, and are permanently refracted.

Fig. 5 is a diagrammatic drawing of this. The ray e i is refracted towards the normal in the glass from i to e, and on emerging to air is refracted again away from the normal in the line e e, and thus an object b would appear to lie in the direction b o. The ray is always refracted towards the thick end or base of the prism. Not only, how-

B i8

O^

Fig. !

ever, is a ray of light refracted in this manner, but it is at the

same time divided up into its component colours, white light being a mixture of various colours. A- threecornered glass lustre will show this excellently. If a candle flame be observed, the refractive beam of light will be seen to be widened out into a band of colours, which is known as the spectrum.

If a lens, with its curved surfaces,, be considered as two circular prisms placed inwards, the formation of a focal

point will be understood.

However, it should be remembered that there are six possible forms of single lens, as shown in fig. 6.

The first three are thickest in the centre, and are known as ' positive ' lenses, while the last three are thinnest in the centre, and are known as ' negative ' lenses.

To consider the formation of an image from a bi-convex

C D E

-Forms of . Lenses

Fig. *l. — Common MaguiFying Glass

lens : Apply the principle of light passing through a prism, and consider two prisms base inwards, but with curved

JiAVS, /MAGES, LENSES iosfcead o£ straight sides. (This is the common form of magnifying glass.) Bays passing along the centre of this lens are undisturbed in their direction, but the refraction increases towards the apex of each of these prisms (fig. 7), and as a result, the rays of light are all brought to a focus at one point. Actually there are several foci, i.e. one for red light, one for yellow light; and one for blue light, etc.; but

these are very close to-

gether. The matter will TZZZZTZ

be referred to further on. -

Kg. 8 shows a parallel "

front of rays striking on a

bi-convex lens. If, however, rays proceed from a source of light at f (fig. 9), they would emerge on the other side of the lens, either as a beam

Fig. 8

Fi&. 9. — Conjugate Foci

of parallel light or, if the point p lies beyond the focus of the lens, they would on traversing it be brought to a focus either side, and vice versa. If these points are interchangeable in this manner, they are known as conjugate foci.

In the case of concave lenses which are thickest at the edges, the action of the lenses is reversed, and light rays are made to diverge. Such lenses have no

Fig. 10 actual focus, but what is known as a negative focus. This is measured from parallel rays in the same manner. If the parallel rays falling on the lenses are refracted (fig. 10) as at A B,' these rays when produced backwards on the other side of the lens, will meet at the point f. This is known as the ' negative focus.'

Lenses and Images. — If a reading glass of 3 inches diameter be.'taken, a simple experiment will demonstrate the property of a lens in forming images. Take a small source of light in a darkened room and set up a white card screen, as described on page 12. Make three or four pinholes in the black card around the first one, but an inch or less from it, in such a manner that the lens more than covers all the holes made. If the reading glass is about 10 inches focus, as is usual, put the black card 8 inches from the candle with the centre pinhole on a level with the flame, and the white card 3 feet beyond. There will now appear five inverted images of the flame on the white card, showing that each pencil of rays forms its own separate image.

Holding the reading glass close to the apertures, note to what extent the refraction of the lens bends in the four outer images towards the centre one. Move the lens gradually towards the white card, and it will be noticed that they approach more and more until there is a point at which the five images become one. This is the focus of the lens. More holes can be made, or the black card removed, but there will only be one image, which will combine the whole of the ray traversing the black card or the lens should the card be removed.

The function of a convex or image-forming lens is to determine a space where the cone of rays diverging from any point in an object shall recombine in one image. Its position marks the place where the rays are refracted to this end, and also the place where the rays from the opposite point of the object cross and invert the image. Thus, the relative sizes of image and object are measured from the optical centre of the lens according to the ratio of the two conjugate foci. The lens itself, however, does no more than thus converge, and combine into one, potential images formed by the bare rays.

Chapter II Peactioal Optics Of The Miceoscope

We como now to certain optical principles belonging to the microscope specially, which should be understood if the best choice is to be made amongst the instruments and accessories offered to our notice.

The Simple Microscope. — The action of this instrument, or a simple ' magnifying glass ' in any form, will be easily understood after the last chapter. Let the arrow a d repre-

A iU^"

Fia. 11. — Action of Magnifying Glass sent an object o, and l a lens held in front of the eye b (fig. 11). From one point a in the object, diverging rays a h and a c proceed, and are so converged by the lens as to enter the pilpil of the eye almost parallel, which is required for distinct vision. The rays thus parallelised are brought to a focus at z on the retina ;e, forming there the visual image of a. The image of d is imaged at x in the same way. But the object being ' seen ' in the direction of the rays which last enter the eye (see page 13) if we produce these till they meet, they appear to proceed from the points A d, which is the apparent image i of the object, as apparently seen at the distance of 10 inches. It is never to be forgotten that the last stage in every optical combination is the formation of this image on the retina at e, and that the final rays which must enter the pupil in order to effect this, have to be rendered so slightly divergent as to be practically parallel.

The Compound Microscope.— In this instrument, known in general as the microscope, and the general plan of which is shown in fig. 12, a real enlarged image of the object o is formed by one lens (called the object glass or objective) in the air. This enlarged image is looked at through another lens called the ocular 'or eyepiece, and by it further enlarged in the manner of the simple microscope.

For reasons to be seen immediately, each of these lenses is always compound, and may be of various forms ; but it is well to notice at once the form of eyepiece shown in the diagram, and' called the Huyghenian or ' negative ' eyepiece. It is called negative because the image looked at is formed within it, or between its two lenses ; and some form of it is more used than any other.

Fig. 12

It consists of a larger planQ-convex lens //, called the field-lens, a smaller one, e e, called the eye-lens, and a diaphragm or stop at h b, in the focus of the eye-lens. Three pencils of rays are drawn from the centre and each end of the object o, which if let alone would form an inverted image at a a. This aerial image ' in space ' behaves in one respect differently from a real object, which sends out wide cones of rays in all directions. The image, on the contrary, is formed by the narrow convergent pencils a, which continue on after meeting or focussing. So if we looked at the image by the lens e e alone, only rays from the centre of thp image could pass through this lens and reach the eye. But the field-lens bends in the pencils fa to fb, also shortening the focus a little, forming a rather smaller image at b b, and so altering matters that the rays fa are now all converged to pass through the eye-lens e e. Besides this, the ' aberrations ' presently mentioned are nearly corrected by this simple form of eyepiece. And lastly, as the rays begin to be converged later by the edges of the lens // than by the centre, the conjugate focus is rather extended there, and so this eyepiece helps to 'flatten the field.'

It will be obvious that the field-lens /"/ belongs in principle to the objective, taking a share in the focal convergence of the image-forming rays. But it is always reckoned as part of the eye-piece.

Power of a Microscope. — This is calculated by multiplying the original magnification of the image a a by the further magnifying power of the eyepiece. If an objective of |-inoh focus forms its image at 10 inches, it will at that distance give a magnification 40 times as ■ large as the object 0. Then, if the eyepiece magnifies 10 times, the power within that objective and eyepiece will be 400 diameters. But it must be remembered that a tube length of 10 inches is very rarely employed. Thus with a shorter tube length the magnification will be reduced. Most modern objectives are corrected for a tube lengtlj of either 6 or 8 inches or 8-10. Thus the magnification is as 6 : 10.

Spherical Aberiation of the Lenses. — If simple lenses did perfectly the refracting and image-forming work described in Chapter I, a microscope would be simple indeed ; unfortunately they do not, failing in three ways.

Firstly, they are ground to spherical surfaces, and these refract too much in proportion as we approach the edges ; thus the rays falling on the edge of the lens come to a focus nearer the lens than the central rays. The image formed by a large reading glass on close examination will be found rather blurred from this cause, which is called ' spherical aberration.'

' Secondly, it will be noticed that print seen through, such a glass appears distorted in the marginal parts. And the image on a card of a flat surface like a lantern slide is not itself flat, but curved hollow towards the lens. These are other effects of spherical aberration.

Chromatic Aberration. — Thirdly, in looking through the glass lustre or prism at the candle-flame (Chapter I), the image was not only refracted, but spread into a band showing all the colours of the rainbow — the coloured band called the spectrum. That is because different colours are differently refracted, blue being the most so and red the least. This being so, clearly a simple lens must bring the blue rays to a focus nearer to it than the red rays ; and this can also be seen, with care, in any large lens. It is called chromatic aberration.

These faults do not prevent much rough useful work with simple lenses, as in a magnifying glass. But they, are fatal to precise work, and most of all to the perfection of images magnified many hundreds of times.

Correction of Aberrations. — Such defects can be more or less corrected in several ways.

(a) All of them are worst at the edge of a lens. Hence, by stopping all but a portion in tbe centre, the image is greatly improved in sharpness. But it is at the expense of light and of other functions of large aperture. Hence this plan can only be used to some extent, and as the cheapest mode of improvement.

(h) Both aberrations differ greatly in amount, according to the inclination of the surfaces to the rays. The spherical aberration of a plano-convex lens in parallel rays is less than a third when the flat side is turned towards the focus, of what it is the other way. And two inclinations may be so disposed that the aberration of one largely corrects that of another. Thus, by using two or more lenses, amongst which the power is divided, instead of one, and proper combination of curves, much may be done. This method is largely employed, and is entirely depended upon in the . Huyghenian eyepiece.

Chiefly of all, while different sorts of glass differ largely in both their refractive power (or optical density) and also in their dispersive power (or degree to which the spectrum is spread out by a prism of given angle), the two qualities do not preserve the same proportion. So it is possible to make, let us say, a plano-convex lens (fig. 13) ,of two glasses, "of which the crown-glass lens, c, has double the curvature of the flint-glass concave lens, p, and so has a large surplus of refractive power, while yet the colour-dispersion of p is enough to counteract all that of c, and so ^"** ^^ brings all colours nearly to the same focus. Such a lens is called achromatic, or ' corrected for colour.' Fortunately it also happens that the concave curve of the flint lenslargely corrects the spherical aberration of c as well ; and so we get a more perfect lens every way.