The microscope; an introduction to microscopic methods and to histology

Front matter

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Preface

IN revising and rewriting this book now for the twelfth time, the aim has been as for all previous editions, to give the student the benefit of the fundamental things which have been worked out in microscopy. The opportunities given by the freedom from teaching have rendered it possible to make this revision more thorough than could be done in any previous edition.

Progress in all that pertains to microscopy has been marked during the last ten years. Any one can see this clearly by comparing the catalogues of manufacturers sent out ten years ago with those sent out at the present time.

Nothing fundamentally new has appeared, but there have been great advances in making practical and usable many processes and much apparatus for which the basic knowledge has existed for a considerable time. Of course there are some. principles and manipulations which a person must become master of if he is to work successfully with the microscope. These have been treated mainly as in the past. Of the new things nothing has been considered in the book which has not been personally tested and found to be workable and helpful.

Among the most important means recently made available, especially for students of biology, are the following:

(1) The single objective binocular for all powers of the microscope from the lowest to the highest. j

(2) The dark-field illuminator for all powers, especially the highest powers with which the finest details in living structures can be seen with marvelous clearness. This makes it possible to compare the living cell with the fixed and stained one.

(3) The perfection of apparatus with which the powerful electric lights recently produced have become: available for demonstrations and for drawing with the projection microscope.

(4) The perfection of photographic light filters and the production of dry plates sensitive to the whole spectrum makes it possible to get good photographs of any microscopic specimen, and indeed of any specimen.

(s) From the numbers who are affected, and the extent of its application, perhaps the greatest improvement of all has been the production of a glass filter which, when used with a gas filled mazda lamp, gives a light of true daylight quality and of sufficient intensity for all powers of the microscope.

In preparing this edition some parts of the previous edition have been omitted. For example, the pages on micro-chemistry and metallography have been left.

: Vv vi PREFACE out because the Micro-Chemistry of Dr. E. M. Chamot, which has recently appeared treats these and indeed all matters pertaining to the chemico-physical side of microscopy in an adequate manner.

As a closing word it may be said that even an elementary book like this depends for its production upon many helps. The work of others must be looked for in a great library; special knowledge in allied departments must be utilized through the help of colleagues; apparatus and ideas can only be put in graphic form by the deft hand of the artist; and perhaps most important of all is the advice and criticism of the friend. All of these helps the author has had in abundance, and he feels grateful to each helper.

SIMON HENRY GAGE CoRNELL UNIVERSITY February 22, 1917

Contents

PAGES FIG TnfrOduch OB ei tee cereen segues vee vo ea aniao es oak I- § 1-3 CHAPTER I ............... 6-35 4-29

The Microscope and Its Parts: Simple and compound microscopes; Visual angle and apparent size of objects; Lenses; Construction of images; Experiments with the simple microscope; Compound microscope and objectives and oculars; Experiments with the compound microscope.

Practical Work with the Parts of the Microscope: Focusing experiments; Working distance; Lighting with daylight; Lighting with artificial light; Artificial daylight with daylight glass; Condensers or illuminators, and experiments with them; Dark-ground illumination with low and with high powers.

Introduction

In dealing with the possibilities and use of any method of investigation, any machine or piece of scientific apparatus, the writer or teacher will naturally proceed as seems to him best from his personal experience, from his general theory of education, and from his conception of the style and method of presentation which will render his book most acceptable to his possible readers.

As stated in the preface to the sixth edition, this book had its origin in the laboratory, and its purpose was, and still is, to give the guidance by which those unfamiliar with the microscope and the methods of work with it can gain an intelligent understanding of the instrument, its limitations, and its possibilities for aiding one to arrive at truth.

In working out the plan the following landmarks have been kept constantly in sight:

(1) To most minds, and certainly to those having any grade of originality, there is a great satisfaction in understanding principles; and it is only when the principles are firmly grasped that there is complete mastery of instruments, and full certainty and facility in using them. The same is true of the methods of preparing objects for microscopic study, and the interpretation of their appearances when seen under the microscope.

Much good work can be and has been done by the rule of thumb method, in which there is no real understanding of the underlying reason for any of the operations; the worker simply knows that good results follow a certain course of action. Probably most of the work of the world is done by rule of thumb. But the originators of the knowledge making rule of thumb possible must have some compre-

I

2 INTRODUCTION [Intro.

hension of principles, and the reasons for what is done. For creative work, then, knowledge of principles is indispensable.

(2) Need of abundant practical work to go with the theoretical part has been shown by all human experience. In all the crafts and in all the fine arts mastery comes only with almost endless effort and repetition, the most common example being the attainment of facility in music. Hence in this work there have been introduced many practical exercises so that the worker might gain the deftness needed. It is also a part of human experience that in successfully going through the manipulations necessary to demonstrate principles

Fic. 1. PRojEcTION MIcROSCOPE WITH ENLARGED REAL IMAGE ON THE SCREEN.

and methods, the principles and methods themselves become more real. That is, comprehension of principles aids in the certainty with which work can be done, and conversely the doing of the work helps. to increase the grasp on the principles.

After observing the work of students in my own and in other laboratories the conclusion was reached and expressed in the third edition of this book (1891) that “simply reading a work on the microscope, and looking a few times into an instrument completely adjusted by another, is of very little value in giving real. knowledge. In order that the knowledge shall be made alive, it must be a part of the student’s experience by actual experiments carried out by the student himself.”

Beale, in his work on the microscope, expresses it thus: “The num-

Intro. ] INTRODUCTION ber of original workers emanating from our schools will vary as practical work is favored or discouraged. It is certain that they who are most fully conversant with elementary details, and most clever at demonstration, will be most successful in the consideration of the higher and more abstruse problems, and will feel a real love for their work which no mere superficial inquirer will experience. It is only by being thoroughly grounded in first principles, and well practised in mechanical operations, that any one can hope to achieve real success in the higher branches of scientific inquiry, or to detect the fallacy of certain so-called experiments.”

And Hon. J. D. Cox, skilled alike in the arts of war, statesmanship, and science, in his notable address upon Systematic Instruction in the Microscope at the University, before the American Microscopical Society, in 1893, says: “I wish to urge the desirability of a somewhat extensive _course of technical training in regard to the microscope. ... Any one who desires to devote himself seriously to investigation with the microscope will find great advantage, as it seems to me, in devoting some time to the study of the instrument itself in all its parts, and the history of their development.” The study of this whole address is urged upon the person interested in the just appreciation of the different parts of the microscope and their successful employment or improvement.

Sir A. E. Wright, in his book “Principles of Microscopy,” says this: “Every one who has to use the microscope must decide for himself the question as to whether he will do so in accordance with a system of rule of thumb, or whether he

~ simple

Fic. 2. A SIMPLE Microscope HELP- ING THE EYE TO Form A_ RETINAL ImMacGE OF A NEAR OBJECT.

Object The object to be seen by the eye.

Lens The double convex lens acting as a magnifier or microscope to aid the eye in seeing a near object.

Cornea The cornea of the eye.

ry The single refracting surface in the schematic eve.

cl The crystalline lens of the eye, also the center of the refracting surfaces or the nodal point of the eye where the secondary axial rays cross.

ri Retinalimage; it is inverted.

will seek to supersede this by a system of reasoned action based upon a study of his instrument and a consideration of the scientific prin-

.

INTRODUCTION [Intro.

ciples of microscopical technique. The present text-book [his “‘Principles of Microscopy”] has no message to those who are content to follow a system of rule of thumb, and to eke this out by blind trial

Axis

OBJECTIVE

--f Object

MIRROR and error. It addresses itself to those who are dissatisfied with the results thus obtained and who desire to master the scientific principles of microscopy, even at the price of some intellectual effort.”

From the observation of ten generations of students and their subsequent career I am confirmed in the belief that for attainment in study with the microscope, as in all other human endeavor, a person must pay for all he gets.

(3) In considering the microscope, it may be looked at as a machine composed of glass and brass complete in itself, or it may be © considered as an artificial aid to the eye, like a spectacle. When complete in itself it is properly called a projection microscope,

Fic. 3. A Compounp Microscope HELPING THE EvE TO Form A RETINAL IMAGE oF A NEAR OBJECT.

Mirror The plane and concave mirror to reflect light through the object.

Object The small object to be seen by the eye.

Objective The objective of the compound microscope to form a real image of the small object.

Axis The principal optic axis of the microscope.

jf Principal focus of the ocular and of the objective.

rim The real image formed by the objective.

Ocular The double convex lens enabling the eye to see the real image formed by the objective.

cr The cornea of the eye.

rs The refracting surface of the schematic eye.

L The crystalline lens of the eye.

vt The retinal image; it is erect with reference to the object, but inverted as compared with the real image.

Intro. ] INTRODUCTION 5 for it produces an image wholly independent of the eye of the observer. This image may be fixed on a photographic plate or used as a basis for a drawing (fig. 1). On the other hand, when used as a microscope in the ordinary way, the eye of the observer is an integral part of the optical combination, just as integral a part as the objective or the ocular (fig. 2, 3). This being the case the optical perfection of the eye is as influencing on the final retinal image as the perfection of the other optical parts.

And finally, quoting again from the preface of the third edition, “Tn considering the real greatness of the microscope and the truly splendid service it has rendered, the fact has not been lost sight of that the microscope is, after all, only an aid to the eye of the observer, only a means of getting a larger image on the retina than would be possible without it, but the appreciation of this retinal image, whether it is made with or without the aid of a microscope, must always depend upon the character and training of the seeing and appreciating brain behind the eye. The microscope simply aids the eye in furnishing raw material, so to speak, for the brain to work upon.”

Chapter I The Microscope And Its Parts

§ 1. Apparatus and material for Chapter I.

1. A simple microscope (§ 3, 14, 6. Stage micrometer (§ 48).

fig. 4, 6). 7. Homogeneous immersion liquid 2. A compound microscope with (§ 25).

nose-piece (§ 4, fig. 25-28). 8. Mounted letters or figures (§ 50). 3. Eye-shade (fig. 33). 9. Ground-glass and lens paper 4. Achromatic (§ 26), apochro- (§ 50).

matic (§ 28), dry (§ 22), immersion to. Black card with pin-hole (§ 7, justable objectives (§ 31). 11. Dissecting spectacles (§ 145).

5. Huygenian or negative (§ 38), positive (§ 39), compensation oculars (§ 40).

§ 2. As the word itself indicates, a microscope is an instrument with which one can see small things (§ 2a).

The microscope makes small things or minute details of larger things visible in two distinct ways, both ways being dependent on an increase of the visual angle (§ 6, 226-227, fig. 75-76).

(1) The first way of increasing the visual angle and thus making small things or details visible is by means of one or more lenses used as a kind of spectacle by which the eye is enabled to form a sharp image on the retina when optically so close to the object that without the artificial aid a sharp image on the retina could not be produced (fig. 2, 3, 6).

(2) The second way of increasing the visual angle under which small things or details are viewed is by means of a projection microscope, which, wholly independent of the eye, produces a sharp, greatly enlarged image of the object upon a white surface. The eye then looks at this image as though it were the object itself and of that size (fig. 1, § 312).

The fundamental difference in the two forms of microscope is that

Ca. I] SIMPLE AND COMPOUND MICROSCOPES 7 in the first only a retinal image is formed, while in the second, a screen image and from that a retinal image.

In this book the first form of microscope is mainly considered except in Ch. VI and VII, where the projection microscope is much used.

§ 2a. The word Microscope is Simple . from two Greek words: pixpds — Lens Lone Convex Convex mikros, small, and cxomety — ene ene skopein, to see. The word was eoaten Subs compounded and given a Latin Convex : form by Giovanni Faber of the tons Lens tens Academy of the Lincei, as shown by a letter of his to Cesi, Presi- ase MAGNIFIER Nee Eee dent of the Lyceum, dated April Microscope M icroscope Microncope 13, 1625. Faber says: ‘‘AsT also Obj : mention his [Galileo’s] new bjective eee occhiale to look at small things Ocutar Ocular Ocular and call it Microscopium. ” Jour.

Royal Microscopical Society, Eye:Paint Tube-Length 1889, p. 578; Carpenter-Dallin- iia ena a ig 2 Fine Adjustment ger, p. 125. Jeeta — ‘Numerical Aperture SIMPLE AND COMPOUND Spectecton Refractive Index atigmatism Microapectroscopo MucroscorEs a cue : . Preabyopla Magic Lantern

§ 3. A simple microscope Daylight ais Beaks Microscope or magnifier is a lens or a Artificial, Daylight Dutch Microscope

2s > Spectro-Photometer Kaplerian Microscope combination of lenses to use Section Knives Free Hand Parrifin Method Book

S a = Reagents Sildes Framo Micro-Chamist with the eye, and with it an — ascent cuturosaitve i tncestaignins enlarged, erect image is seen, dae Cia es Pissaves eaten

‘that is, the enlarged image has all i rts in the sam

ae ts De i ea © Fie. 4. FINE Print SEEN BY THE UNAIDED position as in the object it- EYE AND THROUGH A MAGNIFIER.

self (fig. 4), and but one image is formed, and that is formed upon the retina.

§ 4. Acompound microscope is one in which a lens, or combination of lenses, called an objective, forms a real image, and this real image is looked at, as if it were an object, by the eye and a magnifier or simple microscope known as an ocular. The image seen has the object and its parts inverted. In the compound microscope two images are formed, one by the objective independent of the eye, and the other on the retina by the action of the eye-lens of the ocular and the cornea and crystalline lens of the eye (fig. 3).

8 SIMPLE AND COMPOUND MICROSCOPES (Cu. I

§ 6. Virtual images. —In all diagrammatic drawings showing the microscope when looking directly into it, an enlarged, imaginary

Fic. 5-6. VISION BY THE UNAIDED EYE AND BY THE AID OF A SIMPLE MICROSCOPE.

Fic. 5. UNaripep Ever Vision. Axis, THE PRINCIPAL Optic AXIS OF THE Evr EXTENDED TO THE OBJECT.

Object The object to be seen; it is at a distance of 250 millimeters from the eye. ri The retinal image; it is inverted.

Fic. 6. Vision BY THE AID OF A SIMPLE Microscope. AxIs, PRINCIPAL Optic AXIS OF THE MICROSCOPE AND OF THE EYE.

A! B! The object within the principal focus (F) of the lens.

S M_ A double convex lens acting as a simple microscope.

Cr The cornea of the eye.

R_ Single refracting surface of the schematic eye.

L The crystalline lens of the eve.

B? A? The retinal image; it is inverted.

A’ B’ The virtual image projected into the field of vision at 250 millimeters; it is erect, and the appearance is exactly as if the virtual image were an object as in fig. 4, and no lens were present.

object is shown out in space. This is frequently called a virtual image. Tf there were no microscope and an object of that size were in front of the observer, he would get the same appearance, for a retinal image of the same size would be produced as is produced by the magnifying

Cx. 1] VISUAL ANGLE 9 glass helping the eye (fig. 5-6). In the projection microscope there is an actual or real enlarged image on a screen which the observer looks at as if it were a large object (fig. 1). If one keeps in mind that virtual images are purely imaginary, and that real images are produced by actual rays of light, it will help to avoid confusion and wrong interpretations. In every case where an object is seen, light rays must pass from the object to the eye, and these rays entering the eye must form an image on the retina. It is the retinal image which furnishes the brain the stimulus for vision.

~ APPARENT SIZE OF OBJECTS

Whether one is using a microscope or not, the apparent size of any object seen depends upon the visual angle. Practically the entire purpose subserved by the microscope is that it enables the eye to see objects under a greater visual angle than would be possible without the artificial aid.

§ 6. Visual angle. — This is the angle made by the border rays of light from the object to the retina, and crossing at the nodal point or optical center of the eye (fig. 75-76).

As the visual angle depends upon the distance the object is separated from the eye, any means by which the object can be brought closer to the eye will result in giving a larger apparent size to the object, or in magnifying it. The lenses of the microscope used with the eye enable it to get very close to the object and thus increase the visual angle, and depending on the closeness, finer and finer details of the object are separated, for they subtend an angle of one minute or more (see § 226), and the object as a whole has a much greater apparent size. (For further discussion see Ch. V.)

§ 7. Pin-hole card. — Use a piece of paper about the size of a library card. If the slip is black or of a dark color it makes the experiment a little easier than when white paper is used. Make a hole in this with a needle (fig. 7). If now one holds the slip up close to the eye and gets the hole in the optic axis, the eye can see brilliantly lighted objects very clearly. If, to start with, the object is off about I meter, quite an extent of it can be seen, and it will appear small. Now go up closer and closer, and still the object is clearly seen, and

10 VISUAL ANGLE (Ca. 1 constantly appearing larger. The closer one gets the smaller is the visible field, but the larger will the parts seem to be. If the hole is quite small, one can get the object within 4 or 5 cm. of the eye and still see the image clearly, and see details which could not be seen at a greater distance.

As shown in the figures of the visual angle (fig. 76), the closer the eye gets to the object the greater will be the visual angle, hence details are shown which did not appear at a greater distance. One of the best methods of trying this experiment is to use for object a small mark made with ink or a glass pencil on a window or on a milky or transparent lamp shade. Then there will be plenty of light. The physiological explanation of the power to see clearly through the pinhole at a distance of 5 cm., when, if the eye looks directly at the object, it should be about 25 cm. from the eye, is, that with the pin-hole the beam is so narrow that it affects so narrow a circle on the retina that the appearance is like a good focus. If one takes away the card, the beam gets very wide and the eye has only a blurred

Fic. 7. PrIn-HOLE CarD FoR impression, the diffusion circles are so Viewinc NEaR OBJECTS. large

§ 7a. In case one loses his spectacles or has the accommodation paralyzed by atropin for testing the eyes, it is possible to read fairly well with the perforated card if the print is in a brilliant light. The field which’ can be seen at one time is very small,so one must move the print or the head almost constantly.

LENSES :

The usual and most effective means for increasing the visual angle when examining small objects is by the use of lenses, singly or in combination.

§ 8. Lens. —A lens means a mass of transparent glass or other substance with one plane and one curved, or with two curved sur-

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faces. form, e.g. fluorite, quartz, etc.

REFRACTION AND LENSES II

Natural transparent’minerals may also be given a lenticular

The lens is usually a segment of a sphere or of two spheres (fig. 8). In dealing with lenses mention must frequently be made of the optical center of the lens, the principal axis, secondary axis, and the principal focus.

(1) Optical center. — The point in or near a lens through which, if rays pass, they will suffer no angular deviation, and the emerging ray will be parallel to the _ incident ray (fig. 8 c.l).

(2) Principal axis. — The axis passing through the centers of curvature of the two spheres whose surfaces bound the lens (fig. 8).

(3) Secondary axis. — Any axis oblique to the principal axis, but passing through the optical center of the lens (fig. 11-12).

A ray along a secondary axis.undergoes no angular deviation, although it may suffer displacement as a ray in traversing a piece of plane glass (fig. 51).

(4) Principal focus. —The point where rays of light, parallel to the principal axis, cross after traversing the lens (fig. 10).

Every lens has two principal foci, one on each side (fig. 10).

These are illustrated in fig. 8, 11-12, and are briefly:

Fie. 8. LENS wiTH OvT- LINES OF THE Two SPHERES OF WHICH IT IS SEGMENTS.

Axis The principal optic axis, the line joining the two centers of curvature (¢ c’).

cc’ Centers of curvature, — centers of the two spheres from which the lens is derived.

rr’ Parallel radii.

tt’ Tangents at the terminal points of the radii.

cl Center of the lens, — point where the line joining the radii at the tangential points crosses the principal axis.

With concave lenses the foci are virtual (fig. 9). § 9. Refraction. — By this is meant the change in direction of light in passing from one transparent medium into another.

The possibility of the production of images by lenses depends upon refraction.

The amount of refraction depends upon two things:

(1) The difference in density of the two media.

The greater the difference, the greater the amount of bending of the light in passing from one medium to another.

GEOMETRICAL CONSTRUCTION OF IMAGES

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(2) The obliquity with which the light strikes the second medium. The greater this obliquity the greater the bending of the light, in

ConcAVE LENS

Fic. 9. faci VirTUAL Focus F).

accordance with the law of sines. further discussion see Ch. IX.)

§ 10. Geometrical construction of images. — In this book the lenses shown are thick, but the course of the rays, for simplicity, is shown to be as if the lenses were infinitely thin, that is, they show all the bending at one plane (the refracting plane, fig. 11-12). In reality there is one refraction at the incident or entering surface and one at the emerging surface. With thick lenses like those figured, there will be no

(For angular deviation for rays traversing the optical center of the lens, but there will be a certain amount of displacement, although the emerging ray will remain parallel to the entering or incident ray (fig. 51).

For the construction of images it is necessary to know the position of the principal focus and the optical center of the lens.

§ 10a. Geometrical construction of images. — It should’ be remembered in making the drawings for the geometrical construction of images that there are two fundamental laws which must always be obeyed.

Axis

Axis

Fic. 10. Lens wits A Principat Focus on

Eacu SIDE.

Axis The principal optic axis.

F The principal focus, — the point on the axis at which rays parallel with the principal axis cross.

The arrows indicate the direction of the light.

(x) Light rays extend in straight lines in a transparent medium of uniform density, and whenever the direction is to be changed the light must meet a

Cx. I]

different refracting medium, or a reflecting surface. That is, the direction of a ray of light may be changed by using a mirror, or by putting in its path a transparent medium of greater or less refracting power.

(2) The second law is, that objects are always seen in the direction in which the light reaches the eye, regardless of the actual position of the object. This will be abundantly illustrated in the chapter on drawing; and every one knows that objects seen in a mirror are not where they appear to be in the mirror.

§ 11. Construction of real images.

—(1) The object must be situated at a greater or less distance beyond the principal focal point (fig. rz).

(2) From some point in the object, draw a line to the refracting plane of the lens (§ 10) parallel to the principal axis, and from this crossing point at the refracting plane of the lens to the focus of the lens, and continue the line indefinitely (fig. 11).

(3) From the same point of the object as in (2), draw a secondary axis through the optical center of the lens and extend it indefinitely (fig. 11).

The image of the point in the object from which the two lines were drawn will be located at the point where the two extended lines cross above the lens (fig. 11).

The image of all the other points of the object may be determined by drawing lines from them exactly as just described.

If the image is known one can find the object by reversing the process just described.

GEOMETRICAL CONSTRUCTION OF IMAGES i a / }) eee

Fic. 11-12. GEOMETRICAL Con- STRUCTION OF REAL AND OF VIR- TUAL ImaGEs.

Object, Object The object of which an image is to be formed.

Axis, Axis The principal optic axis extended above and below the lens to the object and image.

S Axis, S Axis Secondary axis passing from the object through the center of the lens.

r-p The plane of refraction (the ideal plane at which all the refraction is made to occur in diagrams of thick lenses).

R. Image Real image.

V. Image Virtual image indicated by broken lines as it has no real existence.

o b,+ m Rays of light indicated by lines passing from the extremities of the object to the extremities of the real image, which is inverted.

ob, 12,3 4,vm_ Lines representing rays of light from the object passing in a diverging manner above the lens, and extended by broken lines below the lens to form a virtual image at their crossing points, » m.

“14 GEOMETRICAL CONSTRUCTION OF IMAGES [Ca. I

§ 12. Construction of virtual images. — (1) For these the object - must be somewhere between the principal focus and the lens.

(2) From some point in the object draw a line to the refracting plane of the lens, parallel to the principal axis, and from this point through the principal focus, and continue it indefinitely.