a tne. | MICROSCOPE a CONRAD | BECK CORNELL |. UNIVERSITY LIBRARY FROM The Fstate of S.F.Gage Ce Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003017401 THE MICROSCOPE THE MICROSCOPE A SIMPLE HANDBOOK CONRAD BECK FIRST EDITION TWO SHILLINGS AND SIXPENCE NET LONDON R. & J. BECK, LTD. 68 CORNHILL, E.C. 1921 Re PREFACE Tus book is intended as a guide to the use of the microscope. The correct use of the instrument follows directly from a know- ledge of the functions of the different parts. It has therefore been found best to develop the method of manipulation in the course of the descriptions of the component portions. Such particulars as are given of the principles of its optical construc- tion are of the simplest character, and their comprehension requires no optical knowledge. Certain theoretical matters are stated in this book, but are not explained. Some of the descriptions may appear to be extremely elementary to experi- enced microscopists, but it is hoped that even they may find useful matter which is not available in the ordinary text-books. The author hopes to publish a further volume at a later date, which will deal with the optical theory and the use of the microscope in greater detail. CONTENTS CHAPTER I PAGE A SIMPLE DESCRIPTION OF THE MICROSCOPE : . dl CHAPTER IT ILLUMINATING APPARATUS AND SOURCES OF ILLUMINATION . 26 CHAPTER III APPARATUS FOR HOLDING SPECIMENS ‘ 50 CHAPTER IV SUNDRY APPARATUS ‘ : 3 ‘ j c . 67 CHAPTER V OBJECT GLASSES AND EYEPIECES . 16 CHAPTER VI THE MICROSCOPE STAND ° - . ‘ , . 92 CHAPTER VII THE MICROSCOPE AS A RECREATION . Z - ‘ . 125 INDEX . F < . . ‘: : : . 143 ZR P ASM ee eo tp a H2pOmMO DESCRIPTION OF FIG. 1 Base to support instrument. Pillar to support instrument. Joint for inclining instrument. Stage for reception of object to be examined. Hole for attaching mechanical stage. Mirror for illuminating transparent objects. Substage for carrying illuminating apparatus. Substage focussing adjustment. Substage condenser for regulating the illumination. Iris diaphragm for varying the light. Limb for holding the body and stage. Body for carrying the observing lenses, Drawtube for lengthening body. . Drawtube stop to prevent reflections from tube entering eyepiece. Eyepiece, combination of lenses nearest observer's eye. Fine adjustment for delicate focussing of body. Coarse adjustment for rapid focussing of body. Object glass, combination of lenses nearest the object. Nosepiece with universal screw for carrying object glasses, Eyepoint position where all emergent light passes through a small area and where observer’s eye should be placed. Position where the primary image formed by the object glass is produced. ‘ Position where final virtual image formed by eyepiece is produced. THE MICROSCOPE CHAPTER I A SIMPLE DESCRIPTION OF THE MICROSCOPE THE microscope is an apparatus for producing an enlarged image of a small object. In its complete form it is an elaborate instrument, but to understand its construction it may be looked upon as a complex form of magnifying lens with the addition of means for making delicate adjustments both for moving the lens and the object and for obtaining special forms of illumination. It consists primarily of three parts—the body, which carries the observing lenses, the stand or framework, and the illumination apparatus. The body (M) carries an object glass (R), which is attached to The body. the object end by a standard size screw thread, and an eyepiece (O), which slips loosely into the tube at the eye end in a standard size fitting. It has a telescopic tube, called a draw-tube (N), for varying the distance between the obiect glass and the eyepiece, and a diaphragm (N1) to prevent reflections from the inner sur- faces of the tubes from entering the eye. The body and its lenses combined form the magnifying apparatus. The object to be examined is placed on the stage (D) of the microscope. The object glass if used by itself acts in the same manner as a lantern lens. It throws an enlarged picture of the object to a position (U) at the upper end of the body, just as a lantern lens throws an enlarged picture of a small lantern slide upon a white screen, but instead of its being thrown upon a white screen it is thrown into space. This image is examined with a magnifying lens called the eyepiece (0), by which it is further magnified. If the primary image were projected upon a lantern screen and one were to cut a hole in the screen and stand behind it with a magnifying lens focussed upon the plane of the screen, one would have the same kind of instrument as a micro- scope on a large and inconvenient scale. ll 12 THE MICROSCOPE i a The object glass (R) in the earliest instruments was a single es double convex lens (Fig. 2); it i an ono but very im- erfect picture of small objects, the outlines were Pirecandesl by coloured fringes, and the details were fuzzy and indistinct. Such lenses were made Fic. 2. several hundred years ago, but in the early part of the nineteenth century is was discovered that the defects of a single lens could be overcome by using several lenses in combination, made of different kinds of glass and of suitable shapes and sizes. . Focal length Modern object glasses are made of different powers to give of object different magnifications in the primary image, and, in general, the more an object glass magnifies, the larger the number of lenses that are required to produce a perfect image- For instance, i i ction of the 2/3-inch (16-mm.), Fig. 3 shows the optical construct: I /6-inch(4-mm.), a and 1/12-inch 3 (2-mm.) object glasses. The name 2/3, 1/6, or 1/12 inch, as applied to an _ object glass, represents its focal length. It indicates its magnifying power. If an oly. ordinary single Fie. 3.—f =focal length; w = working distance. Jeng of 92-inch _ focal length is used as a hand magnifying glass, it has to be placed about 2 inches from an object to give a clear image, and the 2/3, 1/6, and 1/12 inch require to be placed at about these respective distances from the object when in use—thus the higher the magnifying power of a lens, the closer it must be to the object. Working Object glasses are not single lenses, but are composed of distance. + several, and consequently the focal distance is measured from a point about half-way between the front and back surfaces of the component lenses. The distance between the foremost lens and the object is, therefore, always considerably less than the true focal distance. This is called the working distance to signify the space between the end of the microscope and the object when it is so adjusted that a clear picture is obtained, or when it is, as it is called, “in focus.” A list of the working distances of different object glasses is given on page 82. An examination of the diagram (Fig. 1) on page 9 illustrates S54 | 1 —- sem wan ho Bl A SIMPLE DESCRIPTION OF THE;}MICROSCOPE 13 the formation of the images. An enlarged picture of an object placed upon the stage (D) is formed in the neighbourhood of the eyepiece at U, and the eyepiece again magnifies this image, projecting the light into the eye as if it came from an object situated at V. The eye, when placed in a small area (T) through which all light passes, and which is known as the eyepoint, sees the final picture of the object as if it were a real object placed at V, 10 inches from the eye. It is assumed for convenience of measurement that this picture is actually 10 inches away, though it may be formed at a some- what different position according to the adjustment or condition of the observer’s eye. Whether the virtual image is actually at 6, 10, or 20 inches is of no importance. It makes no difference to the size of the picture, because when the virtual image is formed farther away it becomes proportionally larger. In Fig. 4, if E is the eye and O 0’ 0” are objects of different sizes, they produce the same size pictures in the eye if placed at such distances that they subtend the same angle. The magnifying power of the microscope will depend upon the size of this final image formed at V (Fig. 1) compared with the size of the E o oa oO” object being examined. In this Fie. 4, connection it should be under- = stood that if a microscope is said to magnify 100 diameters, it means that the picture that is seen is 100 times as-long and 100 times as wide as the object would appear if it were taken from the stage (D, Fig. 1) and placed in the position V, 10 inches from the eye. In order to express how much larger an object appears when seen through the microscope than when seen by the naked eye, a standard distance must be taken, because an object appears to the naked eye to be of different sizes at different distances. A sixpence is almost invisible at a distance of 100 yards, but it is a large object at 8 inches. Therefore, some standard must be taken for comparison purposes, and 10 inches has been universally adopted. The magnifying power of a micro- scope always denotes the relative size of the picture com- pared with that of the original object when placed 10 inches from the eye. ; If a microscope has a magnifying power of 100, such magnifi- cation may be produced by different methods. The object glass may magnify the object twenty times in the primary image, and the eyepiece increasing the primary image five times will give a total of a hundred. This magnification may also be pro- Eyepiece. Virtual mage. Magnifying power. Different methods of obtaining magni power. Field of view. Aperture. 14 THE MICROSCOPE duced by a lower power object glass which magnifies the object ten times, and a higher power eyepiece which magnifies it again by ten. The same result is obtained as far as magnifying power is concerned, but a different result as regards the quality of the image. Another method of varying the magnifying power is by increas- ing the distance between the object glass and the eyepiece. To enable this to be done the microscope is supplied with a sliding drawtube (N), which allows the tube length to be varied from 140 to 200 mm. The reason for this increase in magnification is well illustrated by reference to the lantern, in which case the lantern lens gives a larger picture when it projects it upon a screen that is at a greater distance. In the same way the micro- scope object glass produces a more highly magnified primary image if by slight adjustment in the focussing of the instrument the picture is formed at a greater distance, and the drawtube of the microscope is extended so as to examine the picture formed at this greater distance. The “‘ field of view” is a term applied to the size of the object that can be seen at one time by means of the microscope. To assist in increasing the size of field an eyepiece is made of two lenses instead of a single one. The lower field lens is situated below the position U (Fig. 1), where the primary image is produced, and increases the field of view while the upper lens does the magnifying. Suppose that the apparent field of view is a circle of about 8 inches diameter at the position V, where the final image seen through the microscope appears to be. It is evident that with a microscope magnifying 100 diameters, the size of the largest object that can be observed at one time is only 1/100 the size of this field, or about 1/12 inch, so that for this reason alone it is important that a microscope should possess a means of varying the magnifying power. It is sometimes desirable to examine a large area of an object with a small magnifying power, at others a small area with a large magnifying power. A table of the fields of view given by different lenses appears on page 82. The question arises as to whether it is preferable to vary this magnifying power by means of changing the eyepiece, by means of changing the object glass, or by means of lengthening the drawtube. This is influenced by an optical consideration of great importance. In the early days, before it was understood how to correct the errors of a single lens, microscopes were constructed in which the object glass was a single lens, the defects of which were reduced by putting a very small aperture—almost a pin- hole—in front or behind this lens. This meant that only an extremely fine cone of light from each point of the object could A SIMPLE DESCRIPTION OF THE MICROSCOPE 15 enter the instrument (see Fig. 5). It was soon found that when this was the case, although great magnifying power could be obtained, fine detail could not be seen, but merely a repre- sentation on a larger scale of the coarse structure which could readily be seen with a lower magnifying power. In order that an advantage should be obtained from the use of higher magnifying power, it was necessary to admit into the microscope a correspondingly larger cone of light from each point of the object, as unless this were done, no advantage could be obtained in the observation of fine details. Such a plan had the further advantage that it collected a larger amount of light and rendered the object more brilliant. The size of the cone of light admitted into the microscope from each point of 4 the object is called the aperture SmallAperture Large Aperture (a, Fig. 5). It is expressed nent sctad either by the angle of the cone F!. 5.—¢ = angular aperture. of light entering the micro- scope or by a figure called the numerical aperture, or N.A. The aperture is of such paramount importance, that the limit rimit ot of what can be seen with the microscope does not depend upon Jaen aent what magnifying power can be obtained, but upon what size on aperture, cone of light can be collected from the object by means of the object glass; and lenses can be made with a much higher magnify- ing power, but they cannot be made with a larger aperture, than those in use at the present time. The aperture, therefore, has a direct bearing upon the best method of increasing magnifying power, because, if an object glass can only admit a certain aperture of light, the use of an eyepiece does not alter this property, and therefore to increase the magnifying power by high eyepieces is of no service, when carried beyond that power which is sufficient to enable the detail that can be shown by the aperture of a particular object glass to be seen. The best method of increasing the magnifying power is, Best method therefore, by changing the object glass. Most object glasses phoma have sufficient aperture to allow of the use of an eyepiece of as power. high a power as 15, but, in general, magnification of more than 10 by means of the eyepiece should only be used in special cases, and the object glass should be changed rather than the eyepiece. The same reason makes it undesirable to depend for increased Standard magnifying power upon extending the drawtube of the micro- pet" scope, and the more so in this case because the object glass can only be constructed to work at its best with a particular length Thickness of cover glass. Apertures suitable for different powers. Table of apertures and powers. 16 THE MICROSCOPE of body. To obtain the most perfect results a tube length of 160 mm. should be used—the drawtube of the microscope is graduated, and can be set at this figure. If a revolving nosepiece is in use, this lengthens the body 15 mm., and the drawtube should be set at 145 mm. instead of 160 mm.; with a Sloan object glass changer measuring 10 mm. it should be set at 150 mm. The thickness of the cover glass used over the object has no effect with an immersion lens and but slight influence with the low powers, but is a matter of importance with a high-power dry lens. A 1/6-inch object glass can only be optically correct for one thickness of cover glass, and it is most important to always use those known as No. 1 thickness. The object glasses, unless otherwise ordered, are always made for a thickness of °007 inch (‘18 mm.), which is the average thickness of No. 1 cover glass. Thicker cover glasses should only be used for objects to be examined with low powers. The delineation of fine structure depends upon the aperture of the object glass being sufficiently large to produce an image of this fine structure, but combined with this it must possess a sufficient degree of magnification to enable this image to be clearly seen. We may know that the finest lines of an etching or steel engraving exist in a print, but it may be necessary to magnify the image in order to make them visible as single lines to the eye. If the print is magnified further, the fine lines appear thicker, but no further fine lines are there to be seen. Thus lines which are invisible require a certain degree of magnification to see them clearly, but extra magnification beyond this point is useless. So with a microscope object glass, it must possess a large enough aperture to produce the detail in the image, and the magnifying power need not be more than enough to enable the eye to see it clearly. Each object glass has a particular aperture, sifficient to form an image of all the detail that can be seen with the magnify- ing power given by it in conjunction with a moderate eyepiece. The following table gives the apertures of standard object glasses : Initial e F ies | oa meatie Me | Mera Power. : 42mm. | 25mm. | 17mm. Ifin. =40 mm. . 19° 16 3 20 34 50 1gin. =32 mm. . 17° 15 4 25 45 65 2/3in. = 16mm. .{ 32° 28 10 62 110 155 1/3in. =8mm, . 60° 5 185 115 200 285 1/6in, =4mm. . | 116° +85 40 285 490 690 1/8 in. oil immer- sion =3mm, . —_ 95 60 427 735 =| 1,015 1/12 in. oil immer- sion =2mm. . — 1:3 90 530 900 |1,275 A SIMPLE DESCRIPTION OF THE MICROSCOPE 17 The 1/6-inch is receiving from the object, cones of light Angle in air of 116°, as shown in Fig. 6. It could not be made to collect compared. a very much larger angle of light because it cannot be used in elas. absolute contact with the object. Sufficient space must be provided for a thin glass cover and a small distance for focussing adjustment. It will be noticed in Fig. 6 that the cone of light, which is 116° as it enters the object glass, is only 68° when it passes through the object. It is spread out by refraction as it enters the air between the cover glass and the lens, If the air space between the cover glass and the lens could be filled up with glass, this spreading out of the cone would not occur, and the cone of light would remain 68° when it entered the lens. As far as the power of depicting detail is concerned it would be equal to a 116° cone in air. It is the same body of light and has just the same properties in this respect. If therefore the space between the object and the lens is glass throughout, a larger angled cone than 68° can be collected by the object glass, and a greater power of depicting detail, what is known as resolution, can be reached, and a further power of seeing fine structure obtained. Cedar-wood oil is a liquid which has the optical properties of Immersion glass, and if a drop of this oil is placed between the front of opie the object glass and the cover glass, the whole distance between the object and the lens is equivalent to glass. A much larger effective aperture can thus be obtained with corresponding increase in resolution. Thus object glasses of higher power than 1/6 inch (4 mm.) are generally what are called immersion object glasses. They are so constructed that a drop of cedar-wood oil must be placed on the front lens so that it connects it to the object being examined. The method of describing the aperture by the term numerical Numerical aperture (N.A.) instead of by the actual angle of the cone is to 7" enable the resolving power of a microscope to be correctly stated. A1/12-inch oil-immersion object glass is generally made to admit an angle in glass of 117°, which corresponds to an angle of more than 180° in air. Itis almost the same actual angle as the 1/6-inch admits from air, but the numerical aperture (N.A.) which gives its true resolving power is 1°3 N.A., while that of the 1/6-inch is only *85 N.A. Dry lenses such as the 1/6-inch cannot be used with cedar- wood oil as immersion lenses, and immersion lenses cannot 2 Immersion fluids. Flatness of field. Depth of focus. Coarse focussing adjustment. 18 THE MICROSCOPE be used without the cedar-wood oil because the lenses must be specially constructed for the conditions under which they are used, No immersion fluid but cedar-wood oil, or a fluid sold for the purpose with exactly the same optical properties, must be used. Water, paraffin, or several other substitutes which are some- times inadvertently employed, entirely destroy the fine quality of the image formed by an oil-immersion lens. In every optical instrument the centre of the field gives the finest definition, and the object being examined should be placed near the centre. An absolutely flat field is incompatible with the finest definition in the centre, and although in certain types of telescopes and photographic lenses the importance of a flat field is so great that a compromise is made, no deterioration of the central image can be allowed in the microscope. The penetration or depth of an object glass or the number of different layers of an object that can -be seen sharply at the same time with a microscope is very small. With lenses of a high aperture, and therefore in general of a high magnifying power, the penetration decreases at a very rapid rate, and the power of seeing different planes sharply must depend on adjusting the instrument. It has been said that the depth of focus of a high-power microscope is really the fine focussing adjustment. The fine adjustment in the hands of a skilled observer is in constant motion, focussing first to one plane and then to another; by this means a perception of depth is obtained which could never be given by an object glass fixed at one focus. The penetration of the microscope may be increased by insert- ing a stop with a small aperture immediately behind the object glass, but such a method reduces the aperture and consequently the detail that can be seen. It is seldom adopted except for photographing certain objects where the image from the upper or lower portion of the object obscures the layer being photographed, or for photographing objects with compara- tively coarse structure. An iris diaphragm is made that will screw into the body of the microscope between it and the object glass for this purpose. There is only one position in relation to the lenses where an object can be placed to give a perfectly clear picture. This. position is generally called the focus, and the microscope is said to be ‘‘in focus” when it is so adjusted that the object is in this position. It is more convenient to effect this adjustment by moving the body which carries the lenses rather than by moving the object. The coarse focussing adjustment is actuated by a helical rack and pinion which moves the body along a slide towards or away from the object. Turning the milled head so that its upper edge moves towards the observer, raises the body ; away from the observer, lowers it. It is a sufficiently delicate A SIMPLE DESCRIPTION OF THE MICROSCOPE 19 motion for focussing with object glasses of lower power than 1/6-inch (4'mm.). The fine focussing adjustment does exactly the same as the coarse adjustment, but the movement is far more delicate: it is actuated by a micrometer screw and a lever moving the whole body along a second slide. A complete turn of the screw moves the body about a quarter of a millimetre. Turning the fine adjustment milled heads moves the body in the same direction as those of the coarse adjustment. In the “Standard” micro- scope the left-hand milled head is twice as delicate a motion as that on the right-hand side. The fine adjustment is required for the focussing of high powers and for examining the different layers of an object. In moving the body of the microscope up and down to obtain the correct focus, care is required to prevent the front of the object glass being forced into contact with the object by racking it too far down. It is easy to break a valuable specimen by this means; and although for its protection the metal mount of the object glass projects slightly in front of the front lens, it is delicate in construction, and can be damaged by being brought into contact with the specimen. Experienced microscopists can focus a lens downwards and stop at the position where the object is sharply seen, but it is unsafe. The correct method is to set the body of the microscope so that the front of the object glass almost but not quite touches the object, and then to rack backwards, turning the milled heads so that the upper portion turns towards the observer, and raise the body until the correct focus is found. With high-power object glasses, especially oil-immersion lenses, this method is not so easy because the distance of the correct focus may be below the point at which the body has been set in the first instance. If, however, the slow motion is used to make the final adjustment, damage is not likely to occur, as it lowers the body very gradually, and the latter is only pressed down upon the object by a spring. When using an oil-immersion lens ajdrop of cedur-wood oil should be placed on the object glass, and the body of the microscope racked down until the drop of oil touches the cover; the final focussing can then be done with the,fine adjustment. Some objects are so transparent that it is quite easy to pass by the focus and miss the correct position. In these cases dust on the cover glass may be focussed first, and thefineadjustmentlowered by an amount representing the thickness of the cover glass. If the slide be moved backwards and forwards on the stage during the process of focussing, the movement will be seen directly the correct position is nearly reached. It may appear absurd to mention that if a slide happens to have been placed on the stage upside down a high-power object glass will not focus through the thick glass slip, but the writer Fine focussing adjustment. The best method of focussing. Revolving nosepiece, Sloan object changer. 20 THE MICROSCOPE has more than once made such a mistake and wondered why he could not focus his specimen. The nosepiece (S) of a microscope is the lower end of the body (M) provided with a screw for attaching object glasses. A revolving nosepiece is an appliance which screws into the nosepiece and which » carries a revolving plate into which two ) or three object glasses can be fixed, @ known as double or triple nosepieces respectively. By rotating the revolving plate each object glass can be rapidly Fia. 7.—No. 3301, Dust-brought into use, being held in the tight Triple Nosepiece. correct, position by a spring clip. The best form is made so that no dust can drop into the back of the object glasses and they can be safely left attached to the micro- scope. The extra length of the body caused by the length of a nosepiece is 15 mm., and the drawtube should be closed by that amount or set at 145 mm. instead of 160 mm. An object glass changer is an apparatus for rapidly changing the object glasses by another method. Each object glass is screwed into a fitting which slips into an adapter that is fixed to the nosepiece of the microscope, and as each fitting is provided with two adjustable abutment screws the object glasses can be individually adjusted so that they exactly register as regards the position of the field of view. Changing an object glass by this means is nearly as rapid and more accurate than that of a revolving nosepiece, and is far more convenient when the object glasses are to be used on different instruments or where more than three are used. It consists of an adapter which has on one side a sloping projection (A), and on the other a clamp screw (B) which actuates a bevelled nut (C). The adapter is clamped to the nosepiece of the microscope by a screwed ring (D), which is provided with slots, into which a half- penny will fit for tightening rye, 8,—No. 3280, Sloan Object it up. Glass Changer. Loose fittings (Fig. 9) are supplied, one of which is screwed on to each object glass. Each fitting has a bevelled gap which fits loosely over the bevelled nut (C) of the adapter and swings round into position when a turn of the milled head (B) forces the fitting against the sloping projection (A) and holds it firmly in position. Each ! A SIMPLE DESCRIPTION OF THE MICROSCOPE 21 adapter has screwed studs with clamping screws, which form the stops in both directions when the object glass is in the correct position. These can be adjusted by means of a spanner supplied for the purpose, so that each lens can be centred with an accuracy that is never possible with a revolving nosepiece, because the error of each individual object glass cannot be com- pensated with the latter. The construction of this apparatus is so simple and rigid, having no slides to wear loose, that it remains in ad- justment permanently. The total extra length of the micro- scope body caused by its use is 10mm., fro, 9No. 3281, Fit- and the drawtube should be set at ting of Sloan Object 150 mm. to obtain the standard tube Glass Changer. length. A box is supplied to carry object glasses with fittings screwed on ready for use, held against dust-tight pads. The illumination of an object seen with a microscope is of tumin- |. almost as much importance as the quality of the lenses. I¢ is 4 interesting to find that the methods worked out by those who were enthusiastic in the use of the microscope as an enjoyment, and to a great extent as an amusement, have been one by one adopted by the more serious scientific worker who has sometimes been ready to consider the time spent on the pure manipulation of the instrument to be of little value. The proper use of the substage condenser to regulate the light in viewing transparent objects is now acknowledged to be of first importance for correct observation. Dark-ground illumination, which has been considered by some to be only useful to show in an attractive manner what could be seen equally well by direct light, has proved to be of paramount importance for the study of living bacteria and colloid particles. The methods devised for illuminating opaque objects have formed the basis for the observation of metallurgical specimens, and the much-criticised study of the markings of diatoms and insects’ scales has proved to be of the greatest value in enabling the images seen by the microscope to be correctly interpreted. A bad lens can never be made to give a perfect image, but a good lens will only give the best image when the illumination is satisfactory. Most objects seen with the naked eye only require that a sufficiently powerful light should fall upon them. They reflect back the light that they receive, or the greater portion of it, in all directions. It is not of importance where the light which illuminates them comes from, although occasionally, when the light falls upon them from one side only, such deep shadows may be formed that it is difficult to recognise the true appearance. 22 THE MICROSCOPE Vasenot The same holds true of opaque objects examined with the objects. microscope, but the greater number of microscope specimens are either transparent or semi-transparent, and must be viewed by sending a beam of light through them from behind. This beam then passes through the microscope into the eye. Natural objects are seldom viewed in this manner, but in order to examine the water mark of paper or a photographic transparency, they must be held between a strong light and the eye, and the ability to see the pattern of the water mark or the view in the transparency depends on certain portions of the light being blocked out which would otherwise enter the eye. In the black portion the whole light is stopped, in others only a portion is absorbed, and thus a complete range of tone in the picture may be obtained. This is the method by which semi-transparent objects are seen with the microscope. The conditions are not the same as ordinary vision, and the direction and character of the beam of light used to illuminate them are a matter of great importance. The mirror. | The mirror of the microscope (F, Fig. 1) is used to direct a beam of light from some source of illumination through the object into the microscope. The mirror swings in gimbals and can be moved in all directions, It has on one side a flat, silvered surface which gives a plane reflection, and on the other a concave surface which -—L- concentrates a more powerful beam upon a small area of the object. The direction of the light should be such that it shines directly along the line that passes through the centre of the microscope—the line that is known as the optic axis. If the light comes from the side it passes obliquely through the object, and even if it does not give an erroneous appearance it prevents a clear image being formed. To illustrate the effect on the object, one should examine the appearance of MMP», fairly thick piece of wood which has fine holes drilled in it: light from one side would not pass through and would not reach the eye. The holes will not Obliquelight. Directlight. be visible unless the light is passing Fie. 10. through them centrally. The effect of an oblique beam of light as it passes through the microscope lenses is shown in Fig. 10. The left-hand diagram in Fig. 10 shows the object glass transmitting oblique light only. The light which actually forms Direction of the light. A SIMPLE DESCRIPTION OF THE MICROSCOPE 23 the image is a fine bundle thrown to the edge of the object glass, so that the object glass acts as if it had only a pinhole aperture at one side, and is consequently no better for depicting detail than the early pinhole lenses which were made before the modern achromatic microscope was discovered. This diagram also shows how the direction of the light can be immediately recognised by focussing the microscope. The only light producing the primary picture is shown in the left-hand diagram of Fig. 10. It is on the right or the left of the axis, according to whether it is above or below the true focus, There- fore, by putting the object in and out of focus with the focussing adjustment, the direction of the light can be observed. When the light which illuminates the object is oblique instead of being truly central, the object will not only become indistinct on either side of the focus, but will appear to move from side to side ; whereas if the light is truly central, the object will become less distinct on either side of the focus, but will not alter its position. The mirror can always be adjusted until the object remains | | stationary as the microscope is being focussed, and the centring of the light is thus assured. Below the stage of the microscope an iris diaphragm (K, Fig. 1) is fitted, and if this is shut down to a small aperture the light will not pass through the microscope at all if the light is very far away from the axis, though this is uot in itself sufficient to make the final adjustment. The nature of the illumination may be varied according to Matarsiot whether it is parallel, divergent, or convergent. If the flat side"™™™™*"°" of the mirror be used and the source of light is at a consider- able distance, a beam of nearly parallel light is obtained (a, Fig. 11). If the source of illu- mination is very close, a diver- gent beam is obtained (c, Fig. 11). If the concave mirror is used, it will be a slightly convergent Fre.11.—Mirror reflecting parallel, beam (6, Fig. 11). By means of convergent, or divergent light. a substage condenser (J, Fig. 1) ; ; with an iris diaphragm below it—described later—the light can be rendered still more convergent and can be regulated with accuracy. As the light which enters the condenser at its margin emerges as the outer portion of the cone, the effect of reducing the aperture of the diaphragm of the condenser is not only to reduce the amount of illumination, but to alter its character by reducing the size of the cone of emergent light. Thus with a very small aperture an almost parallel beam of light can be obtained, and by opening the iris diaphragm a more and more highly convergent cone of light may be used (see p. 27). Tilumina- tion for transparent objects. Scattering of light by object. 24 THE MICROSCOPE As regards the best kind of illumination for transparent objects, the light may be a nearly parallel beam from the flat mirror, or a slightly divergent beam from the flat mirror used with a lamp near the mirror. The light from a lamp may be rendered nearly parallel by placing a bull’s-eye condenser close to the lamp. To find the correct position for the bull’s-eye to give parallel light, an image of the flame or filament of the lamp should be observed on a distant wall and the bull’s-eye moved till the lamp or filament is in sharp focus on the wall. The light is then approximately parallel, and the microscope should be so placed that the mirror is in the beam of light about 8 or 10 inches away from the lamp. , The light may be made slightly convergent if the bull’s-eye be arranged to give parallel light, but the concave instead of the flat mirror be made use of. The light may be condensed by a substage condenser, which not only increases the brilliancy of the illumination, but also gives a strongly convergent beam of light which may be modified to any extent by the use of the iris diaphragm and stops, as described more fully under the description of substage condensers. The question as to whether the best results will be obtained by parallel, divergent, or convergent il- lumination, depends to a great extent on the nature of the object. When light shines through certain kinds of a b objects it is distributed or scattered Fie. 12. in all directions. A cut-glass lamp- ; shade breaks up the light that falls upon it and scatters it all round; the same thing occurs to a lesser extent in the case of a botanical or histological section of tissue when each cell or irregularity acts like a facet of a cut- glass lamp-shade. In this case, whatever the nature of the illumination, there is a sufficiently scattered light to fill the aperture of the object glass, and the general structure of the tissue will be accurately depicted. This, however, does not apply to all kinds of objects. Some do not scatter light, and the question as to whether the aperture of the object glass is filled with light depends on the nature of the illuminating beam. If an object which does not scatter light is illuminated as shown in Fig. 12 (a), the object glass might just as well have nothing but a pinhole aperture; and to make use of the aperture a convergent cone of light must be thrown upon the object, as shown in Fig. 12 (b). All small objects spread the light slightly by diffraction, though in the case of a single dark object on a white field, the A SIMPLE DESCRIPTION OF THE MICROSCOPE 25 amount of such spreading is relatively small and need not be considered. If the object is a regular periodic structure, like a series of dots and lines, the spreading due to this cause may be very considerable, and such an object may not require so large a beam to use the full angle of the object glass. This is very noticeable in the case of the fine periodic structure of diatoms, where the structure may often be shown when the illuminating cone of light is considerably less than that required to fill the whole of the aperture of the microscope. In such cases it will be observed, if the eyepiece of the microscope is removed, that the central direct beam illuminates the central portion of the back lens of the object glass, but the rest of the lens may be illuminated almost as strongly by the large amount of diffracted light scattered by the periodic structure of the diatom. For the use of the microscope with any but the lowest magnifying powers, a substage condenser should be used in order that the nature of the illumination may be completely varied at will. CHAPTER II ILLUMINATING APPARATUS AND SOURCES OF ILLUMINATION A Most important part of the microscope has now to be con- sidered, namely, the substage condenser, which is essential with all higher powers to converge a beam of light upon the object in order to illuminate it brilliantly and to vary the character of the illumination. There are three different kinds of substage condensers. The simple so-called Abbe condenser consists of two lenses with an iris diaphragm close behind the back lens and a tray below for the insertion of patch-stops or colour filters, as shown in Fig. 13. It was in use under various names long before the time of Abbe, who, however, popularised it in a particular form of mounting. It does not focus the rays correctly to one spot (see Fig. 14), Fie. 13.—No. 3286, Abbe Condenser. the oblique rays coming to a nearer focus than the more direct, and does not form a definite image of the source of illumination due to the uncorrected lenses of which it is constructed. It has an aperture of 1 N.A.—that is to say, it will give 180° in air, the maximum aperture obtainable with a dry condenser. A large beam of light from the mirror thrown upon the back lens is concentrated upon a small area (0). This area is illuminated by an imperfect image of the source of light. The condenser fits into the substage of the microscope by which it can be moved up and down, or “focussed,” or can be moved laterally, or “centred,” until the illuminated area (O) coincides with the object being examined. The object is by this means brilliantly illuminated. A powerful illumination is often required to overcome the loss of light due 26 ILLUMINATING APPARATUS 27 to the large magnification obtained with high-power lenses ; but this is not the only advantage gained by the use of a condenser, as illumination might be increased by other means—for instance, by bringing a source of light closer to the object. A substage condenser receiving an approximately parallel bundle of light from the mirror of the microscope converts it into a wide angle cone of light, When this light is centred and focussed, the object is illuminated by light falling upon it in all directions. The achromatic condenser (Fig. 15) has the same aperture as Achromatic the Abbe condenser 1 N.A., but it is corrected almost as care- fully as a microscope object glass, so that the rays come to exact points, and a very perfect image of the source of illumina- tion is formed in the plane of the object, much reduced in size. It is provided also with an iris diaphragm and a tray for patch- stops and filters. Fig. 16 shows a beam of light (A, B, C, D, E, F) passing through this con- denser to the central point of the object at O. The rays A, F, which are at the margin : of the beam of light as it enters, emerge as the most oblique rays falling upon the object O, and the rays C, D, which enter near the centre of the condenser, emerge nearly parallel. Thus, if the iris diaphragm which is placed below the condenser is gradu- ally closed, it excludes more and more of the oblique rays. Fig. 16 shows a large solid cone of light of great angle converged upon the object, the iris diaphragm being fully open. Fig. 17 shows a small- angled cone transmitted by the same condenser, the iris diaphragm being partially closed. Fig. 18 shows the same condenser in which the iris diaphragm is fully open and an opaque patch or stop is placed below the condenser so that the object is being Fic. 15.—No. 3288, Achromatic Condenser. [A SSSHrAggy] LA WL ABCODEF Fia. 16. Fie. 18. Immersion achromatic condenser. Use of a Bubstage condenser. To focus a substage condenser, 28 THE MICROSCOPE illuminated by a hollow cone. Stops or patch-stops with apertures of different shapes, or in different positions below the condenser, used in combination with the iris diaphragm, regulate the illumina- tion so that light in any direction may be passed through the object. ; The dry and immersion achromatic condenser is of even higher quality than the achromatic condenser, being equal in its corrections to a microscope object glass, and has an aperture of 1°3 N.A., so that it can, if used in immer- sion contact with the under- surface of the slide, fill the whole aperture of an oil-immersion lens with light. For transparent objects with object glasses as low in power as a l}-inch (32-mm.) a condenser is not required. The aperture of such a low-power lens is small, and the angle obtained by the sai ees ie Dry and _yse of a concave mirror is usually erry ee sufficient to make the best use of this lens. The same applies to some extent to the 2/3-inch (16-mm.) object glass, but as this lens is so frequently used as a finder for a high-power, it is not always convenient to rapidly remove the condenser, and it is customary to use the condenser, but to put it somewhat out of focus in order to fill the whole field with an even illumination. A condenser should always be used with the 1/3-inch (8-mm.), 1/6-inch (4-mm.), 1/8-inch (3-mm.), or 1/12-inch (2-mm.) object glasses. A substage condenser is only corrected for light which is parallel or slightly divergent ; therefore the flat mirror should be used. The concave mirror giving convergent light is quite un- suitable for use with a condenser. Daylight as a source of light is not recommended with a condenser, as the finest detail cannot be shown by its means. For ordinary microscopic examination of not too critical a nature, daylight is satisfactory, but even then the more delicate details may escape notice. Assuming that the source of light is a paraffin lamp with a flat wick, or other small source of illumination, the condenser must first be focussed and centred. In order to focus the condenser, sufficient light must be thrown through the object to render it visible, and the object glass must be focussed upon the object. The iris diaphragm of the condenser should then be shut down to about one-quarter its size, and it should be focussed up and down until an image of the flame of the lamp is seen sharply in focus at the same time as that of the ILLUMINATING APPARATUS 29 object ; the mirror will require to be adjusted in order to direct the light through the condenser. If the lamp be turned round so that the edge of the flame is opposite the mirror, it makes an easier object to focus, and its image will appear as a slit across the field of view. When focussed the lamp may be turned round, with the flat side of the flame facing the mirror, thus illuminating the whole field. It is found in practice that the best resolution is obtained when the source of light is almost, but not quite, in focus. The accuracy of centring of the simple Abbe condenser is not To centre important. The image that it gives is not accurate, and it is {supsiase generally sufficient to move the mirror slightly till the image of the object does not move from side to side, while the body of the microscope is being focussed up and down in a similar | manner to that described on page 22 for setting the mirror when used alone. Microscopes fitted with this condenser are frequently not provided with a centring adjustment to the substage. When using the achromatic or immersion condenser centring is a matter of importance. The iris diaphragm is placed in a position at such a distance below the condenser lenses that if the condenser be moved downwards the source of light will be put out of focus and an image of the small aperture in the iris diaphragm can be sharply focussed. When doing this the iris diaphragm should be closed to its smallest aperture, so that the image is of a sufficiently small size to be seen in the field of view. The condenser may then be moved by the centring screws until the image of the diaphragm is in the centre of the field of view. If the image of the small aperture is so far out of centre that it is not in the field, the aperture can be enlarged until its edge begins to appear. When the condenser has been thus centred it should be moved upwards till the image of the lamp is again in focus on the object, and the mirror readjusted if the light is not in the centre of the field. The achromatic condenser is now in the best position for use with a 1/3-inch (8-mm.), 1/6-inch (4-mm.), or 1/12-inch (2-mm.) object glass, though slightly better resolution is obtained if the source of light is a little out of focus. If it is required to use a 2/3-inch (16-mm.) object glass, the condenser can be focussed down to give an evenly illuminated field, being brought back into focus when a higher power is used. The substage condenser having been focussed and centred, natect ot the eyepiece of the microscope should be removed, and the a een effect of opening and closing the iris diaphragm be observed by aisphnseu yt looking down the tube of the microscope. This effect is best : observed when an object glass with a fairly large aperture such as 1/6-inch (4-mm.) is used. When the iris diaphragm is fully open the back lens of the object glass will be completely filled with Hffect of focussing a substage condenser. Best aperture of illuminating cone. 30 THE MICROSCOPE light appearing as a uniform circular disc; closing the dia- phragm will at first not make any change in the appearance, because the condenser is coverging upon the object a beam of light of greater angle than can be collected by the object glass, and it will receive the full illumination until the light from the condenser becomes smaller in angle than the aperture of the object glass. If the diaphragm be now closed to its fullest extent, the back lens of the object glass shows a small spot of brilliant light. in the centre; and as the aperture of the iris diaphragm is slowly opened, the spot of light slowly increases in size until the whole of the back lens of the object glass is completely filled with uniform light. As soon as this is done the whole of the aperture of the lens is receiving direct light from the condenser shining through the object as shown in Fig. 16. While the microscope is in this condition, the effect on the appearance of the back lens of the object glass, which is produced by putting the condenser in and out of focus, should be: observed. The iris diaphragm being open to the full extent, it” will be found that unless the condenser is in correct focus the whole area of the back lens will not be equally filled with light, and from the appearance so observed it can be realised why an uncorrected condenser like the Abbe condenser cannot readily be made to fill the whole area of the object glass with uniform | light, the reason being that the light of different obliquity is brought to a focus at different positions. It may be that only one ring of light at the edge of the object glass, or a small area in the centre, or a combination of both, is being illuminated. The importance of the character of the illumination has been referred to, and the question arises as to what aperture cone of light it is best to use. A well-corrected substage condenser centred and in focus gives the means of completely controlling this, and from what has so far been said it might be supposed that the full aperture of the object glass should be always illu- minated; this is not necessarily the case, further research is required before definite rules can be laid down to meet all condi- tions. For the best resolution the diaphragm in the condenser | should never be opened to admit a larger angle of light than that; of the object glass, or “ glare”? may produce a misty appearance which will destroy the crispness of the image. This can be avoided by reducing the size of the diaphragm until the aperture just becomes visible at the edge of the back lens of the object glass. To what extent the angle of light admitted by the condenser should be smaller than the aperture of the object glass depends upon the nature of the object. Full resolution will only be obtained if the whole aperture of the object glass is transmitting light, for reasons previously explained ; and if the light from the condenser were not filling the aperture of the object glass, and there were no object on the stage, the ILLUMINATING APPARATUS 31 aperture condition would not be fulfilled until the iris dia- phragm was open to the desired amount to fill the back lens of the object glass; but the object itself always has some, and often a very great, power of scattering light. Even when the condenser diaphragm is cut down to a minute aperture, such an object as a diatom or a podura scale scatters so much light that on look- Resolution ing at the back of the object glass it is almost as bright over its ett, whole surface as at the spot where the direct light passes through, Py object. and the image is formed by the scattered quite as much as by the direct light. The markings of the podura scale form a good illustration of this point. The scales of this small insect appear to have markings somewhat like small quills. Ifthe aperture of the con- denser is reduced so as to send direct light through only a com- paratively small fraction of the object glass, the best image of these quills is formed somewhat as Fig. 20(A). If the diaphragm be opened beyond a certain amount the clearness of this image is reduced. If a diaphragm is inserted at the back of the object glass to cut off all the scattered light, and only let the direct light from the condenser through, the image will be absolutely fuzzy and indistinct, somewhat as Fig. 20 (B), showing that in the delineation of this object the light scattered by the object is doing the work of resolution, and that it is not done so well if AB the full cone of light is passed through the object by the ieee, 38 condenser, The same applies to a lesser extent to “~~” some diatom structure, though in this case the finest structure is always best shown by a large cone of light from the condenser. The tubercle bacillus embedded in tissue is extremely difficult to see unless a large cone of light is used, and in the early days of its discovery its existence in the tissues themselves was doubted by some of those who were not in the habit of using a condenser to its best advantage. Some delicate semi-transparent objects are quite invisible when a large cone of light is used, but can be seen with a pinhole aperture in the condenser. It should be understood, however, that appearances created by the use of very small apertures may be incorrect representations of. the correct structure. In the case of the podura scale and the structure of diatoms, the nature of the actual structure is not definitely known, and it is not certain that the images obtained of these apparently well-marked structures are even approximately correct. Observers have frequently claimed the discovery of delicate envelopes around bacilli and micrococci which a skilful microscopist can at once refer to the result of incorrect illumina- tion. It is generally best to use the largest aperture in the con- denser that does not produce indistinctness, for the image is more likely to be a correct representation ; and it is well to try 32 THE MICROSCOPE different apertures when it is required to observe very fine struc- ture. Variation There is a difficulty in making use of different apertures because intecuty the light varies so greatly by the change in the aperture of the when iris condenser, that the variation in intensity becomes a serious incon- iaphragm of condenser Venience. The observer should never be tempted to overdo the milion brilliancy of the illumination ; several pieces of ground glass should be at hand to place between the lamp and the mirror to modify the light as the condenser is opened up. Dee The most satisfactory appliance for critical work is an adjust- moderator, @ble pair of neutral tint gluss wedges mounted on a stand which can be placed between the light and the mirror of the microscope to vary the intensity of the light at will. The apparatus consists of a frame which carries two neutral glass wedges which slide in fit- tings and are connected together so that they move over one another in oppo- site directions by sliding a knob. The total thick- ness of the neutral tint glass is varied and the brilliancy of the illumina- tion increased or decreased within very considerable limits. The frame is at- tached by means of a clamp to a rod fixed to a strong stand, Its height from the table may be varied from 2 inches to 8 inches, and two slides are provided in front of the wedges for the reception of colour filters or ground glass. As the illumination is increased by opening the diaphragm of the condenser, it can be reduced by thickening the neutral tint layer of glass by sliding the two wedges over each other. This apparatus can be provided with wedges of different intensities according to the strength of the illuminant with which it is to be used. The use of a small bundle of oblique light directed upon the object at a particular angle has been studied in connection with the delineation of line structure. It is accomplished by cutting a small hole in an opaque sheet of card, metal, or celluloid, and placing this aperture in different positions under the condenser, Fic. 21.—No. 3328, Double Wedge Moderator. ILLUMINATING APPARATUS 33 so that one or more selected beams of oblique light may be used to illuminate the object. An experiment with finely ruled parallel lines shows that if a small oblique beam of light is used to illu- minate them at right angles to their length, finer lines can be distinguished than with direct light; but it is doubtful whether this method is of any advantage for ordinary objects, and it is liable to give rise to quite erroneous impressions of structure. Dark-ground illumination is obtained by throwing light upon the object in such a manner that the object is illuminated, but that none of the light enters the microscope except that reflected by the object itself. The illuminator must be capable of throwing light upon the object at a greater angle than can be received by the object glass in use. The illuminator must have a larger aperture than the object glass. Dark-ground illumination with a substage con- Dark-ground denser in which the object is illuminated by light that is so oblique "we" that it cannot enter the object glass is accomplished by placing substage | a glass with a central black patch below the condenser and by : opening the iris diaphragm to its full extent, as shown in Fig. 18, page 27. With the Abbe form of condenser this method is useful for low powers—14-inch (32-mm.) and 2/3-inch (16-mm.)—but is not sufficiently corrected to cut off the central light with the accuracy required for a high power. It will perform fairly well with a, 1/3-inch (8 mm.) achromatic, which has an aperture of ‘5 N.A., but not for lenses with a larger aperture. With the achromatic or immersion condenser, dark-ground illumination can with care be used with a 1/6-inch (4-mm.) object glass, but it is better to use a special high-power illuminator described later for the 1/6-inch (4-mm.), 1/8-inch (8-mm.), and the 1/12-inch (2-mm.) object glass. This is partly because the high-power illuminator is of shorter focal length and gives more brilliant illumination, and partly because the stop of a substage condenser is some distance below the lenses and allows some light to spread round the stop employed. It does not produce so black a background. For dark-ground illumination with a condenser, an adjustable stop in- vented by Mr. Traviss (Fig. 22), on the principle of a reversed iris dia- phragm, is a very convenient appliance. Fie. 22.—No. 3284, Tra- By moving the handle the size of the viss Patch-stop. central patch is enlarged or diminished. ; ; The high-power dark-ground illuminator is a reflecting device High-power by means of which a very small image of the source of illumina- inletiaie tion is focussed upon the object, and this image is formed by rays of light which fall upon the object at a very oblique angle. Fig. 23 3 Fe Adjustable patch-spot, 34 ‘