The Ultra-Violet Microscope

To that rude scientist in dim historic time who first acquired the knowledge of a simple lens, how strangely must have come the wizardry of magnifying-glass. Before his wondering eyes his hand was doubled, blades of grass grew great, an insect seemed a monster. And when one day a brilliant dragon-fly fluttered within the microscopic field, what startled awe must have arisen in the gazer’s soul to find upon its wings slight cords and filaments, veinings and colorings never before conceived. Yesterday our microscopic lore had reached a boundary, set fast as that which held mankind before those first experiments with light released the world until then hidden from the naked eye. The microscope of glass, with sunlit field, had come as near perfection as the hand and brain of man could reasonably achieve. To-day a single leap into the unknown dark gives new and living empires up to light, doubles the power of any microscope ever produced, revolutionizes the study of the infinitely small. A new aid to scientific discovery has done all this, a microscope with quartz lenses, using light far beyond all visible light, that of the ultra-violet.

Light comes to us in spherical waves which rise and fall like the restless waves of the sea. Still clinging to the old nomenclature, we speak of it as coming in rays as well as in weaves. A convex lens, such as a burning glass, holds from its shape the power to take the light rays of the sun and bring them to a focus. So the camera, by use of a similar simple lens, takes the image of a summer landscape, transfers the light waves which proceed from tree or rock, and prints them on a plate covered with sensitive film. In both these cases the object is comparatively distant from the lens. Used thus, the simple lens converges light and concentrates its power. Change the distance between the object and the convex glass, bring it close, and the simple microscope results, which, like a reading glass above a printed page, diverges light from every letter and magnifies each word to twice or thrice its size. Instead of concentration, such usage gives diversion, and the printed word beneath the lens has, to our eye, swollen, widened and lengthened. If we combine these powers, and, having placed one lens to concentrate our light, use another like it to enlarge the image given by the first, we have a compound visual microscope. This microscope possesses in its simple form two lenses only: one placed directly above the object to be magnified and taking from that cause its name, “objective; ” a second, the “eye-piece,” below the eye, which magnifies yet more the object which the first has thrown into its range.

The light for such a microscope comes from the white light of the sun, or from its substitutes, the artificial lights of daily life. The rays which reach the instrument from window or from arc are caught on a reflector which directs them upward, passing them through a transparent slide on which the specimen rests. This object, being opaque, is seen through the lenses by reason of its contrast with the light around.

Two factors determine what a microscope can do: the light illuminating the object to be seen, the lenses, mirrors, or prisms which transmit its rays. Given the light most favorable for magnification, — given, too, the substance which will most perfectly transmit that light, — and the ideal microscope comes nearest to attainment. The microscope from which have come the vast advances of the years past used sunlight, transmitted through, and by means of, glass. The microscope of the future, the ultra-violet, uses waves invisible to mortal eye, and transmits them through fused quartz.

To recognize the reasons for the change from old to new, we must understand that the crux of the problem, the reason why one microscope is better than another, lies in the resolving power of the instrument, its ability to separate two points closely together. Simple enough this seems, an act which we unconsciously perform every time we read a letter from a friend, separating a from n and d from t. Carry the matter further and new conditions rise. Take a beetle shining with gold and crimson armor. To the naked eye it shows a broad design. A simple lens gives definition to each varied part. Here are ridges, lines, and traceries. More powerful microscopes take up the work. Tiny crevasses now appear. Small differences in size and shape before unnoted suddenly spring into view. Each microscope, more powerful than the one before, presents new wealth of vision clarified! And as each latest, greatest telescope gives myriads of new stars to add to our known universe, so each increase in microscopic power gives realms unknown before, and all because of one supremely simple fact, — two points so close together that man has never seen them as two points before stand now distinct and separate.

Bit by bit, step by step, the cunning polishers of glass, the wise men who by mathematics figured out each lens, advanced on microscopic lines. Decade after decade showed microscopes revealing more, till in these latter years the limit of the instrument which used our common light was practically achieved. To pass that limit, new theories of construction must be opened, using the new beliefs in light transmission for which Abbé and others had done so much. Wonderful indeed are those conceptions which were ready for the worker’s hands.

The gleaming wonder of the spectrum, shown by the filmy rainbow arch or cast by prismatic glass on wall or floor, had sorely puzzled many a philosopher ere Newton, with his master mind, took up the task of elucidation. He placed a prism so that it barred the path of light rays from the sun. They entered white. They emerged broken into a million wavering bands which ranged upward from red through orange, yellow, green, blue, and indigo, to violet. That showed that white light must be made from all those parts. But Newton went still farther. He took the glimmering strands of color just achieved and passed them through a lens. Issuing from the rounded glass, the light came forth pure white as when it first entered the prism. So Newton proved that white light broken down gives all the colors of the spectrum, and conversely showed that all the colors of that spectrum joined produce white light. On the swiftness with which the light of any color reaches the eye depends the color sensation which we feel. A crimsoning sunset and the light green of the spring woods differ only, in color, by the rapidity of the waves. Red is the slowest color of the spectrum. Violet is the swiftest. Down far below the lowest wave of red we know that energy of similar kind exists, a light invisible, the infrared. At the other end of the spectrum, far above the shimmering violet, range yet other invisible light waves, the waves of the ultra-violet. These are the shortest and the swiftest waves of all. It is a fact easily susceptible of proof that the swifter or shorter the wave, the greater the resolving power of any microscope, the closer together the points which it can separate. On that fact rest the principles which underlie the discovery of the new instrument.

White light is far slower than much of the ultra-violet light. It was not long, therefore, before scientific research began to turn towards the upper regions of the spectrum, seeking a source of illumination which should produce a new microscope. The experimenters who entered upon this work were not many in number, but the chief centre of investigation was one known for its microscopes from Tokio to St. Petersburg, and from Buenos Ayres to San Francisco, — the house of Carl Zeiss of Jena. Dr. A. Köhler of their scientific staff designed the apparatus. Dr. M. von Rohr, another member of their staff, worked out the difficult computation of the objectives. At first the work progressed along the lines of violet light, which contains the shortest visible waves of the spectrum. As the research went on, however, it turned gradually but certainly towards the use of the ultraviolet. No simple problem lay before the workers. Microscopes had always depended upon the sun for light, and the use of a new illuminant meant an overturn of the work of many years. To obtain a microscope which should successfully substitute ultra-violet for white light, required a whole series of changes and developed new requirements which appeared in succession.

It was of the first importance to secure a source of energy which would give a constant unvarying supply of ultraviolet waves. Fortunately there was such a source at hand. If we pass an electric current between two poles made up of either metallic cadmium or magnesium, that is to say, if we form either a cadmium or a magnesium arc lamp, there will be given off a number of light waves, visible and invisible. Among these latter waves are some of the shortest and swiftest, and, as a result, some of the most useful of the ultra-violet. One serious difficulty with white light has always been what is known as chromatic aberration, its constant tendency to break down into its component colored parts. Look at an ordinary pin through a common lens, and see the red, yellow, or blue of the spectrum appear around it. The lens, unable to transmit the white light from the pin as a whole, has broken it down into its component parts as it passed through. For that reason, the matter of chromatic aberration has been a fruitful and continual source of trouble in glass lenses. It seemed especially advisable to do away with mixtures of colors in this new microscopic work, and to get rid of illumination made up of waves of different rapidity. That could not be done with white light or composition light, but it could be accomplished by taking a wave of a single length out of the ultra-violet. If that could be achieved, a light of a single tone would be obtained which could not break down when passed through lens or prism, because there was nothing for it to break into.

To separate out such a monochromatic ultra-violet light, to get it where it would illuminate the object below the microscope, and to pass it through the microscope itself, some substance other than glass had to be used. Glass, permeable as it is to white light, is opaque to ultra-violet waves. Place a bit of glass across the path of such rays and you raise as complete a barrier as if you raised a wall of iron. The search for some substance which would transmit these waves was long and arduous. It was ultimately found. Fused quartz would let the waves pass through. With that discovery, the two major difficulties were practically settled. The ultra-violet source of illumination was certain to give a far greater resolving power than white light had ever given. It would pass through quartz as white light does through glass. By quartz prisms the experimenter separated out a single-toned wave of ultra-violet light, passed it up to a quartz reflector, sent it from there through a slide, and finally through quartz lenses. At the slide where the object to be examined rests, be that object what you will, a colony of typhoid germs, or a group of blood corpuscles, opens a whole new set of difficulties. As ultra-violet waves are invisible to the human eye the appearance of an object illuminated by that light makes the word “illuminated ” seem a misnomer. We see nothing but blackness. That lack of visual power might years ago have been a barrier, but now photo-micrography, the art of photographing with a microscope, is so advanced that it offers an easy solution of that question. Photography can be accomplished without white light. Not only can it be accomplished, but it is far easier when done with ultra-violet waves, since the essential rays of white light which break down the chemical salts, and impress the image on a photographic plate, are the so-called actinic rays. These are found in greater quantity in the waves above the violet than in those below it. Photo-micrography, which uses the ultra-violet, gives beautifully sharp, clearly-defined images.

There is, however, another lion in the way before this process can be used. A camera or a microscope must be focused, the lenses must be so placed as to give a clear sharp image on the plate. When the eye can bring a sharp image from a blurred outline, focusing is a simple matter; but here, when the eye is of but little avail, it becomes a tedious and difficult process. An approximate focus can be obtained from that property of the ultra-violet waves by which they impart a glow, fluorescence, to uranium glass. In focusing by this process, a piece of uranium glass is placed on the slide on which the object is to rest, the light is turned on, and the microscope adjusted as the glow sharpens or grows dull. This is, however, more or less of an expedient. For more careful work, a series of films are exposed in succession. The focus is changed and noted for each exposure, and the one giving the sharpest image is chosen.

More than one microscopic tool which has proved a useful servant in the past becomes with ultra-violet light a serious hindrance. The ordinary microscopic slide on which the specimen rests is of glass, sealed with Canada balsam, a substance through which light passes exactly as it does through glass. Such a slide placed beneath an ultra-violet microscope instantly cuts off all illumination. None of the waves will pass through. Therefore the slides, like everything else through which the ultra-violet waves pass, were necessarily made of quartz. The old liquids which held the specimens were likewise impassable to the rays. To obviate this, a mounting fluid, a substance which would afford a food and home for germ growths, was prepared by using a solution of salt with agar (a nutrient), in distilled water. This mounting fluid gave nutrition, non-distortion, and, most of all, transparency to the rays.

When all these changes had been completed, a microscope was obtained of practically the shape and construction of a compound microscope, having eyepiece and objective of fused quartz instead of glass, having quartz prisms for transmission instead of glass mirrors, and slides of quartz instead of glass. The source of illumination had become short, swift, single-toned waves of ultra-violet, instead of long complex waves of white light. What will this instrument do? It will do just double what the old can accomplish. It will show an image of objects just half the size of the smallest the most powerful visual microscope in the world can show. Reduce it to that essential necessity already stated, the ability of the microscope to separate two points near together, and the figures show the tremendous advance. With glass lenses and visible light, the ultimate boundary for separating two such points is one twenty-thousandth of a centimeter. With quartz lenses and ultra-violet waves it is half that, one forty-thousandth of a centimeter. At one bound the new microscope has added one hundred per cent to the power of the old.

If the new microscope did nothing more than that it would be one of the great advances of the century, but there inheres in it another power which promises great future use to medical and biological science, — its action on organic tissue and micro-organic life. The science of microscopy, much as it has given to the world of knowledge which has to do with body-building, with disease, and with the infinitesimal inhabitants of our world, has been at best a science of dead things. As the astronomer gazes at a dead world on the moon, so the microscopist has gazed through his lenses at a bacterial world made up of dead microorganisms, and of organic tissue hardened and distorted from its natural form. With the visual microscope such conditions were inevitable. The light of day would not show tissue properly until it had been so hardened and fixed by the fluids used in preparation that its normal appearance was gravely altered. It would not show cavity or bacterial form until some colored liquid which mapped out the specimen was injected into it. Such liquid perforce killed the specimen, yet it was essential to proper examination. To recognize the necessity for this, suppose for a moment that we desire to study the structure of a jelly fish. As we hold the semi-transparent mass up to the light, the cavities within by no means show their whole structure or extent. If, however, we could inject into them some brilliant crimson fluid which would fill every channel, we can readily imagine that every tube and hollow would stand out, mapped in red upon the yielding surface. So staining, for microscopic use, mapped out the specimen in the slide and gave clear definition, but as it gave that definition it killed or changed the substance.

As the experimenters carried on the new work and at last forced the light of the ultra-violet through every part, they first tried specimens prepared as for the visual microscope, but, to their surprise, they found that every kind of treatment, such as hardening, mounting, staining, made the specimen absolutely opaque to the rays. No result could be obtained. They went from prepared specimens back to fresh and live specimens. Right there developed one of the most interesting features of the whole matter, the fact that ultra-violet light is selectively absorbing. Just what does that mean ? Simply this, that this light is so extremely sensitive to minute changes in the thickness of any substance through which it passes, that the slightest difference in density makes a marked difference on a photographic plate exposed to that light. Every hollow, every cavity of tissue must have a boundary wall to separate it from the flesh or tissue about it, and those walls must be thicker than the surrounding mass. With rays far more potent than those of white light the ultra-violet maps out the whole interior of a specimen, and shows the boundaries of every part. In like fashion a red staining fluid might map our jellyfish example, or would map a microscopic specimen. That opens instantly, not only the world of organic tissue, unhardened, unmounted, and unstained, but it also opens a wide untrodden field, the study of the living bacteria. Sunlight would not reveal the history of the living germ. The ultra-violet can trace that history from its beginning to its end.

With the new microscope we can imbed a typhoid bacillus in a solution where it can live its allotted time of existence under constant observation. We are able to study germ processes of growth, methods of reproduction, the spread of disease, and the effects of inoculation. The winding way of the blood corpuscles, of those myriad travelers which carry with them disease and cure, can be traced as never before. The struggle between the toxins and the anti-toxins opens to our view. Once more, the ultra-violet microscope has not opened up a single road. It has opened up a new world.

No advance in science moves with ordered ease. Each new achievement comes from constant struggle, from a persistent overcoming of obstacles. The new microscope has been no exception to this rule. The effect of ultra-violet light upon living matter seemed at first a barrier which might check the onward movement. It was claimed that these rays, having serious physiological effects, would kill protoplasm and render it opaque to the short waves of light. This theory has now been proved incorrect. In recent experiments typhoid bacilli were exposed to ultra-violet rays without harm for some forty minutes, an exposure distributed through a period of three hours. The focusing still remains a serious obstacle. Whether it be done by fluorescence, or by the taking of a series of photographs, the two methods already described, the process is long and vexing. The preparation of specimens has been difficult in the extreme, and the evolution of satisfactory mounting media, a matter of recent development, is still under discussion.

Until we have one vital piece of evidence, complete proof of what this microscope can do will be impossible. For that we should possess a complete series of comparison pictures, showing numerous instances of the same subject taken by ultra-violet and by white light under precisely similar conditions. An apparatus which will give us such an ocular demonstration is now under process of construction, and we shall in time have plates which show the exact relative values of old and new. Then pictures will tell the tale. Until that time we shall have to do our best with words.

So ends the beginning of the story. Only the beginning, for years of patient labor, tens and hundreds of researches, will not complete the tale. Yet a great thing has been done. The black spots on the earth-maps that stand for unexplored countries are growing smaller and smaller. The light is vastly greater than the shrinking dark. Few strange lands are left for the geographer to chart or for the explorer to search out. But in another world, the world of science, all about the light of the explored hang heavy clouds of the unexplored. Not a science but is inclosed by a blackness of unknown extent, which hides many of the basic truths of each individual branch of the study of nature and her laws. What is electricity ? What is the ultimate composition of matter ? What is life ? How that list could be extended! Every now and then by patient search a key is found that unlocks a door in the black wall, and a great new country is opened to the explorer. Such a key has been found in the ultra-violet microscope.