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Looking For The Small Things In Life – Microscopy And Reef Aquaria
Part 1. Microscopes, Types, and Considerations. By: Ronald L. Shimek, Ph. D.
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It’s A Small World:
As impressive as a beautiful reef aquarium may be, the vast majority of the living things in such a system are obscure or invisible to the aquarist. The oceanic organismal array covers examples of most types of life, from animal to algal to fungal to… well, pretty much you name it. Except for the green plants, represented by a few species of mangroves and sea grasses, and the insects, represented only by a couple of wingless species of both gnats and water striders, virtually all other types of organisms thrive and are diverse in marine systems. This unseen majority of organisms share one obvious characteristic. They are just too small to see clearly, or at all, with the unaided eye.
There are several solutions to “the problem” of not seeing these organisms. The most common of these is simply to ignore them. “Out of sight, out of mind” is a motto practiced by a lot of reef aquarists and, I think, who can really blame them? Well, I can, but that’s another story. Nonetheless, most people set up a reef tank for the visual pleasure it brings and they don’t want to have to spend a lot of extra effort to see what they perceive as “little obscure creatures.” Interestingly enough, however, I have found over the years that I have been discussing the reef aquarium hobby, that a sizeable proportion of aquarists get really and thoroughly “hooked.” They become interested in virtually everything pertaining to their tanks. Often suddenly, and almost always surprisingly, they realize that their interests are not limited to the large and obvious things in their systems. Quite literally, a whole new world, then, opens up to them.
| Figure 1: | 
Figure 1: An ostracode. Ostracodes are common aquarium inhabitants that are just on the “edge” of normal vision. They look like small moving dots or sand grains in aquaria. They have the common name of seed shrimp, but none of their identifying characteristics are visible without significant magnification. This image was taken using a compound microscope at 100x. |
The beauty of many smaller organisms and non-living structures has been recognized and well known for a long time. That is one aspect that drives people into investigating the minute and wonderful miniature life in their captive worlds. But there is for some of us the intrinsic interest in the “small and complex.” For such people, and I rank myself with them, observing a small and elegant solution to a problem is often more aesthetically pleasing and interesting than observing the brute-force and bulky solutions to problems seen in the large world around us. All organisms have to solve the same basic problems in their quest for existence and propagation. These are the fundamental problems of all life, such as how to feed, how to avoid becoming the solution to somebody else’s quest for food, how to sense the world, and how to reproduce. We are often very familiar with how, for example, humans and other large animals solve these problems, but humans are amongst the largest of all animals. We each contain trillions of those subunits of life called cells. Animals containing a few hundred or a few thousand cells also exist and their solutions to our common problems are often elegant and beautifully interesting. Looking at life on those different scales one sure way to appreciate the beauty and the sheer wonder of our world.
For whatever reason, many aquarists become interested in what goes on in the normally unseen world of the minute and invisible that comprises the universe for most of the life in their aquaria. This passion often starts with observations of small moving dots in the water or small gliding dots on the aquarium walls. The first instrument that is used to examine these dots is the “Mark 1 human eyeball,” a remarkable instrument capable of making observations over a phenomenal range of conditions. However, like all instruments, it has limits. When we desire to make observations outside those normal limits, we must use other, additional, instruments. The first ancillary instrument for looking at small things is, generally, a magnifying glass. If the passion for the small stuff really ignites, the magnifying glass soon becomes insufficient; more magnification is needed and the aquarist segues into becoming an amateur microscopist.
I hope that by writing this three-part series as introduction to the science and art of microscopy I will be able to provide enough useful information to allow a reader to navigate out into the waters of what those masters of commerce, the Ferengi, refer to as “The Great Material Continuum,” without encountering the shoals of severely depleted cash reserves or the shipwreck of bankruptcy. Although the previous statement was written as a bit of humor, the cost of a light microscope can range upward from a few hundred dollars to more than a few hundred thousand dollars. Other types of instruments, such as electron microscopes start in the range of a few thousands of dollars and top out in the range of the GDP of a small third world country; I will not discuss these lovely instruments here. I may consider myself a virtuoso at scanning electron microscopy (and I do, and I would love to discuss it in great and complex detail), but such microscopy is truly out of the realm of reef aquarists. The topic of these articles, then, will be affordable light microscopy. As is true of most things, within reason, you get what you pay for, but there are bargains and good buys; but you have to know what you are looking for. I hope that this short series will serve to provide some of the background necessary for any budding microscopist to make a reasonable purchase; that is, one that provides a useful instrument for a reasonable price. This first article will briefly discuss what a microscope is. The second part of the series will discuss how a microscope really functions and how that relates to what to look for when you purchase such an instrument, and the final installment will discuss, in some detail, how to use a microscope and get the most out of it. After these articles, if there is any further interest, I may be able to add more articles on specific aspects of microscopy.
Jargon
Microscopy is the result of the confluence of several sciences, most notably physics and biology. To understand what is necessary in a microscope, one needs to understand a bit of the elementary physics of light. As the living things will be the target of most of our observations, one also has to be able surf over the waves of the biological ocean. The physics, however, come first. Unfortunately, in both disciplines there are technical terms that are necessary for background discussion. In this venue, it is neither necessary to commit such terms to memory nor should they be dwelt on for overly long periods; however, it is necessary to understand how the terms are used and what they mean, at least in theory. Without the understanding of what is present in any given instrument, purchasing a microscope is an exercise more akin to a lottery than to a reasoned purchase. And without the understanding of how to use the instrument, the purchase simply becomes a waste of time. There are not a lot of necessary technical terms, but as I use them, I will try to define or illustrate them and their first appearance will be marked by bold font.
Microscopes
Although there are a wide variety of specialized microscopes available to scientific researchers, because of expense and ease of use, only two types are really applicable for the aquarist who wants to examine the small life in their aquarium. Unfortunately, these two common types of microscopes, the stereoscopic or dissection microscope and the compound microscope, are designed for different functions and are almost non-overlapping in their usage.
Modern light microscopes work by using two lenses to produce a magnified image. The lens nearest the object to be examined, called with amazing alacrity, “the objective lens,” focuses on the object. This lens creates a real, magnified, image inside the microscope’s tube. That image is located about an inch or so below the eyepiece or “the ocular lens,” that you look into. If, after you have a microscope and use it to focus on an object and you then remove the eyepiece and look down into the tube, you will see… nothing. The image is there, but it is small, and it is invisible to the unaided eye. If, however, a piece of ground glass or tissue paper it placed at the right place, that invisible image will be projected upon it and become visible. Give it a try. It takes a steady hand, but it will prove to you that I am not lying.
The ocular lens is designed to focus on that real image and to magnify it creating a second image, the “retinal image,” that is projected upon the observer’s retina. The total magnification of the object as seen in the retinal image is the product of the magnification factors of the two lenses. As a general rule, the standard eyepieces available in most commercially available microscopes magnify ten times, referred to as “10x.” One can, however, often purchase oculars that magnify at other values; 5x, 15x, and 20x are perhaps the next most common values. Objective lenses come in a lot of different magnification values. Generally, the higher the magnification value, the closer to the object that the lens must be. This property has some rather profound ramifications. It is important to note that in the better ‘scopes, the objective and ocular lenses are matched in various ways. It is NOT a good idea to mix and match lenses from different microscopes.
As in a telescope, microscopes are made with the objective and ocular lenses at opposite ends of a light-tight and opaque tube. As far as the functional magnification of the instrument is concerned, such a tube is absolutely unnecessary. One can get exactly the same magnification by just having the lenses aligned and positioned so that the light passes through them properly. However, the addition of the tube prevents unwanted additional light from entering the lenses from the sides and degrading the image. The light path in a standard microscope is often significantly longer than it appears, as it is often directed through prisms internally in the body of the microscope. Making sure that all of the lenses and prisms in a microscope are aligned and positioned properly is fundamental to the proper functioning of the microscope, and is a primary concern if you are purchasing a used instrument.
Dissection microscopes (also called stereomicroscopes because they provide a stereoscopic or 3-D image) are microscopes with a large working distance between the lower or objective lens and the object. As the name implies, one may use the instrument to view a dissection, or more often, just to observe a relatively large object. Stereomicroscopes typically magnify over a range from about 5x to about 60x. Given that they usually have oculars that magnify at about 10x, this means the objective lens range in magnification value from 0.5x to about 6x. The working distance between the objective lens and the object in these microscopes is generally on the order of, at least, several inches, allowing the observer to manipulate the object.
| Figure 2: | 
Figure 2: A diagram of a stereo or dissecting microscope. Note the large working distance between the objective and stage where the specimen is viewed. Dissecting microscopes typically have either 3 lenses in some sort of changeable nosepiece or a zoom capability where magnification is changed by turning a knob. Image quality is generally not consistent across the full range of a zoom microscope, being better at specific values within the range. Such values often have to be determined by trial and error, however. |
Compound microscopes, so-called because the objective lenses are made of several subsidiary smaller internal lenses, are designed to examine very thin objects placed or mounted on glass slides. Such microscopes have a very short working distance between the objective lens and the object. These microscopes typically magnify over the range of 40x to about 1200x. Consequently, the objective lenses may have magnifications of 4x up to around 120x. For the lower powers, the working distances are typically on the order of 3-5mm; but for the higher magnifications, the working distances can be as low as 1mm or so. At high magnifications, often involving oil immersion, the distances may be so narrow that the thickness of a glass cover slip mounted on a slide can cause the slide to crack, and the thickness of the slide, the cover slip, and the specimen must be accounted for if high magnifications are to be used.
| Figure 3: | 
Figure 3: A diagram of a compound microscope. A thorough discussion of the use of these instruments will be given in Part 2 of this series. |
Aberrations
If the only concern was magnification power, it would be easy to construct microscopes that would allow the examination of all sorts of minute things. Unfortunately, it isn’t and it isn’t and we can’t. This is largely a result of the properties of light coupled with the biological process resulting in our perception of colors. We can see objects only as result of a combination of two things. To be visible, objects have to either contrast with the background or differ in color from the background.
Contrast is measured by the power to resolve small items. Consider the situation of driving at night on a long, straight stretch of road. When a car first appears and starts to approach you in the distance, if the road is long enough (over 10 miles or so – and yes, out in the west there are such stretches of road), its headlights first appear as a single bright dot in the distance. At some point, as that car approaches, you will be able to distinguish, or “resolve,” the two point sources of light that are the two headlights of the approaching automobile. If you have normal vision, and it is a clear night, the distance separating your cars will be about eight to nine miles when this occurs. When we use a microscope, in most cases we don’t worry about separating two bright point sources of light against a dark background, rather we worry about separating two dark dots or lines against a bright background. The smaller the distance between two objects that can be resolved into separate objects, the better “the resolving power” of the microscope, and the smaller the object that may be seen.
Although, it is possible to illuminate the object against a dark background (called darkfield illumination), the object is generally illuminated with transmitted light, resulting in a dark object on a bright, and often white, background. Unfortunately, what we often forget is that white light is an illusion; “white” light is simply a human sensory response to the simultaneous illumination of an object of all the wavelengths in the visible spectrum. Human color vision is result of the stimulation of photoreceptors, nerve cells that respond to the stimulus of light. These photoreceptors contain pigments, called photorhodopsins, which change color when light impinges upon them. Humans possess three different photorhodopsins. The color we perceive depends upon the intensity and combination of wavelengths of light hitting these pigments. If the photopigments are all stimulated more-or-less equivalently, the light color we perceive is “white.” Other colors result from differential pigment stimulation. White, then, is an illusion, a color resulting from a “complete” stimulation of our light sensitive cells. It really isn’t a color of its own; there is no color in the solar spectrum that is “white.” Unfortunately, for us, inanimate objects lack our illusions. While we see color as an amalgam or mixture of colors, light passing through the inanimate lenses of a microscope acts as the actual series of separate wavelengths that it is, and that is the root of many problems.
Lenses
Lenses bend light because of the changes in the speed of light that result when light passes through different transparent media. While the speed of light in a vacuum is a universal constant, the speed of light in other media, such as air, water or glass is variable but always less than it is in a vacuum. If a ray of light passes perpendicularly through an interface between any two transparent media, it will change speed, either slowing down or speeding up depending up the properties of the medium. It will not change direction. However, if that same ray of light passes between the same two media at an angle, it will be bent, or refracted, so that its path has changed. The amount that it will be bent depends upon the differences between the speed of light in the two media, and the angle of the interface relative to the pathway of the light ray. Such properties are used to construct lenses. How the various properties interact is really immaterial to this discussion, but such properties are well known and lenses may be constructed with different shapes and out of different materials to alter the paths of light in many ways. What is important to the discussion of microscopy and optics, in general, is that the amount that light is refracted is also dependant upon its intrinsic energy, which in turn, is related to its wavelength. The bottom line is that a lens focuses light of different wavelengths to different points.
| Figure 4: | 
Figure 4: A diagram of a lens showing how blue and red light is focused to different points. This property of differential focusing of light of differing wavelengths is referred to as “chromatic aberration.” |
The focusing of light of different colors to different points results in lenses having a “flaw” or, better, a “property,” called “chromatic aberration.” The result of chromatic aberration may be easily seen with a simple magnifying glass. If white light is used to brightly illuminate a thin dark line on a light background, and a magnifying glass is used to magnify that line into a sharp focus, one side of that line will have a red fringe and the other a violet fringe. Aside from the annoyance of introducing colors, this has the other effect of making the line thicker than it should be. In other words, chromatic aberration discolors the image and reduces the resolving power of a lens.
| Figure 5: | 
Figure 5: The letter “i” photographed against a yellowish background showing the effects of chromatic aberration, note the red fringe on the left and the blue fringe on the right. |
There is yet another property of lenses, “spherical aberration,” that has to be considered. If parallel rays of light pass through a lens at the center of curvature in the middle of the lens, they are not refracted at all and continue on a straight path. Next, consider the path of light slightly to one side of the central axis. At any given point, over a small enough lateral distance, the spherical surface of the lens will approximate being straight and slightly angled relative to the light path. When the light passes through that area, the light path is refracted (“bent”) slightly. If the lens is symmetrical, all the light passing through the lens at a constant distance from the center axis will be refracted to the same point (presuming, of course, it is all monochromatic light). Lateral to this region, light passing through the lens encounters a surface that is angled slightly more than the surface nearer the optical center. The light passing through the lens at this distance lateral to the optical center will be focused to a different point along the axis of focus. And so on out to the edge of the lens. Spherical aberration is the focusing of light rays to different points due to passing through the curved surface of the lens at different distances from the optical center. Spherical aberration also results in the loss of resolving power.
| Figure 6: | 
Figure 6: A diagram showing how light is refracted to differing points due to the curvature of a lens; note there is a “focal region,” along the axis of focus, not a “focal point.” This property of differential focusing of light is referred to as “spherical aberration.” |
There are ways to correct, imperfectly, for both types of aberrations. Specially built objective lenses referred to as “achromatic objectives” are corrected chromatically for red and blue. They are also spherically corrected for one color. “Apochromatic objectives” are color corrected for red, blue and violet, and spherically corrected for two colors. Apochromatic objectives typically require special oculars and substage condensers. Although not without some minor aberrations, a good apochromatic lens is capable of providing an almost error-free image. These corrective lenses are constructed out of several different lens elements, often made of different types of glass (which have differing refractive indices) and different curvatures. The precision with which such lenses are made, and their final characteristics, are what determines the cost of most microscopes. A good apochromatic objective designed for oil immersion microscopy (more about that in the next article in this series) may cost, by itself, several thousand dollars.
Summary and Conclusion
There are two types of microscopes available for an aquarist who is serious about examining tiny organisms. These differ in design, function, quality and cost. Generally, stereo or dissection microscopes are less expensive to design and manufacture and they cost less than compound microscopes. They are designed to be used as relatively low power magnifying instruments with a large working distance. Compound microscopes are designed to examine very small items, usually mounted on a glass slide, at much higher magnifications than the dissection microscope. Here the working distance is much less, and the design tolerances have to be much higher. The complex design and construction of the various objective lenses used in both types of these microscopes is often the single most important factor governing their prices.
The next installment of this series will focus on the choices available to aquarists that may be used as a guide for the aquarist interested in purchasing a microscope. In the final installment of the series, I will discuss the care and use of these instruments.
Suggested Reference:
There are a number of books about microscopy and microtechnique; however there are few of them that are as well written and “easy to digest” as the following tome. Unfortunately, it is out of print, but used copies do show up from time to time. If you can find a copy, it is something to keep at hand during your microscopic safaris.
Galigher, A.E. and E.N. Kozloff. 1971. Essentials of Practical Microtechnique (2nd edition). Lea & Febiger, Philadelphia. 531 pp.
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