Looking For The Small Things In Life – Microscopy And Reef Aquaria
Part 2. Observation With A Compound Microscope.
By: Ronald L. Shimek, Ph. D.
Bells, Whistles And Knobs:
So, Bucky, you read my last column on microscopy and you thought that, maybe, it might be neat to take a look at little things. Besides, microscopes look really cool sitting on your desk. They give an ambience to the place, kinda like you know what you are doing? And that always helps with justifying your hobby expenses to the spouse.
And, if you think that, boy, oh boy, do I have some news for you!
In any case, if you read the column you did your homework, which is really easy enough given the access we all have to the Internet, and after some reflection you have plunked down a sizeable number of ducats, sovereign clamshells, or whatever passes for the coin of the realm these days, and you have purchased a microscope. As you are unpacking this rather unwieldy thing from the box, you notice one distressing thing.
It really doesn’t come with a user’s manual.
Well, that’s no big deal. Since when did any red-blooded aquarist worth their salt ever read an instruction manual? After all, everybody knows how to use a microscope! Heck, if you were lucky you may have had 10 whole minutes of instruction in how to use one, perhaps; back in the murky past when you were wearing high-water pants and your feet didn’t even reach the floor under your school desk. And, I’m sure you remember that instruction, don’t you? Just like you remember Mary Agnes Ducksworth sitting in the desk next to you? Well, I will bet you remember Mary Agnes one whole heckuvalot better than you remember Mrs. Nerdinski’s microscopy instructions.
What’s to remember, right? After all, all you have to do is to put something under the lower lens, turn on the light, and take a look, right? And my answer to you is, “Absolutely!” That is, provided you want guarantee yourself some world class headaches, possibly destroy or damage the instrument you have purchased, and ensure that you always regret spending the money on that ‘scope.
One Step At Time
At this time, it may be worth reflecting again about what, exactly, a microscope does. These instruments have but a single purpose, and that is to make some small object appear larger. In a very real sense, they may be simply thought of as glorified magnifying glasses. The analogy isn’t wrong at all; they actually are a series of magnifying glasses, albeit they are a bit more complicated than the standard reading glass. The lens closest to the object magnifies the object and the remaining ones magnify the image of the object forming a final image on the observer’s retina. That final image is inverted, by the way.
Microscopy, as we have come to know it, got its start in the great blossoming of “Morphology,” the science of animal shape and structure, in Germany in the late nineteenth century. During this period occurred the invention, refinement, and standardization of the instrument we recognize as the modern compound “microscope.” Previous microscopes were generally catch-as-catch-can devices made by avid amateurs. They had to be since there were NO professionals. This had all changed by the beginning of the twentieth century.
As I mentioned in the previous column, an object – any object – is visible only because it differs either in contrast or in color from the background. Microscopes are made and designed to utilize both of these factors, but the major emphasis is upon contrast rather than color. This is important for aquarists who wish to use microscopy, as most small living objects are low in contrast and generally not very colorful. Consequently, to get the best out of a microscope, it is important to learn how to use the instrument properly. I will concentrate on giving practical instructions for the use of the microscopes, and only touch briefly upon the theory behind both the optics and the functionality of the instrument. I refer anybody interested in either the theory or the history of microscopy to:
Galigher, A.E. and E.N. Kozloff. 1971. Essentials of Practical Microtechnique (2nd edition). Lea & Febiger, Philadelphia. 531 pp.
Using A Compound Microscope
One of the more interesting things that nobody comments upon is that the average unspecialized compound microscopes are quite consistent in size and shape. There are little variations in shape and angularity, but these are often relatively less than the differences between the shape of the tail fins on those classics of American consumerism, the 1959 Buick and Caddy. Think about it; there is really no cosmic reason why the tail fins should have been shaped the way they were, and likewise there is no reason – other than convention – that microscopes need be shaped they way they are. However, during the development of microscopy as a scientific tool, the relative shape, size and proportions of microscopes were established, and we have been living with them ever since. There are very good reasons, now, to stick with these standardizations. For example, as with all products that have similar sets of standards, following them significantly assists in the construction of interchangeable parts and additional “support” equipment such as cameras or image analyzers. Although specialized unusual microscopes of quite odd-looking shapes are manufactured and available for purchase, it is really unlikely that any hobbyist will have the opportunity to purchase a microscope that differs dramatically from that shown in the figure in the first part of this series. If you have purchased one of these, however, be forewarned the discussion and the instructions in this essay will probably not work very well for you. Additionally, my discussion assumes that your microscope has an attached or built in light source. If it doesn’t, you will have to adapt your adjustments accordingly.
Figure 1: A diagram of a compound microscope from Part 1 of this series.
Although I promised that I wouldn’t go into detail about the optical principles involved with microscopy, some background is necessary for easy and good observation. First, and foremost, compound microscopes are designed to have their illuminating light rays to be passing though them, and through the object being viewed, parallel to the long axis of the microscope tube. Light that deviates from the parallel pathway causes distortion of one sort or another. The object to be observed causes this parallel light to be disturbed (the light is refracted, absorbed, or reflected), but at a defined place in the light path, and that “disturbed” light forms the image. Extraneous “disturbed” light DEGRADES the image. Your mission as a microscopist, should you choose to accept it, is to minimize that extraneous light. In doing so, you are on a quest for the perfect image. Unlike perfection in life, one may get remarkably close to seeing the perfect image. All it requires is following some rules and procedures.
The destruction of “the perfect image” takes remarkably little extraneous light. This is because remarkably little light – really a very small amount of light – goes to form the image in the first place. Consider that if you are looking at a small protozoan 10 μm in diameter under normal illumination using a compound microscope, all the light that is passing through that protozoan will be passing through a surface area of (πd2/4 * 3.14 x 25 μm) 78.5 square micrometers. At the expense of being a bit obvious, I might add, “You’d need a microscope to see the light at all, let alone form an image of something in it.”
So, you have to do all you can to ensure that extraneous light or misdirected light doesn’t get into the system. You also have to ensure that the main light source and all of the components are aligned properly.
Some rules to follow BEFORE you make any observations.
Rule Number 1: Make sure the lenses and internal prisms are all aligned properly.
Unless you have been trained to adjust the optics within a microscope, this is NOT something you want even to try. Special jigs and equipment are used to do this. Additionally, the proper measurement of alignment typically takes an optical bench (a work bench where all the components may be securely fastened to ensure alignment). If you think that your instrument is out of alignment, contact a manufacturer’s representative and see what they recommend. Often this means that the instrument needs to be sent to a service center. The bad news is that this is often expensive. The good news is that once it is done properly (and most reputable sellers of microscopes will do it on all ‘scopes they sell, prior to shipping them to the customer), the ‘scope will remain aligned unless something really unfortunate happens. Things that might cause misalignment include shipping accidents, dropping or knocking the instrument over and “jiggling” or otherwise improperly carrying or handling the instrument. Generally, the more expensive the ‘scope is, the better made it is and the less likely that misalignment is to occur. This is one place you get what you pay for, at least to a point.
Rule Number 2: Make sure the light source is aligned properly and that the appropriate lamp is used in the light source.
Most microscopes of even moderate quality made after about 1970 have a built in light source. This means that the light is centered in the light path and if the light has been properly attached or emplaced it is automatically in alignment. The use of an attached light source is somewhat of a mixed blessing. Novice users (such as students in a biology course) tend to think that the light source in such instruments is always in alignment. That is not the case. Every time the ‘scope is to be used, the alignment needs to be checked.
· Without any slide or object in the light path:
o Turn on the light,
o Observe the light through the oculars,
o Close down the light source diaphragm.
o Using the coarse focus adjustment,
§ Bring the edges of the light source diaphragm into crisp focus.
§ Then use the focus adjustment to focus on the filament of the light source.
o With the filament in sharp focus,
§ Use the centering screws for the light to move the filament to the center of the field of view.
Even small movements of the instrument, such as changing the nosepiece lenses, may cause the light to vary in position. Consequently, this adjustment may need to be made several times during an observation session. This may seem like an unnecessary exercise in superfluous adjustment; however, a light source, even slightly out of alignment will cause a significant decrease in image quality.
Rule Number 3: Microscopes can never be “too” clean; clean all microscope components in the light path.
The major cause of extraneous light in a microscope is “glare” caused by dust or dirt upon the various pieces of glass in the light path. A small amount of dust or an oily fingerprint can cause a lot of grief, so these must be cleaned off. An ounce of prevention here is worth more than the proverbial pound of cure. Keep the instrument covered and stored in a place that doesn’t allow for the accumulation of dust. Cabinets with doors are good places to store microscopes, as are secure shelves in closets. Remember to carry the instrument securely in a normal orientation when you remove it from or put it into the storage place. Careless handling can cause misalignment to occur.
To clean lenses you will need:
· Some lens cleaning solution, available from photography suppliers.
o Alternatively, a very weak solution of dish soap will work.
· Some lens paper. This is specially manufactured paper that doesn’t have grit and which not leave lint or some other residue on the optical surfaces.
o Note: Tissue paper, although comfy for application to thine hiney or snot locker, often contains microscopic pieces of grit. Such pieces of grit can scratch soft optical glass. The water used in rinsing of this paper during manufacture is not filtered to remove grit or fine sand. Lens paper is manufactured to ensure that the paper is free of such “additives.”
· Cotton-tipped swabs are useful.
· An organic solvent such as acetone or ethanol is also useful.
o Remember such solvents are
§ Hazardous, if inhaled, and
§ Highly inflammable, so
· Take the appropriate precautions when they are used.
Figure 2:
Figure 2: Lens paper, along with other cleaning supplies, is a necessity of microscopy. There are many different manufacturers; I generally get the least expensive brand at the vendor of the moment.
The procedure for cleaning the covering to the light source is rather simple and straightforward.
· First wash your hands and dry them thoroughly.
o You don’t want to shed dirt or flakes of skin on to your newly cleaned surfaces. You can’t stop the shedding, but you can remove loose fragments and excessive dust or dirt.
· Then moisten the lens paper and use it to clean dirt off the light surface.
o Note you may have take the light source apart to get dirt off the inside surfaces.
o Oily smudges, such as caused by a fingerprint may be removed by using a piece of lens paper moistened in acetone.
§ Watch the freshly cleaned surface from an oblique angle as it dries and you will probably be able to see if the surface dries clean. Dirty areas and slight smudges will often show up with this oblique viewing method and can be cleaned.
· When the light source surfaces are cleaned then go on to the lenses.
Objective lenses need special care, as they are often very expensive to replace.
· Objective lenses may be unscrewed from the nosepiece and turned over.
· Place them lens up on a piece of clean lens paper (and take care throughout the procedure that no dust gets into the back aperture of the lens).
· Examine the exterior surface of the lens with a magnifying glass;
o Dirt that needs to be cleaned off will be visible using this method.
· Use a swab moistened in lens cleaner solution and wrapped with lens paper to clean the surface of the lens.
· Re-examine with the magnifying glass.
· Repeat as necessary.
o If the dirt on the lens is excess immersion oil, use a swab dipped in acetone to remove it.
§ You must take care during this procedure; as you might be able to remove the glue holding the lenses in place (such glue is often somewhat soluble in organic solvents).
o When you have removed the final oil layers, rinse the lens once more with lens cleaner (on a swab covered with lens paper).
· If the lens has salt crystals on it, DO NOT simply dislodge the crystals by wiping them off.
o Moving salt crystals across a lens can scratch it. Such scratches will ruin the lens, and scratched lenses cannot be repaired, they need to be replaced.
o Salt crystals need to be dissolved prior to their removal.
§ Use a lens paper covered swab dipped in distilled water (not RO/DI water, such water still has solids in it) and
§ Daub it gently on the lens until the salt is dissolved.
§ Dry the lens with clean lens paper.
§ Rinse and dry it again.
§ Repeat until you are certain that there is no more salt on the lens.
Figure 3:
Figure 3: The oil-immersion lens of my compound microscope removed from the nosepiece. A. The lower-most lens which should be examined regularly and cleaned as necessary is indicated. B. The lens showing the knurled rings used to screw and unscrew the lens from the microscope. Note while the lens in “A” may appear dirty, it is not. This is an old, surplus, university microscope and was corroded in its earlier incarnation. The lens surface, itself, is clean.
Then ask yourself the question, “What idiot allowed this lens to contact salt in the first place?” Find a mirror, look in it, view the idiot and chastise said idiot severely. Salt crystals can cause a lot of problems if they accumulate on lenses, including corrosion and scratching.
The oculars will need cleaning as well. In fact, they may need to be cleaned several times during a single period of observation.
· Unlike the objectives which are screwed into the nosepiece, the oculars are just slid into the tube and may removed by pulling them gently out.
o When you do so, cover the opening to the body tube with a piece of lens paper or some other cap to keep dust and extraneous material out of the tube.
o Although it is possible clean the prisms and such within the body tube or headpiece of a microscope, it is not easy to do and I don’t recommend it. Cover the opening and prevent the dust from entering and you are ahead of the game.
· Otherwise clean the oculars exactly as the objective lenses were cleaned.
o They may need far more frequent cleaning as they are often in contact with eyelashes and eyelashes are covered with oils. When I doing critical work, I often clean my ‘scopes’ oculars several time an hour.
Rule Number 4: Microscopes can never be “too” clean; clean the rest of the instrument as well.
Keep the instrument clean in general, and less time will be spent specifically cleaning the delicate components such as the lenses. When saltwater spills on the microscope, wipe it up immediately! Don’t let it dry on either the stage or the lenses. When saltwater dries, the formation of salt crystals may actually exert sufficient forces to dislodge lens components. All aquarists with microscopes will look at organisms in what are called “wet mounts.” Eventually, and often frequently, the use of wet mounts causes dribbled or spilled water; such small spills are really no big deal IF THEY ARE PROMPTLY dealt with. Be prepared to deal with the consequences of spilt salt water, and have some cleaning cloths on hand.
Rule Number 5: Microscopes can never be “too” clean; cover the instrument when you are finished using it or store it in a place where it will not accumulate dust.
Store the microscope where it is secure from dust and dirt, and perhaps from the inquisitive minds and hands of the more junior members of the household. Children can do things to a microscope that are perfectly explicable if you have the mind of a child. Repair or replacement of microscope parts is often very expensive. Locked cabinets can serve a number of purposes, but preserving the contents of one’s wallet is one of the more reasonable of their functions.
Using It
Okay, now the ‘scope is nice and bright and shiny. And clean. Did I mention it should be clean? What comes next?
With a compound microscope, the object to be viewed has to be on a glass slide (or the equivalent, but I won’t discuss those in this article). Microscope slides and slide cover slips are available from scientific supply houses. The standard microscope slide measures 26mm x 76 mm, or 1 inch x 3 inches and is made of polished plate glass. And, surprise, for best viewing it must be very clean. Most microscope slides available today are pre-cleaned and good to use out of the box. For subsequent uses, they need to be cleaned. Being cleaned with a standard cleaning abrasive powder can work well for cleaning, rinse it in distilled water, and then store the slides in a capped jar filled with 95% denatured alcohol (methanol, ethanol, or isopropyl all work equally well). When you need to use the slide again, remove it from the jar and dry it with a clean lint free cloth and it will be ready to use again.
Figure 4:
Figure 4: Microscope cover glasses generally are purchased by “the ounce.” These are boxes of 22 mm square cover glasses, but they come in many other sizes as well. The size label here is “No. 1 ½.” That is the appropriate thickness for most viewing.
To make a preparation of living material one can simply put the item in a drop of water on the microscope slide, and add a “cover slip” (also known as a “cover glass”). Although omitting the use of the cover glass is a common quick and dirty method of examining material, and we all have done it, for a number of reasons it is not a procedure that will yield good results.
· First, except at the lowest power, the working distance between the objective lens and the object is very small, a few small fractions of a millimeter at best. It is all too easy to immerse the lens into the water containing the item to be observed. Not only is such a practice ruinous for the image formed from such submerged optics, the lens is not designed to be used underwater. Depending upon the construction of the particular lens, it may be altogether possible for capillary action to suck water from around the object up into the lens and between lens components. If this occurs, the lens is irreparably damaged and will have to be discarded. Although they are made for specialized research purposes, few inexpensive microscopes have “water immersion lenses.”
· Second, even if water doesn’t enter the lens (and in most cases, thankfully, it won’t), the water will dry on the lens, and with samples from marine aquaria, that means extra cleaning duties to restore the lens’ functionality.
· Third, if the cover glass is omitted, the upper surface of the water droplet is concave, not flat, and it forms an additional lens in the system. The simple presence of that curved surface can degrade the image formed significantly even if the lens is kept clear of the water surface. Furthermore, a small object, especially a motile object, will more easily move within the droplet making focusing difficult or impossible.
· Fourth, and most importantly, the lens was designed to have a cover glass in place. As with the microscope slides themselves, the cover slips are also polished plate glass. As they go between the object to be examined and the lens, their presence must be considered in the construction of the lenses. Ideally, the cover glasses should have a thickness between 160μm and 170μm. Interestingly enough, cover glass thickness are not directly measured in micrometers, but by arbitrary numerical values such as 1, 1.5, or 2. Number 1 coverglasses are generally too thick for critical work, while number 2 cover slips are generally too thin. Goldilocks would say, “Cover glasses with a thickness of one and half are “just right.” However, for critical microscopy, including such as research photography, the thickness of the coverslip may be measured with special calipers and the tube length of the microscope adjusted accordingly for a precise and proper match. Fortunately, most reef aquarists will not need to go to such extremes; nevertheless, only cover slips with a thickness of 1.5 should be used. Cover glasses should also be clean of any scratches or fingerprints. Because of their thin and breakable nature, I generally discard cover glasses after one use, but they may be carefully cleaned and reused.
Another rule, then, one should NEVER observe an item with compound microscope unless a coverslip of the appropriate thickness in place.
At this point, after innumerable hours of cleaning and recleaning the microscope, our budding aquarist/microscopist has yet to actually see anything with it. An object to be observed and some water has been placed on the microscope slide and a cover glass has been carefully added. Cover glasses should neither be immersed in the fluid covering the specimen nor should they have drops of water or fingerprints on their surface. Now, withdraw as much of the excess water as is possible. This is done for two reasons; first, the thinner the water layer, the better the image, and second, thick layers of water have a distressing habit of abandoning the microscope slide for the safety and stability of the microscope stage, often taking the coverslip and/or object to be observed with them. And that means more clean up. So, fold a piece of tissue paper (it is fine for this function) and touch it to the edge of the slide and coverslip drawing off excess water. It is best to not use too much water to begin with since the wicking of water from under a coverslip can even remove the specimen to be examined.
The microscope slide is now carefully placed on the microscope’s stage and put in the slide manipulator. With the stage lowered (or the objective lenses raised, the method of focusing varies between manufacturers) so that there is the maximum amount of distance between the lens and the specimen, the specimen is centered over the hole in the microscope stage that light passes through. Turn on the light and, while looking in from the side, center the object in the hole.
Select the lens that you want to use for the initial observation. This is generally the lens with the lowest power. Move that lens into position by rotating the nosepiece. Observing from the side, use the coarse focusing knob to gently bring the lens toward the specimen until it almost touches the surface of the cover slip. When you do the adjustments described in the next section, always focus to move the lens away from the specimen. If you go too far and have to focus toward or down into the object be sure you know where the business end of the lens is relative to the specimen or you will crack the slide and possibly ruin the lens. Look from the side, if you are not sure.
Figure 5:
Figure 5: Compound microscope working distances. Notice as the magnifying power of the objective lens increases, the working distance (WD) gets dramatically smaller. With a 100x oil immersion lens, the working distance is so small that to gather enough light for an image to form, a drop of “immersion oil” must be placed between the lens and the coverslip.
I suppose it is in the experience of every biologist who regularly uses a microscope to, at least once, drive a lens through a slide or, at least, crack a cover slip. I have certainly done it and every other microscopist I know has also done it. It is one of those things that one does that makes one feel like a truly exceptionally dumb schmuck. Additionally, it is not something you want to get into the habit of doing. Doing so destroys the preparation and it may easily destroy the lens. At the very least, you are back to the cleaning table… Be prudent, watch where the lens is, and save yourself from this experience.
Observation Using Critical (Kōhler) Illumination
Microscopes are designed to be used without the user wearing glasses. If you normally wear glasses you should remove them during observation. Normally, microscope oculars can be adjusted to correct for vision problems across the whole nearsighted and farsighted range. Unfortunately, microscopes cannot be adjusted for astigmatism.
Even for people without vision problems, the two eyes seldom focus identically. Consequently, the first step in any use of any binocular microscope is to adjust the oculars. One or the other or both of the oculars will move or pivot at their connection to the headpiece of the ‘scope. In the better scopes there is a scale between the oculars that indicates the interpupillary distance, or the distance between the pupils of the observer. If you have glasses, and have your prescription handy, this distance is usually indicated on that. If you have that simply move the oculars apart to the indicated distance.
If not, the method for adjusting the oculars is easy. First, close one eye and look in the appropriate eyepiece with the other. Make sure you can see a complete circle of light. It isn’t important if anything is in focus; at this time, all you want is the complete circle, within the field of view. Then, holding your head steady, close the eye you were just looking through and open the other one. Move the other eyepiece until you can get a complete circle of light in that eye as well. Try to move your head as little as possible. Once that is done, close that eye and open the first one again. Again make sure you can see a complete circle of light in the field of vision. Repeat with the other eye, ensuring that you can see a complete circle of light. When you have the moved the eyepieces so that you can see a complete circle of light in the field of view simultaneously, you have completed this part of the task. Note the interpupillary distance for later, so you won’t have to go through this procedure again.
And, no, you are yet not ready to examine the specimen.
The next step is to ensure that both objectives are focusing on the same object at the same time. One of the oculars typically doesn’t rotate, while one does. Occasionally, both will. Find the one that doesn’t or decide which one you will leave alone. Close the eye over the other ocular. Using the lowest power objective find a small object, such as a grain of dust or a small bubble, in the field of view and focus on it until it is sharply in focus, keeping the other eye closed. When the object is in sharp focus, open the other eye and close the one you have been using. Adjust the eyepiece, using the adjustment ring on the ocular mount until the same object is in focus with the second eye. When that is done, provided you haven’t moved the oculars on the headpiece in the process the object should be in focus with both eyes.
Now you are set to prepare for observation.
Critical illumination, also called Köhler illumination, produces an image with the minimum of glare and the highest resolution possible. In addition to simply viewing the object, such an image can be used to produce the best possible photograph.
Steps in setting up for critical illumination:
1. Put an object on a slide, and focus upon it
2. Close down the light source diaphragm completely or until just a small amount of light is visible.
3. Use the condenser adjustment to move the condenser up or down so that the light source diaphragm edges are in sharp focus.
4. Generally, this means the condenser is moved very close to the underside of the slide.
a. For exceptionally critical viewing,
i. A drop of immersion oil may be placed on the condenser lens and in contact with the underside of the slide. This procedure “brings out the best” in the lens; but it is messy and very seldom used. Personally, I have done it for critical illumination of research slides, but only rarely.
5. Refocus on the object.
6. When the object and the edges of the light source diaphragm are both in focus, use the light source diaphragm adjustment to open the iris until the edges of the diaphragm just coincide with the edges of the field of view.
7. Remove the ocular and look down the tube.
a. The back lens of the objective should be just filled with light.
8. Use the condenser iris diaphragm adjustment to close down the light until a ring constituting about 10% to 20% of the outer part of the back lens of the objective is occluded and dark.
9. Replace the ocular.
10. From this point on, the intensity of the illumination should be adjusted by using the light source rheostat or brightness adjustment.
With the illumination properly set:
· The maximum resolution of the lens will be achieved making the smallest object possibly seen with the lens visible.
· The light intensity across the whole background will be uniform and lacking “hotspots” or “coldspots.”
The condenser diaphragm may be stopped down (= closed a bit) to increase contrast and depth of focus. However, doing so will degrade the image by decreasing the resolving power. A small amount of change in the diaphragm can severely degrade the image. I do this occasionally, but only rarely and never for critical viewing.
Final thoughts on critical illumination:
It should be apparent that setting up the compound scope for optimal viewing is not a haphazard procedure. However, it is also not a cumbersome one. Once learned, the procedure is easy, and if you do a lot of viewing it becomes “second nature.” It has to be repeated each time you change objectives.
Changing objectives to increase or decrease magnification is, of course, very necessary. The field of view at the higher magnifications is very small, it is often necessary to go to lower powers and to move the slide to view an object.
Parfocal and Parcentral Lenses
One might expect that as one changes objective lenses on a compound microscope that the lenses will come to focus on the same place on the slide. Truly, one might expect that… but, only if one pays for it will one’s expectations be fulfilled. When two lenses are aligned so that the same object is in the center of the field of view for each lens those lenses are said to be “parcentral.” Note that parcentral lenses may, or may not, have the object in focus, even if it is centered in field of view. Most medium and virtually all expensive compound microscopes have all their lenses parcentrally mounted. Cheaper instruments… well, as the old saying goes, “You pays your money, and you takes your chances.” Similarly, when two lenses are mounted so that when the observer changes the lenses to increase or decrease magnification and the object being viewed remains in focus, they are said to be parfocal. This is property that occurs, generally, in the better made instruments. It is typically absent on cheap ones. It is also generally absent if the one is using mismatched lenses or lenses from different instruments on the same nosepiece.
Oil-Immersion Lenses
The highest magnification powers of light microscopes are generally reached only with oil-immersion lenses. I won’t go into detail why this is the case, but it involves using oil to bring all the possible light from the condenser to bear on the very small area that is being examined. Unless specifically noted, oil-immersion lenses are not designed to be used with wet mounts. They are designed to be used with mounts of the very narrow thickness that may only be achieved in histological preparations. This is not to say that one cannot use oil-immersion lenses with wet mounts; but to do so and get a good image is difficult. There are some other, potentially, expensive implications, as well. With the oil in place between the first lens surface and the coverslip, focusing movements of the lens will move both the coverslip and the object. In search of a good image, it becomes very easy to “chase” a slightly out of focus object too far as it retreats during the compression of the mount. This can crack the coverslip, sometimes crack the slide itself, and damage the lens in the process. Wet mount observation at high magnification should only be done with extreme caution.
Figure 6:
Figure 6: Immersion oil is a necessity if your microscope has an oil immersion lenses. There are several brands; they are all good. I generally get the least expensive.
Next time
In the next installment of this series, I will finish my discussion of compound microscopes by delving into specimen preparation with hints and suggestions on how to construct a good preparation. I will also discuss making observations with a dissection or stereo microscope.