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There are many simple ways to add functionality to a basic compound microscope. This article describes how to do these techniques without specialized equipment.
These approaches manipulate the light passing through the sample to bring out different features in the sample that we could not otherwise see. Darkfield microscopy (Fig. 1) enhances contrast (differences in light intensity) so that structures that are hard to see otherwise can be observed. Polarization microscopy allows one to see whether and how structures alter the polarization of light, something we cannot see directly with our eyes. While the simple approaches described here may not produce as good results as a microscope built for these techniques, they can still be really informative, and can make lovely images.
This article assumes that the reader has some minimal familiarity with light microscopes, and has access to a compound microscope. Links to more complete discussions about microscopy can be found below.
Brightfield
Brightfield microscopy is the most basic form of microscopy that uses transmitted light (when the sample is illuminated by light passing through the sample). You simply pass the light through the sample, focus on the sample, and observe the image. It works primarily by differences in absorption of the light, so it can be difficult to get enough contrast to see structures in samples that are not strongly pigmented (structures that scatter light can also be observed easily).
One aspect of brightfield microscopy (and all types of transmitted light microscopy) that beginners are often not aware of is just how big a difference features of the illumination make to the quality of the image. It's not just a matter of being bright enough: adjusting the illumination properly strongly affects resolution and contrast. Only one feature of this will be discussed here: the effect of the condenser aperture (Fig. 2). Closing the condenser aperture (Fig. 3) reduces the width of the cone of light focused on the sample, so the light goes straight up through the sample. Closing the condenser increases sample contrast, and often makes features appear that are out of the plane of focus (much like increasing the F-stop on a camera increases the depth of field). Unfortunately there is a trade off: as you close the condenser aperture, the ability to resolve details goes down as diffraction artifacts become more prominent, and the image becomes more confusing as it becomes difficult to differentiate structures at different depths. Somewhere between fully open and fully closed (Fig. 2), there is usually a point where the improvement in contrast is balanced by loss of resolution.
Equipment needed:
- A compound microscope, preferably with an adjustable condenser aperture
- A sample to look at, and a slide and cover slip to mount the sample on.
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Darkfield:
Darkfield microscopy is a useful technique for increasing contrast especially for many transparent structures (e.g. in the image of the jellyfish tentacle in Fig. 1). It is also quite easy to set up a crude but effective version of dark field microscopy. The idea is to block all the light that would go directly from the bulb to the eye, so you only see light that is bent by the sample. It makes some faint structures pop out very nicely against a dark background. Figure 4 shows a comparison of brightfield and darkfield on the same sample (one where both techniques work fairly well).
All one has to do is stick a circle of black tape on a piece of clear glass or plastic to make a "light stop", and put it below the condenser lens which focuses light on the sample (Fig. 3). One has to match the size of the tape circle to the specific objective lens, so that the tape just barely blocks out the light coming straight through to the objective lens. It's easiest to start with a small piece of tape and then use larger pieces until you get the right size. It is also easiest to do with lower power objectives (4x to 20x).
Most importantly, the light stop has to be centered in the light beam. You can see the light blocked as you look in the microscope, and adjust the position of your light stop until the dark spot appears centered. If there is a bright circle around it, the light stop is too small; if the center of the image is dark, the light stop is too wide. Adjust as needed until it matches for your microscope and lens. You may also need to adjust the vertical position of the light stop as well: if the light stop is too low there can be a dark center spot2.
When things are set up just right, the sample should be evenly bright, and the background evenly dark. One thing to remember is that you need to open the condenser diaphragm wide for this to work at all: light has to come to the objective from a wide angle. If the condenser aperture is closed, all the light that is not blocked by the light stop will be blocked by the condenser diaphragm.
Equipment needed
- A compound microscope
- Black tape
- A glass slide, petri dish, etc. to stick the tape to.
- Scissors to cut the tape into a circle
- A sample to look at, and a slide and cover slip to mount the sample on.
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Polarization:
Polarization microscopy can produce beautiful images while giving one clues about the structure of a sample. One can use cheap polarizing filters cut out of sheets of polarizing film. Put one polarizer below the sample (most easily below the condenser lens, which focuses the light on the slide) and one polarizing filter above the sample. One can put the second polarizer right on top of the slide if one is using an objective lens with a long enough working distance. That way it is easy to rotate both polarizers independently. However, one has to be quite careful not to get crud on the lens or squish one's sample, and it does degrade the optical path a bit. Alternatively, one can hold the second polarizer in front of one's eye, or — in some microscopes — put it below the microscope head (between the objective lens and the eyepiece). That last solution seems to be optically the best (putting it in front of they eyepiece seems problematic if there are any plastic pieces or other filters that can affect polarization in the light path).
With this set up one can see things that alter the polarization of light (e.g. are birefringent), including calcium carbonate (e.g. ossicles of the sea urchin tube foot (Fig. 5), or shell in molluscan larvae), and bundles of muscle fibers. In both cases, one orients the two filters until they block the most light, and then rotates the pair of filters relative to the sample. When the axis of the birefringent material (e.g. calcium carbonate, or muscle) lines up just right with respect to the polarizers, it will shift the angle of polarization so that light that would have been blocked by the second filter can now pass through. So you get very nice — often quite colorful — images of bright crystals or muscles against a dark background.
Equipment needed
- A compound microscope
- Two pieces of polarizing film or sheeting
- A sample to look at, and a slide and cover slip to mount the sample on.
 Fig. 5: Polarized light reveals spicules in a sea urchin (Lytechinus variegatus) tube foot. |
Useful websites:
There are many good microscopy resources on the web, aimed at different experience levels and different budgets. Microscopy-UK is geared towards hobbyists or people on a smaller budget. Their "Microscopy Primer" article by F. Sterrenburg gives an introduction to many aspects of microscopy (e.g. the optical effects of the condenser and cover-glass, spherical aberration, etc). They also have lots of guides and articles on techniques, organisms, etc. FunSci.com has a neat series of articles about how to build one's own stereo (dissecting) microscope. Olympus' "Microscopy Resource Center" and Nikon's "MicroscopyU" are quite helpful for improving one's understanding of how different techniques work, and the many forms of optical aberration, among other things. They have in-depth treatments of many techniques, although they sometimes assume one has access to more expensive equipment.
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#Comments: 1
What other optical techniques can be easily and cheaply done using a basic, no-frills compound or dissecting microscope? If you know of some, please suggest them, or add them directly to the article.
If you have a better way to do the techniques described, can improve the descriptions, or have better images, that would be great too!
By the way, one can get a darkfield effect — or a close approximation — with a dissecting/stereo microscope that has an adjustable mirror. And polarization can be setup on a dissecting microscope pretty easily too.