CAROLINA BIOLOGICAL SUPPLY COMPANY
BURLINGTON, NORTH CAROLINA 27215
2700 York Rd. Cable address: Squid April 20, 1989
Mr. Donald L. Ferry
Dear Mr. Ferry:
Jim MacDonald of our facility in Gladston, Oregon, called me yesterday concerning your query about Rheinberg/oblique lighting in photomicroscopy. The technique and name are my own, really a joining of two techniques.
Rheinberg transillumination is achieved through the use of a special filter, Rheinberg, which is positioned in this filter holder of the microscope's substage condenser. The filters, which I make for my use, consist of a central disk such as blue surrounded by an annulus of one or more contrasting colors. I frequently use a blue central disk combined with a yellow annulus. In use, the Rheinberg filter produces a uniform field of the central disk color with the specimen illuminated by the annulus color, i. e., a yellow amoeba on a uniform blue field.
When using the Rheinberg system the lamp diaphragm acts as the field diaphragm. The aperture (substage) diaphragm remains fully open so black reducing rings are used over the central Rheinberg disk to control contrast. I make these of black film disks the same size as the filter disk. The reducing rings have central holes of varying diameter to give me a range of control. In use I simply place a reducing ring directly atop the central disk.
Now for the oblique part which involves decentering the substage condenser laterally or otherwise (mine is rotatable) to achieve oblique transillumination. By decentering the condenser the light is directed obliquely at the specimen so that the near side (nearest the light source} is illuminated while the opposite side is in shadow. It can produce a striking 3-demensionality. In an amoeba, for example, a transparent or translucent specimen becomes 3-dimensional with clearly visible pseudopods, nucleus, vacuoles, crystals, etc. By combining this with Rheinberg filtration one gets a 3-dimensional specimen in one color on a contrasting field color.
Now the fun begins. One can use two or more colors in the annulus, i.e., use 2 colors such as yellow east and west and red north and south to add another dimension. The combinations are many and limited by your imagination. Certain color combinations work better than others but it is fun to try all sorts.
One of my many interests is minerals (native gold, gem faceting, etc.,) and crystal inclusions in minerals such as crystal quartz, emerald, aquamarine, etc., are of special interest. The use of the Rheinberg/oblique technique can really produce some interesting images of such inclusions. For example, I have a small Colombian emerald crystal which has tubular cavities that contain fluid and bubbles and crystals of common salt which has given me some interesting color transparencies.
Many of my specimens are living organisms such as amoebae, planaria, algae, etc. and I use the Rheinberg/oblique system frequently. True, the system ads unnatural color which some purists dislike, but most microscope slides are stained so that unnatural color is routine in studying biology. Visual impact will catch a student's eye when a natural color rendition might not. Perhaps a good example of the subject can be seen in the April, 1989, issue of Natural History Magazine. The feature, The Natural Moment, is my series of the molting/egg laying of a water bear, (tardigrade). The water bear is tiny, almost transparent, and wriggly. In my 5-hour long sequence (with much of the real action happening very quickly) I used physical restraint to hold the live specimen (pressure of the cover slip) and Rheinberg/oblique transillumination or differential color illumination to produce the color contrast and 3-dimensionality. Lots of luck was involved. One of my co-workers in the Cultures Department spotted the tardigrade with unlaid eggs and brought it over. I dropped everything else and spent the rest of the day working with it. I already had the lighting techniques worked out so that part was routine. I had to watch closely and add tiny amounts of water to the edge of the cover slip to prevent cohesion from crushing the specimen. It all came together to result in a sequence I'll not live long enough to repeat.
Refer to Practical Use of the Microscope by George H. Needham, Charles C. Thomas, Publisher, Springfield, Il. Mine is a 1958 copyright but there are probably newer versions. Another source of information is the 2 volume Photomicrography: A Comprehensive Treatise by Roger P. Loveland, Robert E. Krieger Publishing Company, Malabar, Fl., 1981. Both sources will give you detail on how to make your own filters.
I hope the above answers your questions. The techniques are old ones and all I have done is to combine them for my uses.
William R. West
Head, Photography Department
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