Photomicrographics: An overview

For a simple beginning, no apparatus is needed except an ordinary camera and a microscope. Try low powers first and proceed gradually to the higher magnifications. Remove both front and back lenses from the camera, leaving the lens barrel and the shutter; also, remove the eyepiece from the microscope. Bend the microscope to the horizontal position and place the lens of the camera close to the ocular end of the microscope and shut out all light at this point by winding black cloth around the end of the microscope and the barrel of the camera lens. Take great care to have a perfectly straight optical axis through the microscope and camera.

While the camera and microscope can be adjusted so as to secure a perfect optical axis by simply putting both instruments on the table and raising one or the other-according to the size of the cameraby placing a board under it, such an adjustment is extremely unsatisfactory, since the least jar may disturb it, and inserting the plateholder is almost sure to disarrange something. It will save time if you prepare a board to keep both instruments in position. Select a clear board 1 inch thick, about 1 foot wide, and 5 feet long. On the top of this board, screw two pieces 3/4 inch thick, 1 1/2 inches wide, and 5 feet long, so as to form a guideway for the camera. If the camera is so small that it must be raised to bring it into the optical axis of the microscope, fit to the guideway a board of the necessary thickness, and fasten the camera to this board. It is absolutely necessary that the preparation to be photographed and the ground glass of the camera should be perfectly parallel. The board will save time in securing this parallelism. Cut through the board a slot 1/4 inch wide and extending to within 6 inches of each end. By this means the camera can be clamped with the screw used to fasten it to a tripod. Also, a piece of metal or hard wood may be placed over the horseshoe base of the microscope and with a bolt, preferably one with a butterfly nut, the microscope may be held firmly in place. This board, with the long slot, will be useful in making lantern slides.

As an illuminant, direct sunlight, diffuse daylight, a gas-mantle lamp, an acetylene lamp, a Mazda bulb, an arc light, or any strong light may be used. Remove the mirror from the microscope and allow the light to come directly into the optical axis. This mirror will not be needed in any photomicrographic work.

Let us suppose that we are to make a photomicrograph of a vascular bundle and that we are using a 16-mm. objective. If only a part of the bundle is shown on the ground glass, remove the ocular of the microscope. If the illumination is very uneven and shows a "flare spot," look at the inside of the tube of the microscope. Probably it was not blackened and the "flare spot" was due to reflections. Obviate the difficulty by putting apiece of black paper inside the tube. Any modern microscope should have the tube well blackened inside. Move the light back and forth and sidewise to get the best illumination. An easy way to determine whether the light is centered is to close down the iris diaphragm below

##146/MIPH

PHOTOMICROGRAPHS AND LANTERN SLIDES

the condenser until the lighted circle on the ground glass is very small. Then shift the light or the condenser, for it is possible that the condenser is also off center, or both until the illuminated circle is centered. After the light source, the condenser, and the objective have been brought into line, rack the condenser back and forth until the beam of light is focused on the object to be photographed. Then make certain that the cone of light at the point where it enters the objective has a diameter great enough to fill the aperture of the objective. The ordinary form of Abbe condenser is not likely to be satisfactory with objectives of 16-mm. focus, and should not be used at all with objectives of longer focus.

Focus the object upon the ground glass. Even with a 16-mm. objective, the ordinary ground glass is rather coarse for accurate focusing. Always examine the image with a focusing lens. A brilliant view may be obtained by fastening a thin cover-glass to the ground glass with a small drop of balsam. At this spot the image may be examined very critically. Of course, as in any photography, the ground side of the glass should be nearest the object, occupying exactly the place which is to be occupied by the emulsion side of the plate. Do not focus indiscriminately, but be sure that the image is sharp at the level of the ground side of the glass. It is a good plan to make a cross upon the ground glass with a pencil or pen, and then add a drop of balsam and a cover-glass. Focus on this mark and fix the focusing glass at this level. The cheap tripod lenses are good for this purpose.

The time of exposure will vary with the magnification, the intensity of the light, and the speed of the plate. The exposures will be much longer than in ordinary photography. It is better to use artificial light, since one can more quickly learn to estimate the length of exposure when the intensity of the light is constant. A slow plate, even the very slow contrast plate, is likely to prove most satisfactory for the beginner. With a Welsbach lamp, a contrast plate of the same speed as a lantern slide, and a 16-mm. objective used without an ocular, or Abbe condenser, try an exposure of 30 seconds. Develop the negative in whatever solution is recommended in the directions which come with every box of plates. If the negative is too weak, make a longer exposure; if too dense, shorten the exposure. A little experience with your apparatus will soon enable you to estimate the length of exposure with some certainty. We frequently use lantern-slide plates for tests and for small photomicrographs. The Cramer lantern slides and contrast plates of larger sizes have the same speed and, consequently, one can determine the length of exposure by using a cheap lantern slide. In making tests, it will save both time and money to expose for 5 seconds and then push in the dark slide so as to cover a part of the plate; then expose 5 seconds longer and push the slide in a little farther, etc. In this way you can make 4 or 5 exposures on a lantern-slide plate showing exposures of 5, 10, 15, 20, and 25 seconds, the first exposure being 5 seconds and the last, 25 seconds. By making several exposures on 1 plate, with the first probably underexposed and the last probably overexposed, you can note which exposure is best and will waste only 1 plate in getting the time. A print from such a negative is valuable, since it enables one to judge very accurately the printing quality of the various exposures.





Return to Methods in Plant Histology