Chapter 1: The Microscope - Endnotes

  1. For absolute maximum resolution, oil should be used between the condenser and the slide as well, although in practice this is rarely done.

  2. Blue has an additional advantage in that the eye perceives light blue as white . that is the reason for adding bluing to white laundry, to make it look brighter. green light, is nearly as good for resolution as blue and is more pleasing to the eyes. we can withstand longer and more intense observations with green light (note the color of computer monitors and most automobile dash lights). if a green filter is available, replace the blue filter with the green -- be sure to remove the blue -- and note any differences.

  3. Since the electrons are shot down the column, the electron source is known as the electron gun, and the electrons are drawn off of the tungsten wire by a low voltage gun cap, then accelerated by the high voltage anode.

  4. It should be noted that for most routine cell biology, a resolution of about 30 Å is all that is required. Electron microscopes will give resolutions of around 5-7 Å, and the top research microscopes are beginning to approach the theoretical limit of just above 1 Å. as we approach this theoretical limit, however, we enter the can't see the forest for the trees syndrome, or in more perspective, can't see the forest for the chlorophyll molecules syndrome. The cost of the instrument increases exponentially with increased resolution and extremely high resolution electron microscopes are used for research on electron optic theory, rather than for biology. The one advantage of high voltage EM for biologists is the ability to view thicker specimens, even hydrated living cells.

  5. The directions are given for the Nikon Model S-Cb, but will apply to most microscopes, provided allowances are made for variations in manufacturer design.

  6. Exceptions to this occur when you are not attempting to use maximum resolution and are willing to trade resolution for some other purpose, such as ease of operation, or to compensate for the lack of a rheostat.

  7. The Nikon S-Cb is provided with coaxial, coarse and fine focus knobs, both of which are located near the base. Clockwise rotation of either of the focus knobs by the operator lowers the microscope stage. other microscopes may have two separate focus knobs, and may alter the position of the upper lens tubes and/or the objectives rather than the stage.

  8. On the Nikon S-Cb models, the most confusing and potentially damaging part of the microscope is the tension adjustment device for the coarse adjustment, and the preset device. incorrect adjustment of these will result in certain damage to the focus adjustment of the microscope, necessitating complicated repair.

  9. For the Nikon microscopes equipped with a pre-set device, the lever should remain in its counter-clockwise position -- be sure to check this position periodically during the use of the microscope. this device has proven to cause enough problems that many laboratories are resorting to removing the lever from the microscope, to prevent damage to the focusing gears caused by improper use.

  10. Type A immersion has a lower viscosity than Type B and is much preferred. Some laboratories will mix A and B to obtain intermediate viscosities. In any case, the oil should be a non-drying and with low fluorescence.

  11. If the image observed with the 40X appears dull and with a milky halo to it, the first thing to suspect is oil on either the front of the lens or the slide. the second thing to check is the presence of oils and/or make-up from the eye lids on the oculars.

  12. The average is statistically more valid, since there may be differing concentrations within various areas of the slide. A blood smear will definitely have variations since it will be normally a gradient from one end of the slide to the other, due to the means of preparing the smear.

  13. If desired, whole blood may be substituted, diluted 1:200 with saline prior to use.

  14. The coverslip used for the hemacytometer is especially thick, in order to prevent sagging in the middle. A regular microscope coverslip should never be used as a substitute, as it will yield incorrect volumes, often varying with each count.

  15. Most bright field microscopes come with or can be easily fitted with a dark field stop. If your microscope does not have the disk supplied, try taping an ordinary dime into the filter holder below the condenser, and moving the condenser to a position where light can move in a ring around the coin. For many microscopes, this is an economical method of producing a dark field microscope. Alternatively, copy the image of a darkfield disk, size to fit your filter holder and cut it out of cardboard, construction paper or aluminum foil. Place it or tape it in the filter holder, or directly onto the condenser. The important feature is to keep the light moving in a cone around the periphery of the lens, and not through the center of the lens.

  16. Plastic plates are preferred for this purpose, since the plastic has a more uniform thickness than ordinary glass petri plates. Special glass plates are available with ground optically flat glass in the bottom, but these are both expensive and fragile.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 --