Autore Bruno Pacifici

Come usare un microscopio

a campo chiaro

This is the standard setup for bright field, Kohler illumination.

There are two basic methods of using a brightfield compound microscope, critical and Kohler illumination. In critical illumination, the light source is put in focus by the condenser onto the specimen plane. This produces the greatest amount of illumination, however the illumination is not even over the entire field. Kohler illumination produces an even field of illumination by focusing the plane of the field-limiting aperture onto the specimen plane. This is the preferred method to use. The optical corrections made in the objectives lenses are designed to work best with Kohler illumination. In addition, special contrast enhancing microscopes such as phase contrast, DIC and Hoffman modulation must use Kohler illumination to work at all.

1	Clear out the instrument. If the microscope is equipped with phase, differential interference or stop contrast, remove the rings or prisms and set condenser to bright field. Set image tube to the eyepieces. If the microscope has an episcopic condenser set the dichroic prism to diascopic white light. Remove old specimen, if it was left in and destroy it.
2	Select scan objective. Turn on the light. Insert specimen and center it. Stop down iris diaphragm. Focus the specimen.
3	Select 10X objective or higher. Focus specimen.
4	Stop down field-limiting aperture.
5	Focus the edges of the field-limiting aperture with the condenser lens to the blue fringe. Double check that the specimen and the field-limiting aperture are in focus simultaneously.
6	Open field-limiting aperture to just beyond the edge of the image field.
7	Set iris diaphragm to maximum resolution. Note the position of iris diaphragm for future use with that objective. Adjust light level with the rheostat.
8	Adjust eyepiece inter-pupillary distance.
9	Adjust the diopter correction for your eyes.
10	Set contrast & depth of field for your specimen with the iris diaphragm.

1.

2.

When using the scan objective, keep in mind that it is optically different from the other objectives. It does not have the resolution as the others. It use is for the location of the specimen only. In order to view the entire field, you will have to lower the condenser lens to widen the beam of light. Some microscopes have an addition wide field lens that needs to be inserted. The light source may be used at any intensity, But low light is all that's required for low magnification. Be sure to use a specimen that has a clean coverslip #1.0 thickness works best for most objectives. You can tell when the specimen is centered when it glows from the light coming from the condenser. Turn the iris diaphragm all the way down. This will increase the depth of field and the contrast, which will help out finding the focal plane of the specimen when you first focus. Once in focus, the other objectives can be selected. Fine focus is all that will be required to focus the image.

3.

Most microscopes are parfocal. Which means that each objective will focus the image at the same point. So when changing from one objective to another you only need to touch up the fine focus. When setting up a microscope for Kohler illumination any objectives that is 10X or greater may be used.

4.

Many compound microscopes have a field-limiting aperture built into the base. There are two functions of this aperture. First, is to reduce stray light from entering the optical path and second, to allow the operator to set the condenser at the height to give the most even illumination. This is the key point for using Kohler illumination. The condenser will provide nearly perpendicular rays of light. The objective lenses are designed only to be optically correct when this occurs.

If your microscope does not have a field-limiting aperture, then a card with a small hole cut in it will do. Simply place the card onto the illuminator and center it while looking through the scope.

5.

Kohler illumination requires that the condenser be set to a specific point. You do this when the specimen is in focus with a 10X or greater objective. As you adjust the condenser you will see the leaves of the field liming aperture come into sharp focus. At the same time there will be a blue or red fringe on the edge of the field. Set the condenser in between both of the fringes. One fringe will always dominate; in which case you should lean towards the blue. The condenser lens is now set and its position does not have to be changed when using other objectives.

6.

Open the field-limiting aperture to just beyond the edge of the field. This will vary with each objective you use.

7.

Maximum theoretical resolution is the point where the condenser numerical aperture matches the objective numerical aperture. When using apochromat lenses, use the full N.A. If you are using standard achromat objectives multiply the N.A. by 0.7, that will be your best practical resolution. Adjusting the iris diaphragm does this. Some iris diaphragms have calibrated markings to indicate what the condenser numerical aperture is. In which case you simple turn the lever to the objective N.A. value. If your microscope is not so equipped then you will need to remove the eyepiece and look down the tube of the microscope. As shown above, what you will see is the back of the objective lens. There will be no image visible just an illuminated disc. If necessary a telescope can be place into to eyepiece tube to view the objective better. Adjust the iris diaphragm so that the leaves of the aperture match the edge of the lens. This is the point of maximum resolution. If you open the iris diaphragm any further you will just increase flare. If you stop down the iris diaphragm you will trade off resolution for contrast and depth of field. It is up to you to determine the amount of contrast needed for you specimen. For now, however set the diaphragm to maximum resolution.

8.

The distance between each person eyes varies. Most binocular microscopes allow you to adjust the interpupillay distance. On the more sophisticated microscope you will see the eyepieces extend or retract as you make the adjustment. This is so the tube length will be maintained to optimize the optical corrections of the objective lens. The eyepieces should be adjusted so that the images will fuse together. Before they fuse, check to see if the images are separated along the horizontal axis only. If the image appear to vary along the vertical axis the microscope needs to be adjusted by a professional.

9.

It is rare to find two individuals that have the same optical correction for their eyes. As a result each microscope has to be adjusted specifically for each person. This is done with the diopter correction. If you wear glasses and do not suffer from astigmatism you may opt not to use them, while using the microscope. To make the adjustment, be sure the microscope is at its tightest depth of field (maximum resolution). Use high power and focus the specimen onto a tiny spot. Look at the binocular top of the microscope and find the diopter control that will be located just under one of the eyepieces (usually on the left eyepiece). Close the eye that has the diopter control and focus the spot while using only the other eye. Now open the diopter control eye and close the other and focus the same spot, not with the focus knob, but with the diopter control under the eyepiece. Open both eyes.

10.

It is up to the microscopist to determine how much resolution to trade off for contrast and depth of field. This requires an understanding of the specimen and what you want to see. For example, if you are viewing stained bacteria that are fixed to a glass slide. You will not need much depth of field. So you can set the microscope to its maximum resolution. If the specimen was not well stained, when you could trade off some of the resolution for contrast.