A compound light microscope has a white light source emitting rays that travel through the condenser lens to concentrate the light and focus it on the specimen. The specimen is located on a slide, which is placed on the stage of the microscope. The light passes through the specimen into the objective lens where the specimen image is magnified. The light continues up through the body tube and reaches the ocular lens where once again the image is magnified. Our compound microscope oculars have a magnification of 10X. To determine the specimen magnification, multiply the magnification number on the objective lens by 10. Example: When using the 40X objective multiply 40 (objective magnification) by 10 (ocular magnification). The total magnification of the specimen is 400 times. When you take notes about the appearance of a specimen, or when you draw what you see, always record the magnification you used to view it. Determine what magnifications are available on the objective lenses of your microscope. Record in your notebook the total magnifications possible with each lens. The light intensity required for good visual images differs from specimen to specimen. The light source has a switch with several fixed settings for light adjustments.

The iris diaphragm lever, located at the base of the condenser, can also control the light reaching the specimen; move the iris-diaphragm lever back and forth to open and close the aperture. This will not only affect the total amount of light used to view the specimen, but also the contrast; i.e., it can affect your ability to discern a relatively transparent specimen. This may be particularly important when viewing unstained and non-colored organisms or cells. The distance from the specimen to the objective is known as the working distance. The objectives are screwed into the nosepiece at the bottom of the body tube. The proper working distance is achieved when the specimen and objective is in focus.

The working distance decreases as the magnification of the objective used increases. The correct distance for focus is obtained by manipulating the coarse and fine focus knobs located on both sides of the arm. Coarse adjustment will allow you to obtain an approximate focus and the fine adjustment will bring your specimen into sharp focus. Always with the 100X objective, and usually also with the 40X objective, only the fine adjustment should be used. Our microscopes have a feature known as parfocal. Once the area you wish to view is obtained with any of the four objectives, you can switch to another objective without having to make more than a slight focus adjustment with the fine adjustment knob. Therefore, when you begin to observe any slide, start with the 4X objective. Find an area of the specimen that you wish to see in detail and move the slide so that the area is centered in the field of view. Without moving either the coarse or fine adjustment knobs, change to the next higher magnification objective and focus. Repeat the process again to increase the magnification. When finished with a slide, always return to the 4X objective before removing the slide. You will want to begin with the low power on the new slide, and the 4X objective allows more room to replace the slide without scratching the lens or damaging the slide. Both the compound and dissecting microscopes that you will be working with in this laboratory provide a means for adjustment of interpupillary distance interocular scale adjustment to suit the individual’s eye spacing. Look through the eyepieces with both eyes and with one hand on the outside of each eyepiece, push or pull the eyepiece tubes until the fields of vision converge into one. If you see two fields, move the eyepieces until one field is visible. Note the number on the interocular scale for future use. It often takes some practice to adjust the microscope so that it becomes comfortable to use both eyes when focusing through the microscope. It is worth the effort, however, because it will reduce eyestrain and enable you to concentrate on your observations for longer periods. In addition to adjusting the microscope for the interocular distance, it is often necessary to adjust the two eyepieces to accommodate different focusing capabilities between your two eyes. In order to do this, look at a specimen with your right eye through the right eyepiece and focus the specimen first with the coarse adjustment knob; and then with the fine adjustment knob. Next, without moving your head, look at the specimen with your left eye through the left eyepiece and focus the specimen by rotating the diopter adjustment ring, found on the eyepiece itself, without moving the coarse and fine adjustment knobs. Again, practice may be necessary to obtain the best focus possible.

Resolution is the minimum distance two objects can be separated and still be seen as distinct objects. Your eye has a resolving power of 100 microns and the compound microscope has a limit of resolution of 0.2 microns. Check each objective on your microscope for a number other than the magnification number. That number is the numerical aperture (NA) of your objective and the numerical aperture is a constant related to the light gathered by lens at its working distance. When the wavelength is decreased the resolvable distance between two objects is decreased. The wavelength of blue light is 400 nm and the wavelength of red light is 700 nm. The shorter wavelength of blue light has better resolving power. Most of our microscopes have a blue filter that can be moved into the light path to assist in resolving small details at very high magnification. To better understand the concepts, calculate the resolution when using a 40X objective NA = 0.65, condenser NA = 1.25, and wavelength of light used is 400 nm. Repeat the calculation for the resolution when a wavelength of 700 nm is used. Which wavelength will give you better resolution? What color corresponds to that wavelength? Can you understand why we have a blue filter available on our microscopes as part of the condenser lens? When would you expect to use the blue filter? The field size is the actual area you see. As you switch to a higher power objective, the field size decreases. Specimens can be measured by using an ocular micrometer that fits into the ocular tube just above the lens. Another accessory known as a stage micrometer will also aid in measuring a specimen. It is critical to understand depth of focus. Not all specimens are of even height. For instance, two or more objects, such as cells, will often overlap or pile on top of one another. When fine focusing, a sharp image will appear for a cell at one level but not for the cell above or below. For a clear image at another depth the fine focus must be adjusted until the desired viewing level is in focus. Experienced biologists are constantly adjusting the fine focus to ensure that they have a clear picture of the entire specimen, not just a single focal level within that specimen.

The oil immersion objective (100X), when used with a drop of oil, prevents refraction or deflection of angled light from its straight path that would occur if the light were to pass at an angle from glass into air. The degree to which the light is refracted or bent by a substance is formulated as its refractive index. As you might expect, the numerical aperture of a lens, the light-function constant you used to calculate the resolution, is determined in part by the refractive index of the glass. To prevent the light from being bent away on an angled path from the objective lens, allowing the maximum amount of light from the specimen to be gathered by the objective, a drop of immersion oil may be placed on the specimen and the oil immersion objective then lowered into the oil. Immersion oil has the same refractive index as the glass so light traveling up through the slide, the oil and the objective lens is not refracted again until it passes from the convex upper surface of the lens into the air above.That bending, however, is what the lens is designed to do, sending the rays which left the specimen at angles up the tube at new coherent angles to be resolved and magnified by the ocular lens. As light strikes the specimen the qualities of the light are changed in several ways that give the visual image we perceive. It may be scattered or reflected away from a path leading to the objective, darkening the image; it may be completely occluded by solid structures that appear black to the observer; specific wavelengths of the light may be partially absorbed by certain substances (including stains), giving a characteristic color to structures containing them.