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UCL Home  /  Geography  /  Resources  /  Laboratory  /  Light Microscopy  /  Bright Field Microscopy

Bright Field Microscopy

With a conventional bright field microscope, light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. Most microscopes will have a built-in illuminator. The condenser is used to focus light on the specimen through an opening in the stage. After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. The magnification of the image is simply the objective lens magnification (usually stamped on the lens body) times the ocular magnification.

 

Most people are usually aware of the use of the coarse and fine focus knobs, used to sharpen the image of the specimen. They are frequently unaware of adjustments to the condenser that can affect resolution and contrast. Some condensers are fixed in position others are focusable, so that the quality of light can be adjusted. Usually the best position for a focusable condenser is as close to the stage as possible. The bright field condenser usually contains an aperture diaphragm, a device that controls the diameter of the light beam coming up through the condenser, so that when the diaphragm is stopped down (nearly closed) the light comes straight up through the center of the condenser lens and contrast is high. When the diaphragm is wide open the image is brighter and contrast is low.


Using the Bright Field Microscope:

Mount the specimen on the stage

Make sure you are using the correct type of coverslip. High magnification objective lenses can't focus through a thick glass slide; they must be brought close to the specimen, which is why cover slips are so thin. The stage may be equipped with simple clips (less expensive microscopes), or with some type of slide holder. The slide may require manual positioning, or there may be a mechanical stage that allows precise positioning without touching the slide.


Optimize the lighting

A light source should have a wide dynamic range, to provide high intensity illumination at high magnifications, and lower intensities so that the user can view comfortably at low magnifications. Better microscopes have a built-in illuminator, and the best microscopes have controls over light intensity and shape of the light beam. If your microscope requires an external light source, make sure that the light is aimed toward the middle of the condenser. Adjust illumination so that the field is bright without hurting the eyes.


Adjust the condenser

In most cases the condenser will already be set to it's optimum position. However there are times when you will want to check this adjustment. To adjust and align the microscope, start by reading the manual. If no manual is available, try using these guidelines. If the condenser is focusable, position it with the lens as close to the opening in the stage as you can get it. If the condenser has selectable options, set it to bright field. Start with the aperture diaphragm stopped down (high contrast). You should see the light that comes up through the specimen change brightness as you move the aperture diaphragm lever.


Focus, locate, and center the specimen

Start with the lowest magnification objective lens, to home in on the specimen and/or the part of the specimen you wish to examine. It is relatively easy to find and focus on samples that are fixed and stained, as with most prepared slides. However it can be very difficult to locate living, minute specimens which move around.
Use dark field mode (if available) to find unstained specimens. If not, start with high contrast (aperture diaphragm closed down). Start with the specimen out of focus so that the stage and objective must be brought closer together. The first surface to come into focus as you bring stage and objective together is the top of the cover slip.
If you are having trouble, focus on the edge of the cover slip or an air bubble, or something that you can readily recognize. The top edge of the cover slip comes into focus first, then the bottom, which should be in the same plane as your specimen.
Once you have found the specimen, adjust contrast and intensity of illumination, and move the slide around until you have a good area for viewing.


Adjust eyepiece separation and focus

With a binocular microscope you need to adjust the eyepiece separation just like you do a pair of binoculars. One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it. Since very few people have eyes that are perfectly matched, most of us need to focus one eyepiece to match the other image. Look with the appropriate eye into the fixed eyepiece and focus with the microscope focus knob. Next, look into the adjustable eyepiece (with the other eye of course), and adjust the eyepiece, not the microscope.


Select an objective lens for viewing

The most frequently used objective lens is the 10x lens, which gives a final magnification of 100x with a 10x ocular lens. For very small objects and for details in prepared slides you will need a higher magnification. Typical high magnification lenses are 40x and 100x. The 100x is used exclusively with oil in order to improve resolution.
Move up in magnification by steps. Each time you go to a higher power objective, re-focus and re-center the specimen. Higher magnification lenses must be physically closer to the specimen itself, which poses the risk of jamming the objective into the specimen. Be very cautious when focusing. Good quality sets of lenses are parfocal, that is, when you switch magnifications the specimen remains in focus or close to focused.
Bigger is not always better. All specimens have three dimensions, and unless a specimen is extremely thin you will be unable to focus with a high magnification objective. The higher the magnification, the harder it is to "chase" a moving specimen.


Adjust illumination for the selected objective lens

The apparent field of an eyepiece is constant regardless of magnification used. So it follows that when you raise magnification the area of illuminated specimen you see is smaller. Since you are looking at a smaller area, less light reaches the eye, and the image darkens. With a low power objective you may have to cut down on illumination intensity. With a high power you need all the light you can get, especially with less expensive microscopes.