Bright Field versus Dark Field
Bright field and dark field illumination techniques are some of the most common and affordable lighting options for using a microscope. Bright field is usually the default illumination technique in ordinary light microscopes while dark field is considered the more affordable option to phase contrast microscopy in viewing translucent samples and specimens.
What is Bright Field Microscopy?
Bright field microscopy utilizes incandescent light to illuminate a sample or specimen under observation. Once the light box releases light, this will travel through the substage condenser lens. From there it will hit the specimen then travel through the microscope’s objective lens before finally reaching the eyepiece. As long as the sample or specimen has natural pigments or colors as well as adequate thickness that can absorb light then they’re sure to be visible under bright field illumination. Staining may make translucent or thin samples and specimens visible in bright field illumination.
To use a bright field microscope, start with mounting the specimen or sample on the stage. Use stage clips or the slide holder to keep the glass slide in place. If your microscope is equipped with a mechanical stage then adjust it accordingly to obtain the ideal position for the slide without having to touch it.
Your microscope should be able to operate at low and high power magnification. It’s also better if your microscope has a built-in illuminator. With it, you can then control light intensity and even choose the shape of your microscope’s light beam. The light level should be bright enough to make all parts and details of your sample or specimen clear but not so that it would hurt your eyes.
If your microscope’s condenser is adjustable, place it as near as possible to the aperture opening. Now, start adjusting the focus. Set the microscope at low power first. This will provide you with the greatest field of view. Make sure that the sample or in the center of your focus. If you’re using a monocular microscope, just make sure that the eyepiece is clean. If you’re using a binocular microscope, adjust the eyepieces to ensure smooth viewing.
Adjust the objective lens next. Try out the objective lenses in the nosepiece one by one until you attain the ideal total magnification level. Every time you change objective lenses, you will have to refocus. Use the coarse adjustment knobs to elevate or lower the stage then use the finer adjustment knobs for more precise adjustment.
What is Dark Field Microscopy?
In dark field microscopy, an opaque disk may either be inserted or incorporated with the condenser lens itself. The disk causes only scattered light beams to reach the eyepiece. Dark field illumination is used to make invisible samples and specimen as well as those unable to absorb light due to insufficient thickness discernible. Do remember also that dark field illumination works better with high power magnification. For more details about this type of microscope, visit http://www.darkfield-microscope.com.
The process for using dark field illumination with a microscope is virtually the same with that of bright field illumination.
Bright Field versus Dark Field Illumination
Bright field illumination is, as mentioned before, best used for samples and specimens with natural colors or have been subjected to staining. Although it is generally useless in studying live transparent specimens like bacteria, there are certain situations when using bright field illumination for such specimens is possible.
Live specimens taken from aquatic environment may be made visible with bright field illumination as long as they’re placed in wet mounts and of adequate size. Those that are stained or smeared may also be made visible such as bacteria and organelles. Thickly cut sections of tissues can also be seen. In all cases, specific magnification levels are prescribed.
The main advantage of using bright field illumination is its ease of setup and operation. Its greatest disadvantages, however, are its seemingly low optical solution and contrast levels.
Dark field illumination is best used for initial studies of prepared and lightly stained specimen, single-celled cultures, hay or soil infusions, samples taken from aquatic environments, and certain types of cells and tissue fractions. Dark field illumination is also effective in determining motility in cell or tissue cultures.
Dark field is also relatively simple to setup and operate. Pitted against bright field optics, its main disadvantage is its relatively lower light levels, requiring greater light intensity to be used. Light that’s too intensified, however, can damage the specimen or sample.
Ultimately, one really shouldn’t have to make a choice between the two because they’re polar opposites of each other. If competition is necessary, it’s better to pit dark field illumination with phase contrast microscopy as both excel in making the “invisible” visible.
