What is Bright Field Microscopy?
Bright-field microscopy (BF) is the technique that most people think of when they think of microscopy. We can recognize it by the background (field) being bright white and the sample being highly contrasted against it.
Bright-field microscopy (BF) is one of the simplest of all the optical (light) microscopy illumination techniques. In bright-field microscopy, illumination light is transmitted (i.e., illuminated from below and observed from above) through the sample, and the contrast is generated by the absorption of light in dense areas of the specimen.
Bright-field microscopy is the simplest of a range of techniques used for the illumination of samples in light microscopes, and its simplicity makes it a popular technique. The typical appearance of a bright-field microscopy image is a dark sample on a bright background, hence the name.
The name “brightfield” is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field. Simple light microscopes are sometimes referred to as brightfield microscopes.
Working Principle Bright Field Microscopy
Microscopy is used to visualize objects that are too small to see with the naked eye. In biology, this technique enables us to examine things like bacteria and cells at a magnification of up to 10000 times their original size.
In light microscopy, visible light is used to detect such small objects – with bright field microscopy being the most common form of light microscopy.
In bright field microscopy, a specimen is placed on the stage of the microscope and incandescent light from the microscope’s light source is aimed at a lens beneath the specimen.
Light travels from the source of illumination through the condenser, through the specimen, through the objective lens that magnifies the light and transmits it to an oracular lens or eyepiece and from their into the eye of the observer.
The light thus gets transmitted through the specimen and it appears against an illuminated background. The observer can see objects in the light path because some of the light is absorbed by stains, natural pigmentation, or dense areas of the sample and this contrast allows you to see the specimen.
They are just thick enough (but not too thick) to absorb a significant amount of light despite being colorless.
For good results with this microscopic technique, the microscope should have a light source that can provide intense illumination necessary at high magnifications and lower light levels for lower magnifications.
How Are microbes Illuminated With Bright Field Microscopy
A bright field microscope forms its image when light is transmitted through the specimen. The specimen, being denser and more opaque than its surroundings, absorbs some of this light, and the rest of the light is transmitted directly up through the ocular into the field. As a result, the specimen will produce an image that is darker than the surrounding brightly illuminated field.
For example, plant cells would appear darkest at the nucleus and central region where cellular matter is most dense, and lighter in the cytoplasm void of the ribosome, the endoplasmic reticulum, and other intracellular components. Animal cells are more difficult to image with this technique without staining of the sample, which ultimately kills live cells.
When to Choose Bright Field Microscopy
- BF gives the user structural information of the cell (membrane, cytoplasm, and nucleus), and structural and organizational information in tissue samples. The user will be able to see the overall shape of the cell and the organization of the cells within the tissue.
- BF is the most common technique used in pathology and histology.
- BF is mostly used for imaging fixed cells and tissues.
- If imaging live cells using BF, the user should use as low a light level as possible and image quickly to reduce stress on the live sample.
Uses and Advancements
To some extent, bright field microscopy is used in most disciplines requiring microscopic investigation. Because it is a simple method, this is the first type of microscopy students learn in schools.
The life sciences, particularly microbiology and bacteriology, have always relied on the brightfield technique.
This technique can be used to view fixed specimens or live cells. Since many organic specimens are transparent or opaque, staining is required to cause the contrast that allows them to be visible under the microscope.
Different stains and staining techniques are used depending upon the type of specimen and cell structure being examined.
For example:
- Fuchsin is used to stain smooth muscle cells
- Methylene blue is used to stain cell nuclei
- Gram stain is used on bacteria and gives rise to the name gram-negative or gram-positive bacteria based on the reaction of the bacteria to the stain. In fact, many scientific journals will not accept microbiological research for publication that is not supported by gram staining and bright field illumination methodology. Most routine medical microscopic examination of blood and tissue is performed using this illumination technique.
Different complementary techniques can be used to augment brightfield microscopy. By using a polarizing filter this illumination technique can be used in geological microscopic research and will reveal details not visible using white light.
Properly stained, microorganisms may be magnified to 1200x; utilizing an oil immersion objective will increase resolution at this high magnification.
Advantage
Bright field microscopy is very simple to use with fewer adjustments needed to be made to view specimens. Some specimens can be viewed without staining and the optics used in the brightfield technique don’t alter the color of the specimen.
It is adaptable with new technology and optional pieces of equipment can be implemented with bright field illumination to give versatility in the tasks it can perform.
Disadvantage
Certain disadvantages are inherent in any optical imaging technique.
- By using an aperture diaphragm for contrast, past a certain point, greater contrast adds distortion. However, employing an iris diaphragm will help compensate for this problem.
- Bright field microscopy can’t be used to observe living specimens of bacteria, although when using fixed specimens, bacteria have an optimum viewing magnification of 1000x.
- The limitations of bright-field microscopy include low contrast for weakly absorbing samples and low resolution due to the blurry appearance of out-of-focus material.
Bright field microscopy has very low contrast and most cells absolutely have to be stained to be seen; staining may introduce extraneous details into the specimen that should not be present.
Also, the user will need to be knowledgeable in proper staining techniques.
Lastly, this method requires a strong light source for high magnification applications and intense lighting can produce heat that will damage specimens or kill living microorganisms.
