The bright-field microscopy produces an image with a coloured specimen having lightened background. Generally, the microorganisms do not absorb light but after staining, the organisms get the ability to absorb the light.
The optical lens, i.e. condenser lens and the path of light coming from the light source illuminator produce a bright-field image with higher contrast and magnification.
Bright-field microscope can produce a magnified image about 1,000 X to 2,000 X, and the image magnified more than the 2,000 X will become unclear or fuzzy.
Content: Bright Field Microscopy
- Definition of Bright Field Microscopy
- Steps of Bright Field Microscopy
- For Better Results
Definition of Bright Field Microscopy
Bright field microscopy can define as the optical microscopy, which is the simplest of all the illumination techniques, wherein a smear, the stained or the dense part appear darker with a white or brighter background.
It is a type of light microscopy, where a path of light is very simple, which requires a light source like a halogen lamp, condenser lens, objective lens and ocular lens. Bright-field microscopy can use either critical or Koehler magnification or illumination system to add contrast to the image.
- Critical illumination: It was the old method where an image formed by the condenser, but gives uneven illumination.
- Koehler illumination: The image formed by this requires addition optic systems like condenser diaphragm, a condenser lens, collector diaphragm and collector lens. It gives an even illumination.
It is the power of the optical lens, which distinguishes between the two particular bodies that held very close to each other. The highly magnified image will not give better results as the picture becomes gauzy.
The resolving power increases, as the lines per unit area, appears as distinctive lines. When there is a small distance between the two distinct objects, the resolving power can be best known.
The resolution power can give 1000-1500 times magnified image. Resolving power of the microscope decides the quality of the picture by the objective lens. Higher is the magnification power, higher will be the resolution of the microscope.
The resolution of the bright field microscope is represented as ‘r’ which is equal to the half of the light wavelength by the numerical aperture. Thus, the resolution of the bright field microscope depends upon the two factors:
- Numerical aperture
- Wavelength of light
The numerical aperture also refers to as “Object side aperture” which is equal to the product of refractive index ‘n’ and the magnitude of the angular aperture represented as ‘sinƟ’.
Steps of Bright Field Microscopy
To visualize the specimen, a bright field microscope includes the following steps:
Preparation of smear
Firstly, the smear is prepared on a glass slide by mixing the inoculum with a drop of distilled water. A sample is then heat fixed and followed by staining, the specimen is stained.
For the identification of the bacteria, one can perform gram staining, and for the identification of the fungus, lactophenol cotton blue stain is widely used. Therefore, when the specimen is placed on the glass slide, the process will refer to as “Mounting of specimen”.
Optimization of light
After air drying of the glass slide, add oil immersion for the better resolution. Then place a slide on the stage of a light microscope and adjust the slide by the stage clips. Observe the specimen through the eyepiece, whether the organism of desire is visible or not.
Adjustment of the Condenser lens
To visualize the magnified image of the specimen first, adjust the condenser lens of the light microscope. A condenser lens plays a significant role in transferring the incident light from the illuminator to the specimen. The condenser lens must be near the specimen.
Focus the specimen
To get the clear picture of the specimen, focus the object or specimen through the “Iris diaphragm” which play a pivotal role to control the diameter of the light coming from the condenser to the specimen.
When the diaphragm is nearly close to the condenser lens, it adds contrast to the specimen. When the diaphragm is farther apart from the condenser lens, it adds brightness to the specimen.
The positioning of the specimen
After focusing, locate the specimen prior to the eyepiece via stage control. The stage control consists of coarse and fine adjustment knobs, which help in the movement of the stage to move up, down, left and right directions. The coarse and fine knobs also sharpen the image.
Adjust the separation between an eyepiece and objective lens
The distance between the eyepiece and the objective lens is the separation distance, which is adjusted to view the image.
Selection of objective lens
Select one type of objective lens which can give a best-magnified image via objective revolver. The objective revolver holds three or four types of objective lens with different magnification power like 10x, 40x, 100x etc.
Adjust the Illuminator
Bright field microscopy is a technique used in the light microscope which gives a magnified image of the dark specimen with the colourless background. To accomplish the bright field microscopy, transfer the glass slide having a stained specimen onto the microscope stage.
The luminous light will come through the source of light or we can say through a source of illumination. The light coming from the illuminator is aimed at the condenser lens, which is present beneath the specimen. The aperture diaphragm helps to focus and control the light source coming from the light source illuminator on the specimen.
Some of the light will reflect out of the specimen, which is then collected by the objective lens. The objective lens first magnifies the light and then transmits it to the ocular lens. During this whole method, some of the light will get deflected, and some will get absorbed by the stain and the dense areas in the sample.
The intense illumination and can increase the magnification of the image. Therefore, in bright field microscopy, those which have absorbed the part of the light will appear darker and the remaining, i.e. the background will appear brighter.
- Bright field microscopy is a simple method to perform.
- It can easily produce a magnified image of the fixed specimens and live cells.
- The bright field microscopy produces low contrast to the image.
- It can give magnification only up to 1300 X.
- Bright field microscopy has a low apparent optical resolution.
For Better Results
For the better results, we can adjust the iris diaphragm, which can reduce or increase the amount of light source coming to the specimen. The use of oil immersion can improve the resolution of the image by using an objective lens of power 100X. The use of staining methods adds contrast to the picture. One can also use a coloured or polarizing filter on the light source to highlight the features of the mineral samples.