The following points highlight the three main principles of photomicrography. The principles are: 1. Basic Principle 2. Microscope Configuration for Photomicrography 3. Taking the Photomicrograph.
Principle # 1. Basic Principle:
The basic principle of photomicrography involves the use of classical microscopy techniques of bright field and cross polarized illumination. Most microscopes used in biological laboratories are of transmitted light variety and operate in the bright field mode. Placing a polarizing element into the light path restricts the passage of light thus reducing the amount of transmitted light to apprimately 30% of the emitted value.
To obtain cross polarized illumination from bright field microscope, two individual polarizing elements (one is called polarizer and the other one analyser) are inserted into the light path with their vector proportion planes crossed at a 90° angle with respect to each other.
When a sample is placed in the light path between the polarizer and the analyser (i.e., crossed polarizers), the only light emitted will be that which is refracted by the sample until it can pass unimpeded through the analyser.
Principle # 2. Microscope Configuration for Photomicrography:
The commonly used bright field microscope in which an external light source is reflected into the sub-stage condenser through a mirror, can easily be converted for use with polarizing elements (Fig. 15.6).
The polarizer responsible for polarizing the light is taped onto the bottom of the condenser. The analyser (the second polarizer) is inserted inside the body of the microscope between the main body tube and the eyepiece tube.
Attaching a camera to the microscope is the last step. Microscope viewing heads come in three varieties: monocular (one eyepiece), binocular (two eyepieces) and trinocular (two eyepieces and a photography lube).
A camera can be adapted to each of these viewing heads. A simple camera back will be sufficient for photomicrography as the camera is needed only to store, expose, and advance the film. The microscope itself acts as a camera lens.
Principle # 3. Taking the Photomicrograph:
After both the polarizer and the analyser are in place and the camera is fitted on the microscope, the specimen is placed on the stage and is viewed directly into the eyepiece. The image of the specimen is brought into focus and the polarizer is rotated until the view-field becomes very dark (maximum extinction).
At this point, the polarization direction is perpendicular between the polarizer and analyser i.e. they are in the position of cross polarizers as described above. This results in cross polarized illumination which is needed for photomicrography. Now the photomicrograph of the specimen is taken with the help of camera already adapted to viewing head of the microscope.