Principles and characteristics of dark field microscopy
1. The principle and structural characteristics of dark field microscope
The difference between a dark field microscope and a general bright field microscope lies in their different concentrators. A dark field microscope is equipped with a centrally shaded concentrator, which prevents light from passing through the concentrator and only allows it to be obliquely projected onto the specimen on the glass slide from the four edges of the concentrator and the unshielded areas. Due to the oblique nature of light, it cannot enter the objective lens, resulting in a dark field of view for observation. However, the light emitted by the condenser at an oblique angle onto the bacterial cells emits a bright light due to light scattering, which is reflected back into the objective lens. As a result, the bright object image in the dark field of view can be seen in the microscope.
2. Operation method
① Use a research dark field microscope or remove the condenser from a regular optical microscope and replace it with a dark field condenser.
② Whether using a dry objective lens or an oil immersed objective lens, a large drop of tar should be added to the upper lens of the condenser during mirror inspection.
③ Place the prepared hanging droplet specimen on the stage and raise the condenser to the top to allow the oil to come into contact with the slide.
④ Amplify the light source.
⑤ Adjust and focus the optical axis of the condenser. Use a 10 * objective lens to find the image of the object being inspected. Turn down the iris aperture of the condenser until the outline of the field of view aperture can be seen in the field of view. Then slowly adjust the condenser up and down to make the image of the field of view aperture clearer. If the field of view aperture is not in the center, use the two adjustment buttons on the outside of the condenser to adjust it. When the bright light point is in the center of the field of view, turn it up again to observe.
3. Precautions
① The numerical aperture of the objective lens used for dark field observation should be around 1.00-1.25. If it is too high, the effect may not be good. It is better to use an objective lens with a field stop and rotate the adjustment ring in the middle of the animal mirror to freely change the size of the numerical aperture.
② The required glass slides and cover slips must be scratch free and dust-free, and the front lens of the objective lens must also be clean and dust-free. The thickness of glass slides and cover slides should meet the standard. The glass slide is too thick, and the focus of the condenser will fall inside the slide, unable to reach the plane of the object being tested; When using an oil lens, due to the short working distance of the objective lens, it is even impossible to focus, resulting in the inability to see or clearly see the object being inspected.
③ During mirror inspection, the room should be dark. If such conditions are not available, shading devices should be used as much as possible to prevent light from entering around the eyepiece.
