Imaging methods for bright and dark fields of fluorescence microscopy
(1) Bright field imaging (BFI), which only allows the transmitted electrons in the paraxial region to pass through the objective aperture, forming a dark image on a bright background. The smaller the aperture of the objective lens, the greater the contrast of the bright field image; (2) Dark field imaging (DFI), which only allows a portion of a large angle scattered beam or a diffraction beam of a crystal to pass through the objective lens aperture, while blocking the transmitted beam. This creates a bright graphic image on a dark background. This dark field imaging method can improve the contrast of images and is an important imaging method.
There are roughly four methods for fluorescence microscopy to achieve dark field imaging: (1) maintaining vertical illumination along the optical axis while moving the objective aperture; (2) The use of oblique illumination to obtain scattered beams in the optical axis direction is called Center Dark Field Imaging (CDFI); (3) An objective lens aperture with a central beam blocking and a circular transparent area; (4) Use a circular transparent spotlight to detect light and an objective aperture with a central circular hole. The * methods here are simple and convenient, but they utilize electronic imaging in the far sleeve region, resulting in large aberrations and poor image quality. The second method can avoid the above drawbacks, but the objective aperture of the Leica fluorescence microscope only receives a small portion of scattered diffracted electrons, resulting in lower efficiency. The side of the aperture is often bombarded by a large number of electrons, which can easily cause asymmetric pollution and affect image quality. The circular objective has been improved in this regard, but the disadvantage is that it is difficult to make and it is difficult to achieve complete axial symmetry. Given the relatively relaxed requirements for the lighting system, the latter solution can be adopted, which involves adding a circular aperture at the condenser to provide hollow illumination for the sample. The effect of this device. Its image resolution can be close to that of bright field images, while the contrast is greatly improved.
