Two Commonly Used Microscopic Observation Methods
1, Dark field observation
Dark field of view is actually dark field illumination Its characteristics are different from bright field of view, as it does not directly observe the illuminating light, but rather observes the reflected or diffracted light of the object being inspected Therefore, the field of view becomes a dark background, while the object being inspected presents a bright image
The principle of dark field is based on the optical Tyndall phenomenon, where dust particles cannot be observed by the human eye when exposed to strong light due to diffraction caused by strong light If the light is obliquely projected onto it, the particles appear to increase in volume due to the reflection of light, making them visible to the human eye
The special accessory required for dark field observation is a dark field spotlight Its characteristic is that it does not allow the beam of light to pass through the object from bottom to top, but changes the path of the light to be obliquely directed towards the object, so that the illumination light does not directly enter the objective lens, and uses the bright image formed by the reflection or diffraction light on the surface of the object being inspected The resolution of dark field observation is much higher than that of bright field observation, reaching up to 0.02-0.004
2, Phase contrast mirror inspection method
The successful invention of phase contrast microscopy in the development of optical microscopes is an important achievement in modern microscopy technology We know that the human eye can only distinguish the wavelength (color) and amplitude (brightness) of light waves. For colorless and transparent biological specimens, when light passes through, the wavelength and amplitude do not change much, making it difficult to observe the specimen in a bright field
The phase contrast microscope uses the difference in optical path length of the object being inspected for mirror inspection, effectively utilizing the interference phenomenon of light to transform the phase difference that cannot be distinguished by the human eye into a distinguishable amplitude difference. Even colorless and transparent substances can become clearly visible This greatly facilitates the observation of living cells, therefore phase contrast microscopy is widely used in inverted microscopes
The basic principle of phase contrast microscopy is to convert the optical path difference of visible light passing through the specimen into amplitude difference, thereby improving the contrast between various structures and making them clear and visible After passing through the specimen, the light undergoes refraction, deviating from the original optical path and being delayed by 1/4 λ (wavelength). If the optical path difference is increased or decreased by another 1/4 λ, the optical path difference becomes 1/2 λ, and the interference between the two light beams increases or decreases after the axis is combined, improving the contrast In terms of structure, phase contrast microscopes have two
special differences from ordinary optical microscopes:
1. An annular diaphragm is located between the light source and the condenser, and its function is to form a hollow cone of light that passes through the condenser and focuses on the specimen
2. Angular phase plate: A phase plate coated with magnesium fluoride is added to the objective lens, which can delay the phase of direct or diffracted light by 1/4 λ. It can be divided into two types:
(1) . A+phase plate: Delay the direct light by 1/4 λ, and add the two sets of light waves after combining the axes. The amplitude increases, and the specimen structure becomes brighter than the surrounding medium, forming a bright contrast (or negative contrast)
(2) . B+phase plate: Delay the diffracted light by 1/4 λ, and subtract the light waves after combining the axes of two sets of light rays, resulting in a decrease in amplitude and forming a dark contrast (or positive contrast). The structure becomes darker than the surrounding medium
