Seven observation modes of microscope
Bright field microscopy is a familiar microscopic examination method, which is widely used in pathology, inspection, and observation of stained sections. All microscopes can perform this function.
2. Dark field DF
Darkfield is actually darkfield illumination. Its characteristics are different from those of bright field. It does not directly observe the light of illumination, but observes the light reflected or diffracted by the object under inspection. Therefore, the field of view becomes a dark background, while the object under inspection presents a bright image.
The principle of dark field is based on the Tyndall phenomenon in optics. When the dust is directly passed by strong light, the human eye cannot observe it, which is caused by the diffraction of strong light. If the light is cast obliquely on it, due to the reflection of the light, the particle seems to increase in size and is visible to the human eye.
A special accessory required for darkfield observation is a darkfield condenser. Its characteristic is that it does not allow the light beam to pass through the object from bottom to top, but changes the path of the light so that it shoots obliquely towards the object, so that the illuminating light does not directly enter the objective lens, and uses the reflection or diffraction light formed by the surface of the object Bright image. The resolution of dark field observation is much higher than that of bright field observation, up to 0.02—0.004
3.Phase contrast PH
During the development of optical microscopes, the successful invention of phase contrast microscopy 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 the light passes through, the wavelength and amplitude change little, and it is difficult to observe the specimen in bright field observation. .
The phase contrast microscope uses the optical path difference of the object to be inspected, that is, effectively uses the interference phenomenon of light to change the phase difference that cannot be resolved by the human eye into a resolvable amplitude difference, even for colorless and transparent substances. become clearly visible. This greatly facilitates the observation of living cells, so phase contrast microscopy is widely used in inverted microscopes.
The basic principle of the phase contrast microscope is to change the optical path difference of the visible light passing through the specimen into an amplitude difference, thereby improving the contrast between various structures and making various structures clearly visible. The light is refracted after passing through the specimen, deviates from the original optical path, and is delayed by 1/4λ (wavelength) at the same time. If it is increased or decreased by 1/4λ, the optical path difference becomes 1/2λ, and the two beams interfere after the optical axis Strengthen, increase or decrease the amplitude, improve the contrast. In terms of structure, phase contrast microscopes have two special features different from ordinary optical microscopes:
1. The annular diaphragm is located between the light source and the condenser, and its function is to make the light passing through the condenser form a hollow light cone and focus it on the specimen.
2. Annular phaseplate A phase plate coated with magnesium fluoride is added to the objective lens, which can delay the phase of direct light or diffracted light by 1/4λ. Divided into two types:
A phase plate: Delay the direct light by 1/4λ, add the light waves after the two groups of light waves combine, and increase the amplitude, the specimen structure is brighter than the surrounding medium, forming a bright contrast (or negative contrast).
Phase B plate: Delay the diffracted light by 1/4λ, the light waves of the two groups of light are subtracted after the axis is aligned, and the amplitude becomes smaller, forming a dark contrast (or positive contrast), and the structure is darker than the surrounding medium
Four. Differential interference contrast DIC
Differential interference microscopy appeared in the 1960s. It can not only observe colorless and transparent objects, but also show a three-dimensional sense of relief, and has some advantages that phase contrast microscopy cannot achieve. The observation effect is even better. lifelike.
principle;
Differential interference called microscopy is the use of a special Wollaston prism to split the light beam. The vibration directions of the split beams are perpendicular to each other and the intensity is equal, and the beams pass through the object at two points that are very close to each other, and there is a slight difference in phase. Since the split distance between the two light beams is extremely small, there is no double image phenomenon, so that the image presents a three-dimensional three-dimensional feeling.
