Five observation modes of microscope
1. Bright field BF
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 observation
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 slantly shoots towards the object to prevent the illuminating light from directly entering the objective lens. 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 microscopy
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 difference in the optical path 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 (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. Phase plate (annular phase plate) 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:
1. Phase A plate: Delay the direct light by 1/4λ, add the light waves after the combination of two sets of light waves, and increase the amplitude. The structure of the specimen becomes brighter than the surrounding medium, forming a bright contrast (or negative contrast).
2. B phase plate: delay the diffracted light by 1/4λ, after the two groups of light are aligned, the light waves are subtracted, and the amplitude becomes smaller, forming a dark contrast (or positive contrast), and the structure is darker than the surrounding medium.
4. Differential Interferometry Microscopy
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.
5. Polarizing microscope
Polarizing microscope is a kind of microscope to identify the optical properties of the fine structure of matter. All substances with birefringence can be clearly distinguished under a polarizing microscope. Of course, these substances can also be observed with dyed hair, but some are not possible, and a polarizing microscope must be used.
The characteristic of the polarizing microscope is the method of changing the ordinary to polarized light for microscope inspection to identify whether a certain substance is single refraction (isotropic) or birefringence (anisotropy).
Birefringence is a fundamental property of crystals. Therefore, polarizing microscopes are widely used in mineral, chemical and other fields. It also has applications in biology and botany.
