Microscopy classification and working principle for cell research
The microscope is the primary tool for observing cells. According to different light sources, it can be divided into two categories: optical microscopes and electron microscopes. The former uses visible light (UV microscopes use ultraviolet light) as the light source, while the latter uses electron beams as the light source.
-,Optical microscope
(1) Ordinary optical microscope
Ordinary biological microscopes are composed of three parts, namely: ① illumination system, including light source and condenser; ② optical magnification system, composed of objective lens and eyepiece, which is the main body of the microscope. In order to eliminate spherical aberration and chromatic aberration, both the eyepiece and the objective ③Mechanical device, used to fix the material and facilitate observation (Figure 2-1).
Whether the image of the microscope is clear is not only determined by the magnification, but also related to the resolution of the microscope. The resolution refers to the ability of the microscope (or the human eye to be 25cm away from the target) to distinguish the smallest distance between objects. The size of the resolution is determined by The wavelength and aperture ratio of light and the refractive index of the medium are expressed by the formula:
In the formula: n = refractive index of medium; α = aperture angle (the opening angle of the specimen to the objective lens aperture), N.A. = lens aperture (numeric aperture). The lens angle is always less than 180°, so the maximum value of sina/2 must be less than 1.
The refractive index of the glass used to make the optical lens is 1.65 to 1.78, and the refractive index of the medium used is closer to the glass, the better. For dry objective lenses, the medium is air, and the lens aperture ratio is generally 0.05 to 0.95; for oil lenses, cedar oil is used as the medium, and the lens aperture ratio can be close to 1.5.
The wavelength of ordinary light is 400-700nm, so the resolution value of the microscope will not be less than 0.2μm, and the resolution of the human eye is 0.2mm, so the maximum magnification of the general microscope design is usually 1000X.
(2) Fluorescence microscopy
Some substances in cells, such as chlorophyll, can fluoresce after being irradiated by ultraviolet rays; some substances themselves cannot fluoresce, but if they are stained with fluorescent dyes or fluorescent antibodies, they can also fluoresce when irradiated by ultraviolet rays, and the fluorescence microscope (Fig. 2-2, 3, 4) is one of the tools for qualitative and quantitative research on such substances.
Fluorescence microscopes and ordinary microscopes have the following differences:
1. The illumination method is usually epi-illumination, that is, the light source is projected on the sample through the objective lens (Figure 2-3);
2. The light source is ultraviolet light, the wavelength is shorter, and the resolution is higher than that of ordinary microscopes;
3. There are two special filters, the one in front of the light source is used to filter out visible light, and the one between the eyepiece and the objective lens is used to filter out ultraviolet light to protect the eyes.
(3) Laser scanning confocal microscope
Laser confocal scanning microscope (laser confocal scanning microscope, Figure 2-5, 6) uses laser as scanning light source, and scans imaging point by point, line by line, surface by surface, and the scanning laser and fluorescence collection share an objective lens, and the focus of the objective lens is The focal point of the scanning laser is also the object point of instantaneous imaging. Because the wavelength of the laser beam is short and the beam is very thin, the confocal laser scanning microscope has a higher resolution, which is about 3 times that of an ordinary optical microscope. The system is focused once and the scan is limited to one plane of the sample. When the focusing depth is different, images of different depth levels of the sample can be obtained. These image information are stored in the computer. Through computer analysis and simulation, the three-dimensional structure of the cell sample can be displayed.
Confocal laser scanning microscopy can be used not only to observe cell morphology, but also for quantitative analysis of intracellular biochemical components, optical density statistics and measurement of cell morphology.
(4) Dark field microscope
The dark field microscope (dark field microscope, Figure 2-7) has a light sheet in the center of the condenser, so that the illumination light does not directly enter the human lens, and only the light reflected and diffracted by the specimen is allowed to enter the objective lens, so the background of the field of view is black, The edges of objects are bright. Using this microscope, particles as small as 4-200nm can be seen, and the resolution can be 50 times higher than that of ordinary microscopes.
(5) Phase contrast microscope
Phasecontrast microscope (phasecontrast microscope, Figure 2-8, 9) by P. Zernike was invented in 1932 and won the Nobel Prize in Physics in 1953 for it. The biggest feature of this microscope is that it can observe unstained specimens and living cells.
The basic principle of phase contrast microscopy 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. After passing through the specimen, the light is refracted, deviated from the original optical path, and delayed by 1/4λ (wavelength). Strengthen, increase or decrease the amplitude, increase the contrast. In terms of structure, phase contrast microscopes have two special features that are 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 on the specimen.
2. The phase plate (annular phaseplate) adds a phase plate coated with magnesium fluoride in the objective lens, which can delay the phase of direct light or diffracted light by 1/4λ. There are two types:
①A+ phase plate: The direct light is delayed by 1/4λ. After the two groups of light waves are combined, the light waves are added together, and the amplitude is increased. The structure of the specimen is brighter than the surrounding medium, forming a bright contrast (or negative contrast).
② B+ phase plate: The diffracted light is delayed by 1/4λ. After the two sets of rays are combined, 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.
(6) Polarizing microscope
Polarizing microscope is used to detect substances with birefringence, such as filaments, spindles, collagen, chromosomes and so on. The difference from ordinary microscopes is that there is a polarizer (polarizer) in front of the light source, so that the light entering the microscope is polarized light, and there is an analyzer (a polarizer whose polarization direction is perpendicular to the polarizer) in the lens barrel. The stage of this microscope can be rotated. When a single-refractive substance is placed on the stage, no matter how the stage is rotated, since the two polarizers are vertical, no light can be seen in the microscope, and the light is not visible in the microscope. When entering birefringent materials, the rotating stage can detect such objects because light is deflected as it passes through such materials.
