Principle and structure of fluorescence microscope
发表时间:2019-11-29     阅读次数:     字体:【

The fluorescence microscope uses a point light source with high luminous efficiency, and emits light of a certain wavelength (such as 3650 ultraviolet light or 4200 ultraviolet light) through the color filter system as excitation light, and the fluorescent substance in the excitation sample emits various colors. After fluorescence, it is observed through the magnification of the objective lens and eyepiece. In this way, under a strong contrasting background, it is easy to recognize even if the fluorescence is very weak, and it is highly sensitive. It is mainly used for the study of cell structure and function, and chemical composition. The basic structure of a fluorescence microscope is composed of an ordinary optical microscope plus some accessories (such as a fluorescent light source, an excitation filter, a two-color beam splitter, and a blocking filter, etc.)

Fluorescent light source-Usually uses ultra-high pressure mercury lamps (50-200W), which can emit light of various wavelengths, but each fluorescent substance has an excitation light wavelength that produces the strongest fluorescence, so an excitation filter ( Generally, there are ultraviolet, purple, blue and green excitation filters), which only allows excitation light of a certain wavelength to be irradiated onto the specimen, while absorbing other light. After being irradiated with excitation light, each substance emits visible fluorescence with a longer wavelength than the irradiation wavelength in a very short time. Fluorescence is specific and generally weaker than the excitation light. To observe the specific fluorescence, a blocking (or suppression) filter should be added behind the objective lens. It has two functions: one is to absorb and block the excitation light from entering the eyepiece, so as not to disturb the fluorescence and damage the eyes, and the other is to select and allow specific fluorescence to pass through, showing a specific fluorescence color. The two filters must be used together. There are two types of fluorescence microscopes in terms of their optical path:

1. Transmission fluorescence microscope:

The excitation light source excites the fluorescent material through the specimen material through the condenser. Commonly used dark-field light collectors can also be used ordinary light collectors, adjust the reflector to make the excitation light be transmitted and bypassed to the specimen. This is a relatively old fluorescence microscope. The advantage is that the fluorescence is strong at low magnification, and the disadvantage is that the fluorescence decreases with increasing magnification. So it is better for observing larger specimen materials.

2. Epi-fluorescence microscope

This is a new type of fluorescence microscope developed in modern times. The difference from the above is that the excitation light falls from the objective lens to the surface of the specimen. That is, the same objective lens is used as the illumination condenser and the objective lens for collecting fluorescence. A two-color beam splitter needs to be added to the optical path, which is 45 degrees to the optical uranium. At the angle, the excitation light is reflected into the objective lens and concentrated on the sample. The fluorescence generated by the sample and the excitation light reflected by the objective lens surface and the cover glass surface enter the objective lens at the same time, and return to the two-color beam splitter to make the excitation light Separated from fluorescence, the residual excitation light is then absorbed by the blocking filter. For example, the combination of different excitation filter / dual-color beam separator / blocking filter can meet the needs of different fluorescent reaction products. The advantage of this type of fluorescence microscope is that the illumination of the field of view is uniform, the imaging is clear, and the greater the magnification, the stronger the fluorescence.

(2) How to use the fluorescence microscope

1. Turn on the light source, the ultra-high pressure mercury lamp needs to be preheated for a few minutes to reach the most bright spot.

2. Transmission fluorescence microscope needs to install the required excitation filter between the lamp source and the condenser, and install the corresponding blocking filter behind the objective lens. Epi-fluorescence microscopes need to insert the required excitation filter / two-color beam splitter / blocking filter insert into the slot of the optical path.

3. Observe with a low magnification lens, adjust the center of the light source according to the adjustment device of different types of fluorescence microscopes, so that it is located in the center of the entire illumination spot.

4. Place the specimen film and observe after focusing. During use, please pay attention: do not directly observe the final filter, so as not to cause eye damage; when observing the specimen with an oil lens, a special non-fluorescent oil lens must be used; after the high-pressure mercury lamp is turned off, it cannot be reopened immediately. Restart after 5 minutes, otherwise it will be unstable and affect the life of mercury lamp.

(3) Observing the blue-violet light filter under the fluorescence microscope on the teaching stand, it can be seen that the cells stained with o.01% acridine orange fluorescent dye, the nucleus and cytoplasm are excited to produce two different colors of fluorescence (dark Green and orange-red).

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