Principle and Application of Fluorescence Microscopy
(1) The principle and structural characteristics of the fluorescence microscope: the fluorescence microscope uses a point light source with high luminous efficiency to emit light of a certain wavelength (such as ultraviolet light 3650 in or purple blue light 4200 in) through the filter system as the excitation light to excite the specimen. After the fluorescent substance inside emits fluorescence of various colors, it is observed through the magnification of the objective lens and eyepiece. In this way, under a strong contrast background, even if the fluorescence is very weak, it is easy to identify and has high sensitivity. It is mainly used for the research 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 - generally use ultra-high pressure mercury lamp (50-200W), which can emit light of various wavelengths, but each fluorescent substance has an excitation wavelength that produces the strongest fluorescence, so it is necessary to add an excitation filter ( Generally, there are ultraviolet, purple, blue and green excitation filters), which only allow excitation light of a certain wavelength to pass through and irradiate the specimen, while absorbing other light. After each substance is irradiated by excitation light, it emits visible fluorescence with a longer wavelength than the irradiation wavelength in a very short time. Fluorescence is specific and generally weaker than excitation light. In order to observe specific fluorescence, a blocking (or suppressing) filter is required 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; the other is to select and let the specific fluorescence pass through, showing a specific fluorescent color. The two filters must be used together.
There are two types of fluorescence microscopes in terms of their optical paths:
1. Transmission fluorescence microscope: The excitation light source excites fluorescence through the specimen material through the condenser lens. A dark field collector is commonly used, and an ordinary collector can also be used to adjust the mirror so that the excitation light is transmitted and bypassed to the specimen. This is an old-fashioned fluorescence microscope. The advantage is that the fluorescence is strong at low magnification, and the disadvantage is that the fluorescence decreases with the increase of magnification. Therefore, 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 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 dichroic beam splitter needs to be added in the light path, which is 45° away from the light uranium. The excitation light is reflected into the objective lens and collected on the sample. The fluorescence generated by the sample and the excitation light reflected by the lens surface of the objective lens 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, residual excitation light is absorbed by blocking filters. If you change to a combination of different excitation filters/two-color beam splitters/blocking filters, the needs of different fluorescent reaction products can be met. The advantage of this kind 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 warm up for a few minutes to reach the brightest point.
2. For the transmission fluorescence microscope, the required excitation filter should be installed between the light source and the condenser, and the corresponding blocking filter should be installed behind the objective lens. Epi-fluorescence microscopes need to insert the required excitation filter/dual color beam splitter/blocking filter inserts into the slots in the light path.
3. Observe with a low-magnification lens, and 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 sheet and observe after focusing. Attention should be paid during use: Do not observe directly with the end filter, so as not to cause damage to the eyes; when observing the specimen with an oil lens, a special oil lens without fluorescence must be used; after the high-pressure mercury lamp is turned off, it cannot be turned on again immediately, and it needs to be tested. It can be restarted after 5 minutes, otherwise it will be unstable and affect the life of the mercury lamp.
(3) Observation Using a blue-violet light filter under a fluorescent microscope on the teaching platform, 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).
