Fluorescence microscope principle application and use
(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 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 is passed through the specimen material through a condenser lens to excite fluorescence. 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 redirected and bypassed to the specimen. This is an older fluorescent microscope. The advantage is that the fluorescence is strong at low magnification, but the disadvantage is that the fluorescence decreases with the increase of magnification. Therefore, it is better to observe larger specimen materials.
2. Epi-fluorescence microscope 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. Such as changing to a combination of different excitation filter/two-color beam splitter/blocking filter, it can meet the needs of different fluorescent reaction products. 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, and the ultra-high pressure mercury lamp needs to warm up for a few minutes to reach the brightest point.
2. The transmission fluorescence microscope needs to install the required excitation filter between the light source and the condenser, and install the corresponding blocking filter 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 so that it is located in the center of the entire illumination spot according to the adjustment device of different models of fluorescence microscopes.
4. Place the specimen piece and observe after focusing. Attention should be paid during use: do not observe directly with the end filter, so as not to cause eye damage; when observing specimens 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, 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) Observe the cells stained with 0.01% acridine orange fluorescent dye under the fluorescent microscope on the teaching platform with a blue-violet light filter. The nucleus and cytoplasm are excited to produce two different colors of fluorescence (dark green and orange-red).
