Principle and structural characteristics of fluorescence microscope
The fluorescence microscope uses a point with high luminous efficiency to emit light with a certain wavelength (such as ultraviolet light 3650 or violet-blue light 4200) through the color filter system as excitation light, and then excites the fluorescent substances in the sample to emit fluorescence with different colors, and then observes them through the amplification of the objective lens and eyepiece. In this way, under the 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 study of cell structure and function and chemical composition. The basic structure of fluorescence microscope is composed of ordinary optical microscope and some accessories (such as fluorescent light source, excitation filter, dichroic beam splitter and blocking filter, etc.). Fluorescent light source —— Ultra-high-pressure mercury lamp (50-200W) is generally used, which can emit light with various wavelengths, but each fluorescent substance has an excitation light wavelength that produces the strongest fluorescence, so it is necessary to add excitation filters (generally ultraviolet, purple, blue and green excitation filters), so that only a certain wavelength of excitation light is transmitted to the specimen, and all other light is absorbed. After each substance is irradiated by excitation light, it emits visible fluorescence longer than the irradiation wavelength in a very short time. Fluorescence has specificity, which is generally weaker than excitation light. In order to observe the specific fluorescence, it is necessary to block (or suppress) behind the objective lens and must be used together.
Difference between fluorescence microscope and common microscope
1. The illumination mode is usually episodic, that is, the light source is projected on the sample through the objective lens;
2. The light source is ultraviolet light with short wavelength and higher resolution than ordinary microscope;
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 people's eyes.
Fluorescence microscope is also a kind of optical microscope, the main difference is that their excitation wavelengths are different. This determines the difference between fluorescence microscope and ordinary optical microscope in structure and usage.
Fluorescence microscope is the basic tool of immunofluorescence cytochemistry. It is composed of light source, filter plate system and optical system. It uses light with a certain wavelength to excite the specimen to emit fluorescence, and magnifies it through the objective lens and eyepiece system to observe the fluorescence image of the specimen.
