The principle and structural characteristics of fluorescence microscopy
Fluorescence microscopy uses a point with high luminous efficiency to emit a certain wavelength of light (such as ultraviolet light 3650 in or violet blue light 4200 in) through a color filtering system as excitation light, and excites the fluorescent substances in the sample to emit various colors of fluorescence. After that, it is observed through magnification of the objective lens and eyepiece. In this way, it is easy to recognize even when the fluorescence is very weak against a strong background, with high sensitivity. It is mainly used for the study of cell structure and function, as well as chemical composition. The basic structure of a fluorescence microscope is composed of an ordinary optical microscope plus accessories such as a fluorescent light source, excitation filter, dichroic beam separator, and blocking filter. Fluorescent light sources generally use ultra high pressure mercury lamps (50-200W), which can emit light of various wavelengths. However, each fluorescent substance has an excitation light wavelength that produces the strongest fluorescence, so excitation filters (typically ultraviolet, purple, blue, and green excitation filters) need to be added to allow only a certain wavelength of excitation light to penetrate and irradiate the specimen, while absorbing all other light. After being irradiated by excitation light, each substance emits visible fluorescence longer than the irradiation wavelength in a very short time. Fluorescence has specificity and is generally weaker than excitation light. In order to observe specific fluorescence, it is necessary to add blocking (or suppression) behind the objective lens and use it in combination.
Difference between fluorescence microscope and ordinary microscope
1. The lighting method is usually a falling beam type, in which the light source is projected onto the sample through an objective lens;
2.The light source is ultraviolet light, with a shorter wavelength and higher resolution than 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 rays to protect the human eye.
Fluorescence microscopy is also a type of optical microscopy, with the main difference being the different excitation wavelengths. This determines the differences in structure and usage between fluorescent microscopy and ordinary optical microscopy.
Fluorescence microscopy is a basic tool for immunofluorescence cytochemistry. It is composed of main components such as a light source, a filter plate system, and an optical system. It uses a certain wavelength of light to excite the sample to emit fluorescence, and magnifies it through an objective lens and eyepiece system to observe the fluorescence image of the sample.
