Release date: 2019/7/11 10:30:30 　Click volume: 1100

Fluorescence microscopy uses a point source with high luminous efficiency to emit a certain wavelength of light (such as ultraviolet light 3650 into or violet blue 4200) as excitation light, and the fluorescent substance in the excitation sample emits various colors. After fluorescence, it was observed by magnification of the objective lens and the eyepiece. In this way, in the strong contrast background, even if the fluorescence is very weak, it is easy to recognize and has high sensitivity, and is mainly used for research on cell structure and function as well as chemical composition. The basic structure of a fluorescence microscope consists of a common optical microscope plus some accessories (such as a fluorescent light source, an excitation filter, a two-color beam splitter, and a blocking filter). Fluorescent light source - generally uses ultra-high pressure mercury lamp (50-200W), which can emit light of various wavelengths, but each fluorescent substance has a wavelength of excitation light that produces the strongest fluorescence, so it is necessary to add an excitation filter ( Generally, there are ultraviolet, purple, blue, and green excitation filters), and only a certain wavelength of excitation light is transmitted through the irradiated specimen, and other light is absorbed. After each substance is irradiated with excitation light, it emits visible fluorescence longer than the irradiation wavelength in a very short time. Fluorescence is specific and generally weaker than the excitation light. In order to observe the specific fluorescence, a blocking (or suppression) filter is added after the objective lens. It has two functions: one is to absorb and block the excitation light into the eyepiece to avoid disturbing the fluorescence and damage the eye, and the other is to select and allow the specific fluorescence to pass through, showing a specific fluorescent color. Both filters must be selected for use.

Fluorescence microscopy has two types of light paths:

1. Transmissive fluorescence microscope: The excitation source is excited by the concentrating mirror through the specimen material. Commonly used dark field concentrators, can also use ordinary concentrators, adjust the mirror to make the excitation light and side-by-side 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 is weakened as the magnification is increased. Therefore, it is better for the observation of the larger specimen material.

2. Epi-fluorescence microscope This is a new type of fluorescence microscope developed in modern times. The difference is that the excitation light is emitted from the objective lens to the surface of the specimen, that is, the same objective lens is used as the illumination concentrator and the objective lens for collecting fluorescence. A two-color beam splitter is added to the light path, which is 45 with the uranium. The excitation light is reflected into the objective lens and is collected on the sample. The fluorescence generated by the sample and the excitation light reflected by the surface of the objective lens and the surface of the cover glass enter the objective lens at the same time, and return to the two-color beam splitter to make the excitation light. Separated from the fluorescence, the residual excitation light is then absorbed by the blocking filter. For example, a combination of different excitation filter/two-color beam splitter/blocking filter inserts can meet the needs of different fluorescent reaction products. The advantage of such a fluorescence microscope is that the field of view illumination is uniform, the imaging is clear, and the larger the magnification, the stronger the fluorescence.