Principles, characteristics, and use of fluorescence microscopy
The principle and structural characteristics of a fluorescence microscope: A fluorescence microscope uses a high luminous efficiency point light source, which emits a certain wavelength of light (such as ultraviolet light 3650 in or ultraviolet blue light 4200 in) through a color filtering system as an excitation light. The fluorescence substance in the sample is excited to emit various colors of fluorescence, and then observed through magnification of the objective and eyepiece. In this way, even with weak fluorescence, it is easily recognizable and highly sensitive under strong contrasting backgrounds, mainly used for studying cell structure and function, as well as chemical composition. The basic structure of a fluorescence microscope is composed of an ordinary optical microscope and some accessories (such as a fluorescent light source, excitation filter, bichromatic beam separator, and blocking filter, etc.). Fluorescent light sources - usually 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 (usually ultraviolet, purple, blue, and green excitation filters) need to be added to allow only a certain wavelength of excitation light to pass through and illuminate the specimen, while absorbing all other light. After being irradiated by excited light, each substance emits visible fluorescence with a longer wavelength than the irradiation in a very short period of time. Fluorescence has specificity and is generally weaker than excitation light. In order to observe specific fluorescence, a blocking (or suppressing) filter needs to be added behind the objective lens.
It has two functions: firstly, it absorbs and blocks excitation light from entering the eyepiece to avoid disturbing fluorescence and damaging the eyes; secondly, it selects and allows specific fluorescence to pass through, displaying a specific fluorescence color. Both types of filters must be selected for use together.
