Principles and structural features of fluorescence microscopy
Fluorescence microscope is the use of a high luminous efficiency of the point light source, through the colour filtering system to emit a certain wavelength of light (such as ultraviolet 3650 or violet-blue 4200) as the excitation light, the excitation of fluorescent material within the specimen emits a variety of different colours of fluorescence, and then magnified through the objective and eyepieces for observation. In this way, in the strong contrast background, even if the fluorescence is very weak, it is easy to identify, high sensitivity, mainly used for the study 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 fluorescence light source, excitation filters, dual-colour beam separator and blocking filters, etc.). Fluorescent light source - generally used ultrahigh-pressure mercury lamp (50-200W), which can emit a variety of wavelengths of light, but each fluorescent material has a strongest fluorescence excitation wavelength, so the need to add excitation filters (generally ultraviolet, violet, blue and green excitation filter), so that only a certain wavelength of excitation light through the irradiation of specimens, while the other light is absorbed. So it is necessary to add an excitation filter (usually ultraviolet, violet, blue and green excitation filters), so that only a certain wavelength of excitation light through the specimen, while other light is absorbed. When each substance is irradiated with excitation light, it emits visible fluorescence of a longer wavelength than that of the irradiated light within a very short time. Fluorescence has a specificity, generally weaker than the excitation light, in order to be able to observe the specific fluorescence, in the objective lens behind the need to add a blocking (or suppression) filter.
Its role is twofold:
One is to absorb and block the excitation light into the eyepiece, so as not to disturb the fluorescence and eye damage.
The second is to select and let the specific fluorescence through, showing a specific fluorescent colour. The two types of filters must be selected and used in conjunction.
