The optical system of fluorescence microscopy must perform two main functions:
(1) To excite the fluorescent reactant in the sample effectively, the emission wavelength Zui is close to its absorption peak.
(2) Must collect as much fluorescence as possible in order to make the visibility Zui in the microscope appropriate.
The imaging quality of fluorescent optical system depends mainly on the contrast and brightness of the image. The contrast of the image is determined by the ratio of the fluorescence emitted from the sample to the light observed in the background. Background light includes spurious excitation light through a filter blocking the excitation light, spontaneous fluorescence of sample tissue composition and spontaneous fluorescence and stray light of optical system. In fluorescence microscopy, all efforts should be made to obtain good image contrast of Zui while maintaining sufficient brightness of image, which are often contradictory. For example, very narrow band filters only use very little light energy to excite, and the background is dark but the fluorescence brightness is weak. In the application, we can only take into account the reasonable trade-offs to decide the choice.
The optical system of fluorescence microscopy should give priority to:
1. Material caused by optical system should be selected as non-fluorescent material in the excitation light wave. It means not only the glass of optical system but also the cementing agent, slice, cover glass, dipping solution and sealant in the gluing manufacture. When there is spontaneous fluorescence, the background light is added to the quilt, and sometimes the fluorescence image is disturbed.
2. In the parameter selection of optical system, the Zui large light energy should be taken into account.
For example, in the illumination system, the collector should choose Zui large inclusive angle, Kohler illumination and filling the pupil with filament or arc image, in order to achieve uniform illumination rate.
For example, the objective for collecting fluorescence imaging should have a larger value as a fluorescence objective because the illumination of the image plane is proportional to the numerical aperture.
If the binocular barrel is used as fluorescence, it should have a higher transmittance in the visible spectral band. The coating technology can greatly reduce the loss of light energy. It can double the transmittance of the general binocular barrel, which is necessary for the observation of weak fluorescent substances.
3. The spectral characteristics of the components of the fluorescent optical system are also very important. In particular, the transmission coefficients of the violet region should be paid attention to when ultraviolet light passes through the components such as lighting system, concentrator and objective lens. For example, the material of concentrator should be made of ultraviolet radiation. For example, when the reflecting mirror turns the light path in the lighting system, the surface of aluminium plating is better than that of silver plating, because the reflectivity of aluminium is more than 90%, while that of silver is only 70%.