The optical system of fluorescence microscope must fulfill two main functions:
(1) To effectively excite the fluorescent reactant in the sample, the emission wavelength Zui is close to its absorption peak
(2) It is necessary to collect as many fluorescence as possible so that the visibility Zui in the microscope is appropriate.
The image quality of the fluorescence optical system mainly depends on the contrast and brightness of the image. The contrast of the image is determined by the ratio of the fluorescence excited in the sample to the light observed in the background. The background light includes the stray light through the filter, the spontaneous fluorescence of the sample tissue components, and the spontaneous fluorescence and stray light of the optical system. In the fluorescence microscopy, what we try our best to solve is to obtain the best contrast of Zui on the one hand, while maintaining the enough brightness of the image, which are often contradictory. For example, when excited by a very narrow band filter, only a small amount of light energy is used. The background is dark, but the fluorescent brightness is also weak. In the application, we can only take into account the reasonable trade-off to determine the choice.
The optical system of fluorescence microscope needs to give priority to:
1. The materials caused by the optical system shall be non fluorescent materials in the excitation light wave, which not only refers to the optical system glass, but also includes the adhesive, slice, cover glass, immersion solution and sealant in the adhesive manufacturing. If there is spontaneous fluorescence, background light will be added to the sheet, and sometimes the fluorescence image will be observed.
2. In the parameter selection of optical system, the Zui large light energy should be taken into consideration.
For example, in the lighting system, the light collecting mirror should be Zui with large containment angle; Kohler lighting should be used and the filament or arc image should be filled into the pupil to achieve uniform lighting with the desired rate.
For example, if the objective lens for fluorescence imaging is collected, because the illumination of the image surface is directly proportional to the numerical aperture, it should have a larger value as the objective lens for fluorescence.
For example, when the binocular tube is used for fluorescence, it should have a high transmittance in the visible spectrum. The loss of light energy can be greatly reduced by using the coating technology, which can double the transmittance of the general binocular tube, which is necessary for the observation of weak fluorescent substances.
3. As the components of the fluorescence optical system, the spectral characteristics are also very important, especially for the components such as the lighting system, the condenser and the objective lens through which the ultraviolet light passes, we should pay attention to the transmission coefficient of the purple light area. For example, the material of condenser should be made of UV light. For example, when the reflector turns the light path in the lighting system, aluminum plating is better than silver plating, because the reflectivity of aluminum is more than 90%, while that of silver is only 70%