Skills and methods of using fluorescence microscope
Technical parameters of fluorescence microscope 1. Wide-angle eyepiece 2. Achromatic objective 3. Four-hole objective converter 4. Emitting fluorescence device blue (b) green (g) excitation system 100W mercury lamp 5. Coaxial coarse micro-focusing mechanism: focusing range 15 mm, micro-grid value 0.002 mm 6. Longitudinal moving range of double-layer mechanical workbench: 70 mm, lateral moving range: 50 mm.
Skills and methods of using fluorescence microscope
(1) glass slides
The thickness of the slide should be between 0.8～1.2mm and 1.2 mm. Too thick a slide will not absorb much light on the one hand, and will not make the excitation light gather on the specimen on the other. The slide must be smooth, uniform in thickness and free of obvious spontaneous fluorescence. Shi Ying glass slides are sometimes needed.
(2) Cover glass
The cover glass is about 0.17mm thick and smooth. In order to enhance the excitation light, interference cover glass can also be used, which is a kind of special cover glass with several layers of substances (such as magnesium fluoride) which have different interference effects on different wavelengths of light. It can make the fluorescence pass smoothly and reflect the excitation light, and this reflected excitation light can excite the specimen.
(3) Specimens
Tissue sections or other specimens should not be too thick. If it is too thick, most of the excitation light is consumed in the lower part of the specimen, while the upper part directly observed by the objective lens is not fully excited. In addition, cell overlap or impurity masking affects judgment.
(4) sealing agent
Glycerol is commonly used as the sealing agent, which must be colorless and transparent without spontaneous fluorescence. The brightness of fluorescence is bright at pH 8.5 ~ 9.5, and it is not easy to fade quickly. Therefore, the equivalent mixture of glycerol and 0.5 mol/l PH9.0 ~ 9.5 carbonate buffer is often used as the sealing agent.
(5) mirror oil
Generally, mirror oil must be used when observing samples with dark-field fluorescence microscope and oil mirror. It is best to use special non-fluorescent mirror oil, or the above glycerin can be used instead. Liquid paraffin can also be used, but the refractive index is low, which has a slight impact on image quality.
