Fluorescence microscope tips and tricks
(i) Slides
The thickness of the slide should be between 0.8 and 1.2 mm, too thick slopes, on the one hand, more light absorption, on the other hand, can not make the excitation of light in the specimen gathered. The slide must be smooth, uniform thickness, no obvious autofluorescence. Sometimes quartz glass slides are required.
(ii) Coverslips
Coverslip thickness of about 0.17mm, clean. In order to enhance the excitation light, can also be used to interfere with the coverslip, which is a special surface plated with a number of layers of different wavelengths of light play a different role in the interference of substances (such as magnesium fluoride) of the coverslip, which can make the fluorescence through the smooth and reflective excitation of the light, the reflected excitation of the light can be excitation of the specimen woman.
(iii) specimens
Tissue sections or other specimens can not be too thick, such as too thick excitation light is mostly consumed in the lower part of the specimen, and the objective lens directly observed in the upper part of the insufficient excitation. In addition, cell overlap or impurities masking, affecting judgement.
(D) sealing agent
Sealing agent commonly used glycerol, must be free of autofluorescence, colourless and transparent, the brightness of fluorescence in pH 8.5 ~ 9.5 is brighter, not easy to fade away quickly. Therefore, commonly used glycerin and 0.5mol/lpH9.0 ~ 9.5 of carbonate buffer equal amount of mixture for sealing agent.
(E) mirror oil
General dark-field fluorescence microscope and oil mirror specimen observation, must use mirror oil, preferably using special non-fluorescent mirror oil, can also be used in place of the above-mentioned glycerol, liquid paraffin can also be used, but the refractive index of the lower, slightly affecting the quality of the image.
