Requirements for the production of specimens for the fluorescence microscope
(1) Glass slide
The thickness of the slide should be between 0.8 and 1.2mm. If the slide is too thick, on the one hand, it will absorb more light, and on the other hand, it will not allow the excitation light to gather on the specimen. The glass slide must be smooth, uniform in thickness, and have no obvious spontaneous fluorescence. Sometimes quartz glass slides are needed.
(2) Cover glass slide
The thickness of the cover glass is about 0.17mm and it is smooth. In order to enhance excitation light, interference cover glass can also be used. This is a specially designed cover glass with several layers of substances (such as magnesium fluoride) that have different interference effects on light of different wavelengths. It can allow fluorescence to pass smoothly, while reflection excitation light can excite the specimen.
(3) Specimen
Tissue slices or other specimens should not be too thick. If they are 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 can affect judgment.
(4) Sealing agent
Glycerin is commonly used as a mounting agent, which must have no spontaneous fluorescence, colorless and transparent. The fluorescence brightness is relatively bright at pH 8.5-9.5 and is not easy to fade away quickly. Therefore, an equal mixture of glycerol and 0.5mol/lpH9.0-9.5 carbonate buffer is commonly used as a mounting agent.
(5) Mirror oil
When using a dark field fluorescence microscope and an oil microscope to observe specimens, it is necessary to use lens oil. It is best to use a specially made non fluorescent lens oil, which can also be replaced with glycerol as mentioned above. Liquid paraffin can also be used, but the refractive index is low and has a slight impact on image quality.
