Requirements for the production of fluorescent microscope specimens
（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 absorbs more light, and on the other hand, it cannot cause excitation light to gather on the specimen. The slide must be smooth, uniform in thickness, and free from obvious spontaneous fluorescence. Sometimes quartz glass slides are needed.
（2） Cover glass slide
The thickness of the cover glass is about 0.17mm, which is smooth and clean. In order to enhance excitation, 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 different wavelengths of light. It can allow fluorescence to pass smoothly, while reflection excitation can excite the specimen.
（3） Specimen
Tissue slices or other specimens should not be too thick, as the excitation light is mostly 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, and it must be free from spontaneous fluorescence, colorless and transparent. The brightness of the fluorescence is brighter at pH 8.5~9.5, and it is not easy to fade quickly. Therefore, an equal mixture of glycerol and 0.5mol/l carbonate buffer at pH 9.0-9.5 is commonly used as a sealing agent.
（5） Mirror oil
When observing specimens using a dark field fluorescence microscope and an oil microscope, 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.
Precautions for using a fluorescence microscope
(1) Strictly follow the factory manual requirements of the fluorescence microscope and do not change the program arbitrarily.
(2) Inspection should be conducted in a dark room. After entering the darkroom, connect the power supply and light the ultra-high pressure mercury lamp for 5 to 15 minutes. After the light source emits strong light and stabilizes, the eyes fully adapt to the darkroom before starting to observe the specimen.
(3) To prevent UV damage to the eyes, protective glasses should be worn when adjusting the light source.
(4) The inspection time should be 1-2 hours each time. If it exceeds 90 minutes, the luminous intensity of the ultra-high pressure mercury lamp gradually decreases and the fluorescence weakens; After being exposed to ultraviolet radiation for 3-5 minutes, the fluorescence of the specimen also significantly decreased; So, it should not exceed 2-3 hours at most.
(5) The lifespan of the fluorescent microscope light source is limited, and the specimen should be inspected centrally to save time and protect the light source. When the weather is hot, an electric fan should be added to dissipate heat and cool down. When replacing a new light bulb, the usage time should be recorded from the beginning. When the lamp is turned off and wants to be used again, it must wait for the bulb to fully cool before being ignited. Avoid igniting the light source multiple times.
(6) Observe the specimen immediately after staining, as the fluorescence gradually decreases over time. If the specimen is stored in a polyethylene plastic bag at 4 ℃, it can delay the fluorescence attenuation time and prevent the evaporation of the mounting agent.
(7) The judgment standard for fluorescence brightness is generally divided into four levels, namely "one" - no or visible weak fluorescence. +- Only clearly visible fluorescence can be seen. ++- Visible with bright fluorescence. '+++' - Visible and dazzling fluorescence.
