Fluorescence microscope basic operation
a. Turn off the lights in the room and turn on the microscope mercury lamp;
b. Select the corresponding filter according to the fluorescein labeled by the sample;
c. Place the sample and find a suitable field of view;
d. If you need to take pictures, please confirm that the color film is installed in the camera;
e. Turn on the Selfie device, select the manual gear, usually the shooting speed is within 0.5-10 seconds;
f. After use, turn off all power and make a record of use. the
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3. Recording method of fluorescence image
The fluorescence image seen by the fluorescence microscope has both morphological characteristics and fluorescence color and brightness. When judging the results, the two must be combined for comprehensive judgment. The results are recorded based on subjective indicators, that is, visual observation by workers. As a general qualitative observation, basically reliable. With the development of technology and science, objective indicators are used to record judgment results to varying degrees, such as cell spectrophotometers, image analyzers and other instruments. However, the results recorded by these instruments must also be combined with subjective judgments. the
Fluorescence microscope photography is very necessary for recording fluorescence images. Since fluorescence is easy to fade and weaken, it is necessary to record the results in real time. The method is basically the same as that of ordinary photomicrography. Just need to use high-speed photosensitive film. Because ultraviolet light has a strong effect on fluorescence quenching, such as FITC markers, the fluorescence brightness will decrease by 50% when irradiated with ultraviolet light for 30s. Therefore, if the exposure speed is too slow, the fluorescent image cannot be taken. General research fluorescence microscopes have semi-automatic or fully automatic photomicrography system devices. the
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4. Precautions for Using Fluorescence Microscopy
a. Operate strictly in accordance with the requirements of the fluorescence microscope factory instructions, and do not change the program at will. the
b. The inspection should be carried out in a dark room. After entering the darkroom, connect the power supply and light the ultra-high pressure mercury lamp for 5-15 minutes. After the strong light from the light source is stable, the eyes are completely adapted to the darkroom, and then start to observe the specimen. the
c. To prevent the damage of ultraviolet rays to the eyes, wear protective glasses when adjusting the light source. the
d. The inspection time is preferably 1-2 hours each time. If it exceeds 90 minutes, the luminous intensity of the ultra-high pressure mercury lamp will gradually decrease and the fluorescence will weaken; after the specimen is irradiated with ultraviolet light for 3-5 minutes, the fluorescence will also be significantly weakened; therefore, it should not exceed 2-3 hours at most. . the
e. The life of the fluorescent microscope light source is limited, and the specimens should be checked centrally to save time and protect the light source. When the weather is hot, fans should be added to dissipate the heat, and the new bulbs should be recorded from the beginning of their usage time. When you want to use it again after the lamp is extinguished, you must wait until the bulb is sufficiently cooled before lighting it. Avoid lighting the light source several times a day. the
f. Observe the specimen immediately after staining, as the fluorescence will gradually weaken over time. If the specimen is stored in a polyethylene plastic bag at 4°C, the fluorescence weakening time can be delayed and the mounting agent can be prevented from evaporating. the
g. Judgment standard of fluorescence brightness: it is generally divided into four levels, that is, "-" means no or weak fluorescence can be seen. "+" means only clearly visible fluorescence was seen. "++" means bright fluorescence is visible. "+++" means visible bright fluorescence.
