Stereo Fluorescence Microscope Usage and Precautions
Instructions:
(1) Place the material sheet on the stage.
(2) Turn on the voltage stabilizer of the fluorescent microscope. Then press the start group to ignite the UV lamp and do not turn it off within 15 minutes, and do not start it again within 3 minutes after it is turned off.
(3) Turn the excitation filter to v, the color separation filter to v, and select a 495 or 475nm blocking filter.
(4) Choose uvFL40, uvFL100 fluorescent objective microscope for microscopic examination.
(5) Add non-fluorescent oil on the slide, first use 40 x, and then use 100 x focusing microscope to check. Fluorescence appears.
(7) Since the fluorescence of fluorescent substances gradually weakens with time when they are irradiated by ultraviolet light, the field of view should be changed frequently during microscopic examination.
(8) uvFL10 or uvFL20 low-magnification microscopes can also be used to inspect materials (no fluorescent oil is added when using low-power lenses).
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.
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 such as ASA200 or above or 24. above. 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.
Precautions:
(1) Operate strictly in accordance with the requirements of the fluorescence microscope factory instructions, and do not change the program at will.
(2) 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.
(3) To prevent ultraviolet rays from damaging the eyes, protective glasses should be worn when adjusting the light source.
(4) 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 rays for 3-5 minutes, the fluorescence will also be significantly weakened; therefore, it should not exceed 2-2 hours at most. 3h.
(5) The life of the light source of the fluorescence microscope is limited, and the specimens should be inspected collectively 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.
(6) 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.
(7) Judgment standard of fluorescence brightness: it is generally divided into four levels, that is, "one" - no or weak fluorescence can be seen. "+"—Only clearly visible fluorescence can be seen. "++"—Visible and bright
