Fluorescence microscope use methods and precautions
Method of use:
(1) Place the material sheet on the carrier stage.
(2)Switch on the fluorescence microscope voltage regulator . Then press the start group to ignite the UV lamp shall not be turned off for 15min, and shall not be restarted for 3min after it is turned off.
(3)Turn the excitation filter to v, dichroic film to v, choose 495 or 475nm blocking filter.
(4) Choose uvFL40, uvFL100 fluorescence receiving objective lens microscopy.
(5) Add non-fluorescent oil on the slide, first 40 x, and then 100 x focusing microscopy. Show fluorescence.
(7) Because the fluorescent material by ultraviolet irradiation with the growth of fluorescence gradually weakened over time, so the microscopic examination should be frequently change the field of view.
(8) can also use uvFL10 or uvFL20 low-magnification microscopy material (with low-magnification without adding non-fluorescent oil).
Fluorescence image recording method:
Fluorescence image seen by fluorescence microscope, one is with morphological characteristics, the second is with the colour and brightness of fluorescence, in judging the results, the two must be combined with a comprehensive judgment. The results are recorded according to subjective indicators, i.e., by visual observation by the worker. As a general qualitative observation, basically reliable. With the development of technology and science, to varying degrees, the use of objective indicators to record the results of the judgement, such as the use of cell spectrophotometer, image analyser and other instruments. However, the results recorded by these instruments must also be combined with subjective judgement.
Fluorescence microscope photography technology is very necessary for recording fluorescence images, because fluorescence is easy to fade and weaken, to instantly record the results of photography. The method is basically the same as ordinary micrography. Just need to use high-speed photographic film such as ASA200 above or 24. above. Because the ultraviolet light on the fluorescence burst effect, such as FITC markers, in the ultraviolet light irradiation 30s, fluorescence brightness reduced by 50%. Therefore, the exposure speed is too slow to capture the fluorescence image. General research fluorescence microscopes have semi-automatic or fully-automatic microphotography system devices.
Precautions:
(1) Operate in strict accordance with the requirements of the fluorescence microscope factory manual, do not change the procedure arbitrarily.
(2) The inspection should be carried out in the darkroom. After entering the darkroom, connect the power supply, light the ultra-high-pressure mercury lamp for 5-15min, wait until the light source emits a strong light to stabilise, and the eyes are completely adapted to the darkroom, and then start observing the specimen.
(3) To prevent damage to the eyes from ultraviolet light, protective glasses should be worn when adjusting the light source.
(4) check the time each time to 1 ~ 2h is appropriate, more than 90min, ultra-high-pressure mercury lamp luminous intensity gradually decreased, fluorescence weakened; specimens irradiated by ultraviolet light 3 ~ 5min, fluorescence is also significantly weakened; therefore, * shall not be more than 2 ~ 3h.
(5) fluorescence microscope light source life is limited, the specimen should be centralised inspection, in order to save time and protect the light source. When it is hot, should be added fan cooling, new bulb should be recorded from the beginning of the use of time. When the lamp is extinguished and wants to be used again, the bulb must be sufficiently cooled before lighting. The light source should be avoided several times.
(6) Observe the specimen immediately after staining, as the fluorescence will gradually diminish over time. If the specimen is placed in a polyethylene plastic bag stored at 4 ℃, it can delay the time of fluorescence weakening and prevent the evaporation of the sealing agent.
(7) Judgement criteria for fluorescence brightness: generally divided into four levels, that is, 'one' - no or visible weak fluorescence. '+' - only clear visible fluorescence. '+ +' - visible with a bright
