1. Basic operation steps
(1) Install the UV shield.
(2) Turn on the power control box switch of the high pressure mercury lamp.
(3) Insert the light barrier to interrupt the optical path.
(4) Preheat for 5-10 minutes.
(5) Place the slide containing the sample on the stage.
(6) Select the objective lens (in order of low magnification, high magnification).
(7) Rotate the beam splitter assembly turntable to select the desired beam splitter assembly: “O†for observation of transmitted light; “WU†for observation of blue fluorescence (eg DAP1); “WB†for observation of green fluorescence Use (such as FITC); "WG" is used when observing red fluorescence (such as TRITC).
(8) Use a blocking filter (currently most blocking filters are built into a fluorescence microscope).
(9) Adjust the focal length by a coarse and fine spiral.
(10) Open the computer connected to the microscope and click on the digital imaging system software to collect digital images.
2. Precautions
(1) The light source used for observing fluorescence is a high-pressure mercury lamp, in which the emitted light contains ultraviolet light, which has a detrimental effect on the human eye, so an ultraviolet protective cover must be installed.
(2) In order to extend the life of the mercury lamp, the mercury lamp cannot be turned off immediately after opening it, so as to prevent the mercury from evaporating incompletely and damaging the electrode. It usually takes 15 minutes to turn off.
(3) After the mercury lamp is extinguished, it can be restarted after being completely cooled. Otherwise, the mercury vapor in the lamp has not recovered to the liquid state, and the internal resistance is extremely small. When the voltage is applied again, a short circuit may occur, causing the mercury lamp to explode. This will not only damage the appliance, but more importantly, the mercury vapor will overflow, which will cause studio pollution. Therefore, after the mercury lamp is turned off, it cannot be turned on again immediately and must wait at least 30 minutes.
(4) High-pressure mercury lamps emit a lot of heat when working. Therefore, the working environment temperature should not be too high, and there must be good heat dissipation conditions.
(5) The service life of the mercury lamp is about 200-300 hours. There is a time accumulating counter on the power control box. The user has to record the accumulated hours to reach 300 hours. The new bulb needs to be replaced, otherwise the brightness is not enough, which will affect the observation.
3. Fluorescence microscopy specimen production characteristics and observation requirements
(1) Slide: The thickness of the slide should be between 0.8 and 1.2 mm. The slide is too thick to absorb more light, and the excitation light is difficult to accumulate on the specimen. Slides must be smooth, uniform in thickness, and free of autofluorescence. Quartz glass slides are sometimes used.
(2) Cover slip: The thickness of the coverslip should be about 0.17mm, smooth and clean. In order to enhance the excitation light, an interference cover glass can also be used. The surface of the special cover glass is coated with several layers of substances (such as magnesium fluoride) which interfere differently with different wavelengths of light, which can make the fluorescence pass smoothly and reflect. Excitation light, this reflected excitation light excites the fluorescence in the specimen.
(3) Specimen: Tissue sections or other specimens should not be too thick, generally 5 to 20 pm. If the slices are too thick, most of the excitation light is consumed in the lower part of the specimen, and the upper part of the specimen directly observed by the objective lens cannot be excited. Slice folding or excessive impurities can affect the observation of fluorescence.
(4) Sealing tablets: The sealing tablets must be free of autofluorescence and transparent in five colors. Add anti-quenching agent and then use nail polish (the actual operation of the nail polish to seal the film is very good, can be thin and uniform) to seal the cover glass around, not only keep the cover slip does not slide, but also prevent resistance The quencher evaporates. In the absence of an anti-quenching agent, PBS containing 30% glycerol may also be used, and a mixture of glycerol and 0.5 mol/L carbonate buffer (pH 9.0 to 9.5) may be used as a tablet.
(5) Cleaning of the lens: After the oil mirror is used, wipe it with a mixture of absolute ethanol or 3:7 alcohol ether.
(6) Timely observation: The specimen should be observed in time after staining, and the fluorescence will gradually weaken due to the long time. If the specimen is stored in a polyethylene plastic bag at 4 ° C, the fluorescence decay time can be delayed.
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