Fluorescence microscopy detection method
fluorescence microscope
Fluorescence microscopy is a fundamental tool for immunofluorescence cytochemistry. It is composed of main components such as light source, filter plate system, and optical system. It uses a certain wavelength of light to excite the specimen and emit fluorescence, which is magnified through an objective and eyepiece system to observe the fluorescence image of the specimen (Figure 1).
(1) Light source
Nowadays, a 200W ultra-high pressure mercury lamp is commonly used as the light source. It is made of quartz glass, with a spherical shape in the middle and a certain amount of mercury filled inside. During operation, the discharge between the two electrodes causes the mercury to evaporate, and the pressure inside the sphere rapidly increases. When the mercury completely evaporates, it can reach 50-70 standard atmospheric pressures, which generally takes about 5-15 minutes. The luminescence of ultra-high pressure mercury lamps is the result of the discharge between electrodes, which continuously dissociates and reduces mercury molecules and emits photons. It emits strong ultraviolet and blue violet light, which is sufficient to excite various fluorescent substances, therefore, it is widely used in fluorescence microscopes.
Precautions for using a fluorescence microscope
(1) Strictly follow the instructions of the fluorescence microscope factory and do not change the program arbitrarily.
(2) Inspection should be conducted in a dark room. After entering the darkroom, connect the power and light the ultra-high pressure mercury lamp for 5-15 minutes. After the light source emits strong light and stabilizes, the eyes should 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; Specimens exposed to ultraviolet radiation for 3-5 days
After min, the fluorescence also significantly decreased; So, the maximum duration should not exceed 2-3 hours.
(5) The lifespan of the fluorescent microscope light source is limited, and the specimen should be centrally examined to save time
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 light is turned off and you want to use it again, you must wait for the light bulb to cool sufficiently before igniting 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 ℃, it can delay the fluorescence attenuation time and prevent the evaporation of the mounting agent.
(7) The judgment criteria for fluorescence brightness are generally divided into four levels, namely "one" - no or visible weak fluorescence. "+" - Only visible fluorescence is visible. ++- Visible with bright fluorescence. "+++" - Visible and dazzling fluorescence.
