Structure and main components of a fluorescence microscope
Fluorescence microscopy is an essential tool in immunofluorescent cytochemistry. It is composed of main components such as light source, filter plate system and optical system. It is to use a certain wavelength of light to excite the specimen to emit fluorescence, and to observe the fluorescence image of the specimen by amplifying the objective lens and eyepiece system.
Structure and main components of a fluorescence microscope
(1) Light source
Nowadays, 200W ultra-high pressure mercury lamps are often used as light sources. They are made of quartz glass, with a spherical shape in the middle, and a certain amount of mercury is filled inside. During work, the discharge between the two electrodes causes mercury to evaporate, and the air pressure in the ball rises rapidly. When the mercury is completely evaporated, it can reach 50-70 standard atmospheric pressure, and this process generally takes about 5-15 minutes. The luminescence of the ultra-high pressure mercury lamp is the result of the emission of light quanta during the continuous dissociation and reduction of mercury molecules by the discharge between the electrodes. It emits strong ultraviolet and blue-violet light, which is enough to excite various fluorescent substances, so it is widely used in fluorescence microscopy.
Ultra-high pressure mercury lamps also emit a lot of heat. Therefore, the lamp house must have good heat dissipation conditions, and the working environment temperature should not be too high.
The new ultra-high pressure mercury lamp can be ignited without high voltage at the beginning of use. After a period of use, it needs to be started at high voltage (about 15000V). After starting, the maintenance working voltage is generally 50-60V, and the working current is about 4A. The average lifespan of a 200W ultra-high pressure mercury lamp is about 200 hours if it is used for 2 hours each time. The shorter the working time, the shorter the lifespan. If it is only used for 20 minutes, the lifespan will be reduced by 50%. Therefore, minimize the number of starts when using it. During the use of the bulb, its luminous efficacy is gradually reduced. After the light goes out, wait for it to cool down before restarting. Do not turn off the bulb immediately after igniting it, so as not to damage the electrode due to incomplete mercury evaporation. Generally, it needs to wait for 15 minutes. Due to the high pressure of the ultra-high pressure mercury lamp and the strong ultraviolet rays, the bulb must be placed in the lamp chamber before it can be ignited, so as not to damage the eyes and cause operation in the event of an explosion.
The circuit of the ultra-high pressure mercury lamp (100W or 200W) light source and several parts including transformer, ballast and start. There is a system for adjusting the luminous center of the bulb in the lamp chamber. An aluminum-plated concave reflector is installed behind the bulb bulb, and a light-collecting lens is installed in the front.
The domestic ultra-high pressure mercury lamp GCQ-200 has good performance and can replace imported bulbs such as HBO-200. The average lifespan is more than 200h and the price is relatively low.
A simple and portable high color temperature bromine tungsten fluorescent light source device developed in my country has small size, light weight, low power, AC and DC dual-use (with DC power supply), easy to carry, easy to use, and has been popularized and applied.
(2) Color filter system
The color filter system is an important part of the fluorescence microscope, which consists of an excitation filter plate and a pressing filter plate. The filter plate model and the name of each manufacturer are often not uniform. The filter plates are generally named after the basic color, the letters in front represent the color, the letters in the back represent the glass, and the numbers represent the characteristics of the model. olympus microscope
(3) Objective lens
Various objective lenses can be used, but achromatic objective lenses are used because their autofluorescence is minimal and their light transmission properties (wavelength range) are suitable for fluorescence. Since the fluorescence brightness of the image in the microscope field of view is proportional to the square of the aperture ratio of the objective lens and inversely proportional to its magnification, in order to improve the brightness of the fluorescence image, an objective lens with a large aperture ratio should be used. Especially at high magnification its effect is very obvious. Therefore, for specimens with insufficient fluorescence, an objective lens with a large aperture ratio should be used with an eyepiece as low as possible (4×, 5×, 6.3×, etc.).
(4) Mirror
The reflective layer of the reflector is generally aluminum-plated, because aluminum absorbs less in the blue-violet region of ultraviolet light and visible light, and the reflection is more than 90%, while the reflection of silver is only 70%. Generally, a flat reflector is used.
(5) Condenser
Condensers designed for fluorescence microscopy are made of quartz glass or other UV-transparent glass. There are two types of clear field condensers and dark field condensers. There are also phase contrast fluorescence concentrators.
(6) Epi-light device
The new type of epi-light device is that after the light from the light source hits the interference spectroscopic filter, the short wavelength part (ultraviolet and purple blue) is reflected due to the properties of the coating on the filter. When the filter faces the light source, the angle is 45. When it is tilted, it shoots to the objective lens vertically, and shoots to the specimen through the objective lens, so that the specimen is excited. At this time, the objective lens directly acts as a light collector. At the same time, the long part of the filter (green, yellow, red, etc.) is transparent to the filter, so it does not reflect in the direction of the objective lens, and the filter acts as an excitation filter plate, because the fluorescence of the specimen is in the long-wavelength region of visible light , can pass through the filter and reach the eyepiece for observation, the brightness of the fluorescent image increases with the increase of the magnification, and is stronger than the transmitted light source at high magnification. In addition to the function of a transmission light source, it is more suitable for direct observation of opaque and translucent specimens, such as thick slices, filter membranes, colonies, and tissue culture specimens. The new fluorescence microscopes developed in recent years mostly use epi-light devices, which are called epi-fluorescence microscopes.
