Principle and application of fluorescence microscope
(i) The principle and structure of fluorescence microscope: fluorescence microscope is a point light source with high luminous efficiency, the filtered color system emits a certain wavelength of light (such as ultraviolet light 3650 or violet Blue 4200 into) as excitation light, stimulates the fluorescent material in the specimen to emit the fluorescence of various colors, and then through the enlargement of the objective lens and the eyepiece. In this case, even though fluorescence is very faint and sensitive, it is mainly used in the study of cell structure and function and chemical composition in the strong contrast. The basic structure of fluorescence microscope is composed of general optical microscope and some accessories (such as fluorescent light source, excitation filter, double color beam separator and blocking filter). Fluorescent light Source--the use of ultra-high pressure mercury lamps (501,200 W), it can emit a variety of wavelengths of light, but each fluorescent material has a maximum fluorescence to produce the excitation light wavelength, therefore, it is necessary to add the excitation filter (usually ultraviolet, purple, blue and green excitation filter), so that only a certain wavelength of excitation light through the specimen, and the other light absorption. After each material is stimulated by light irradiation, the visible fluorescence with longer irradiation wavelength is emitted in a very short time. Fluorescence has specificity, generally is weaker than the excitation light, to be able to observe the specificity of fluorescence, in the objective need to block (or suppress) the filter. Its role has two: one is to absorb and block the excitation light into the eyepiece to avoid disturbing fluorescence and damage the eyes, the second is to select and let the specific fluorescence through, showing a single-minded fluorescence color. The two filters must be selected to be used with each other.
Fluorescent microscopes are divided into two types of light paths:
1. Transmission fluorescent microscope: The excitation light source is through the condenser through the specimen material to excite the fluorescence. Commonly used in the dark field of vision collector, can also be used as a general optical collector, adjust the reflector so that the excitation light and side shot on the specimen. This is a more old-fashioned fluorescent microscope. The advantage of this method is that the fluorescence is strong when the macro is low, while the disadvantage is that the fluorescence decreases with magnification. So it is better to observe the larger specimen material.
2. This is a new type of fluorescence microscope developed in modern times, which is different from the objective to shoot the surface of the specimen, that is to use the same objective as the illumination concentrator and the collecting fluorescence lens. A two-color beam separator is added to the optical path, which is 45 as light uranium. Angle, the excitation light is reflected into the objective lens, and gathered on the sample, the fluorescence produced by the sample, as well as the lens surface, the surface of the cover glass reflection of the excitation light at the same time into the objective lens, back to the two-color beam splitter, so that the excitation light and fluorescence separation, residual excitation light is blocked by the filter absorption. If the combination of different excitation filter/double color beam separator/block filter is used, the need of different fluorescence reaction products can be satisfied. The advantages of this kind of fluorescence microscope are that the field illumination is even, the image is clear, the larger the magnification is, the stronger the fluorescence is.
Precautions for the use of fluorescent microscopes:
1, in strict accordance with the fluorescent microscope appearance requirements of the operation, do not change the program.
2, preferably in the darkroom for testing. Enter the darkroom, connect the power, ignite ultra-high pressure mercury lamp 5~15min, waiting for the light source to emit strong light and stability, the eyes fully adapted to the darkroom, and then began to observe the specimen.
3, to prevent ultraviolet rays on the eye damage, and then adjust the light source, should take protective eyes.
4, check the time to a Times 1-2hrs advisable, if more than 90min, ultra-high pressure mercury lamp luminous intensity gradually decreased, fluorescence weakened; the specimen was irradiated by ultraviolet Ray 3~5min, and the fluorescence of specimen was obviously weakened. So do not exceed 2~3hrs.
5, fluorescent light source life is limited, specimens should be concentrated inspection to save time, protect the light source. When hot weather, should turn on the fan or air-conditioning cooling cooling. The new light bulb should be recorded from the start of use. After the extinction to be used, the bulb should be fully cooled before ignition. The light source should be avoided several times in one days.
6, the specimen was stained immediately after the observation, the fluorescence will gradually weaken for a long time. If the specimen is kept in a polyethylene plastic bag for 4 degrees, it can delay the fluorescence weakening time.
7, fluorescence intensity judgment standard; generally divided into four levels, 1: No or visible faint fluorescence, 2: Only visible fluorescence can be seen, 3: visible fluorescence, 4: visible bright fluorescence.