Introduction to Fluorescence Microscopy
Fluorescence microscopy is to irradiate the object stained with fluorescein with short-wavelength light, so that it is excited to produce long-wavelength fluorescence, and then observed. In a fluorescence microscope, the excitation light of a specific wavelength must be selected from the illumination light of the specimen to generate fluorescence, and then the fluorescence must be separated from the mixed light of the excitation light and fluorescence for observation. Therefore, the filter system plays an extremely important role in selecting a specific wavelength. Fluorescence microscopes are widely used in biology, medicine and other fields.
1. The main components of the fluorescence microscope:
(A) Light source: The light source radiates light of various wavelengths (from ultraviolet to infrared). the
(B) Excitation filter light source: through the light of a specific wavelength that can cause the specimen to produce fluorescence, while blocking light that is useless for exciting fluorescence. the
(C) Fluorescent specimens: generally stained with fluorescent pigments. the
(D) Blocking filter: block the excitation light that is not absorbed by the specimen and selectively transmit the fluorescence, and some wavelengths are selectively transmitted in the fluorescence.
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2. Classification of fluorescence microscopes:
Fluorescence microscopes are generally divided into two types: transmission and epi-emission:
a. Transmission type: the excitation light comes from the bottom of the object to be inspected, and the condenser is a dark field condenser, so that the excitation light does not enter the objective lens, but the fluorescence enters the objective lens. It is bright at low magnifications, but dark at high magnifications. It is difficult to operate in oil immersion and centering. It is especially difficult to determine the lighting range at low magnifications, but it can get a very dark field of view background. The transmission type is not used for non-transparent objects. the
b. Epi-type: The transmission type is almost eliminated at present. Most of the new fluorescence microscopes are epi-type. The light source comes from above the object to be inspected, and there is a beam splitter in the optical path, so it is suitable for both transparent and opaque objects to be inspected. Since the objective lens acts as a condenser lens, it is not only easy to operate, but also can achieve uniform illumination of the entire field of view from low magnification to high magnification. the
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3. Precautions for Fluoroscopy
a. Long-term irradiation of excitation light will cause fluorescence attenuation and quenching, so shorten the observation time as much as possible, and use a baffle to cover the excitation light when not observing temporarily. the
b. When observing with an oil immersion lens, use "non-fluorescent oil". the
c. Fluorescence is almost always weak and should be carried out in a dark room. the
d. It is best to install a voltage stabilizer in the power supply, otherwise the unstable voltage will not only reduce the life of the mercury lamp, but also affect the effect of the microscope inspection. the
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4. Use of Fluorescence Microscopy
1. Preparation requirements for fluorescence microscope specimens
a. Glass slide
The thickness of the glass slide should be between 0.8 and 1.2mm. A slide that is too thick will absorb more light on the one hand, and on the other hand, the excitation light cannot be concentrated on the specimen. Slides must be smooth, uniform in thickness, and free from obvious autofluorescence. Sometimes quartz glass slides are used. the
b. Cover glass
The thickness of the cover glass is about 0.17mm, smooth. In order to strengthen the excitation light, a dry cover glass can also be used, which is a special cover glass coated with several layers of substances (such as magnesium fluoride) that have different interference effects on light of different wavelengths, which can make the fluorescence go smoothly. Excitation light is passed through, and the reflected excitation light excites the specimen. the
c. Specimen
Tissue slices or other specimens should not be too thick. If it is too thick, most of the excitation light will be consumed in the lower part of the specimen, while the upper part directly observed by the objective lens cannot be fully excited. In addition, cell overlap or impurities cover up, affecting the judgment. the
d. Mounting agent
Glycerin is commonly used as a mounting agent, which must have no autofluorescence, colorless and transparent, and the brightness of the fluorescence is brighter at pH 8.5-9.5, and it is not easy to fade quickly. Therefore, an equal mixture of glycerol and 0.5mol/L carbonate buffer solution with a pH of 9.0 to 9.5 is commonly used as a mounting agent. the
e. mirror oil
Generally, immersion oil must be used when observing specimens with dark-field fluorescence microscopes and oil immersion immersion objectives. It is best to use special non-fluorescent mirror oil, which can also be replaced by the above-mentioned glycerin, and liquid paraffin can also be used, but the refractive index is low, which slightly affects the image quality.
