There are two types of fluorescence microscopes based on their light paths.
1. Transmission fluorescence microscope: The excitation light source passes through the specimen material through a condenser to excite fluorescence. A dark field collector is commonly used, or an ordinary collector can be used, and the reflector is adjusted to divert and side-radiate the excitation light to the specimen. This is an older fluorescence microscope. The advantage is that the fluorescence is strong at low magnification, but the disadvantage is that the fluorescence weakens as the magnification increases. Therefore, it is better to observe larger specimen materials.
2. Epi-fluorescence microscope is a new type of fluorescence microscope developed in modern times. The difference from the above is that the excitation light falls downward from the objective lens to the specimen surface, that is, the same objective lens is used as the illumination condenser and the objective lens for collecting fluorescence. A two-color beam splitter needs to be added to the optical path, which is 45% with the optical uranium. Angle, the excitation light is reflected into the objective lens and concentrated on the sample. The fluorescence generated by the sample and the excitation light reflected by the objective lens surface and the cover glass surface enter the objective lens at the same time and return to the dichromatic beam splitter, so that the excitation light Separated from the fluorescence, the residual excitation light is then absorbed by the blocking filter. If you use different excitation filter/dual-color beam splitter/blocking filter combination inserts, you can meet the needs of different fluorescence reaction products. The advantage of this kind of fluorescence microscope is that the field of view is uniformly illuminated, the image is clear, and the greater the magnification, the stronger the fluorescence.
(2) How to use fluorescence microscope.
1. Turn on the light source, and the ultra-high-pressure mercury lamp needs to warm up for several minutes to reach its brightest point.
2. Transmission fluorescence microscopes require the required excitation filter to be installed between the light source and the condenser, and the corresponding blocking filter to be installed behind the objective lens. Epi-fluorescence microscopes need to insert the required excitation filter/dual-color beam splitter/blocking filter insert into the slot of the light path.
3. Use a low-magnification microscope to observe, and adjust the center of the light source according to the adjustment devices of different models of fluorescence microscopes so that it is located in the center of the entire illumination spot.
4. Place the specimen piece and adjust the focus to observe. Please pay attention during use: Do not observe the filter directly with your eyes to avoid eye damage; when observing the specimen with an oil lens, you must use a special non-fluorescent oil lens; the high-pressure mercury lamp cannot be reopened immediately after it is turned off. It can be started again after 5 minutes, otherwise it will be unstable and affect the life of the mercury lamp.
(3) Observe under the fluorescence microscope on the teaching platform using a blue-violet light filter. It can be seen that the passage is o. In cells stained with 01% acridine orange fluorescent dye, the nucleus and cytoplasm are excited to produce two different colors of fluorescence (dark green and orange-red).
