There are two types of fluorescence microscopes based on their optical path:
1. Transmission fluorescence microscope: The excitation light source is transmitted through the specimen material through a condenser to excite fluorescence. Commonly used dark field light collectors, or ordinary light collectors, can be used to adjust the reflector to convert excitation light and side light onto the specimen. This is a relatively old-fashioned fluorescence microscope. Its advantage is that the fluorescence is strong at low magnification, while its disadvantage is that the fluorescence weakens as the magnification increases. Therefore, it is better for observing larger specimen materials.
2. Falling beam fluorescence microscope This is a new type of fluorescence microscope developed in modern times. Unlike the previous one, the excitation light falls down from the objective lens to the surface of the specimen, using the same objective lens as the illumination condenser and the objective lens for collecting fluorescence. A dual color beam separator needs to be added to the optical path, which is at a 45 degree angle to the light uranium. The excitation light is reflected into the objective lens and concentrated on the sample. The fluorescence generated by the sample, as well as the excitation light reflected from the surface of the objective lens and cover glass, enter the objective lens simultaneously and return to the dichroic beam separator, separating the excitation and fluorescence. The residual excitation is then absorbed by the blocking filter. If different excitation filters/dual color beam separators/blocking filter combinations are used, they can meet the needs of different fluorescent reaction products. The advantage of this fluorescent microscope is that the field of vision illumination is uniform, the imaging is clear, and the larger the magnification, the stronger the fluorescence.
（2） Usage of fluorescence microscope
1. Turn on the light source, and the ultra-high pressure mercury lamp needs to be preheated for a few minutes to reach its peak.
2. The transmission fluorescence microscope requires the required excitation filter to be installed between the lamp source and the condenser, and the corresponding blocking filter to be installed behind the objective lens. The falling beam fluorescence microscope needs to insert the required excitation filter/dual color beam separator/blocking filter block into the slot of the optical path.
3. Observe with a low-power microscope and adjust the center of the light source according to the adjustment devices of different models of fluorescence microscopes, so that it is located at the center of the entire illumination spot.
4. Place the specimen slices and focus them to observe. During use, it should be noted that the final filter should not be directly observed with the eyes to avoid eye damage; When observing specimens with an oil microscope, a special non fluorescent oil microscope must be used; After the high-pressure mercury lamp is turned off, it cannot be immediately turned on again. It needs to take 5 minutes to start again, otherwise it will be unstable and affect the lifespan of the mercury lamp.
（3） Observing under a fluorescence microscope on the teaching platform using a blue purple light filter, it can be seen that cells stained with 0.01% acridine orange fluorescent dye, the nucleus and cytoplasm are stimulated to produce two different colors of fluorescence (dark green and orange red).
