There are two types of fluorescence microscopes with respect to their optical path:
1. Transmission fluorescence microscope: the excitation light source is through the condenser mirror through the specimen material to stimulate fluorescence. Commonly used dark-field collector, can also be used ordinary collector, adjust the reflector so that the excitation light transmitting and bypassed to the specimen. This is the older type of fluorescence microscope. The advantage is that the fluorescence is strong at low magnification, and the disadvantage is that the fluorescence is weakened with the increase in magnification. So it is better to observe the larger specimen material.
2. Falling-type fluorescence microscope this is a new type of fluorescence microscope developed in recent times, and on the difference is that the excitation of light from the objective lens down to the surface of the specimen, that is, the same objective lens as the illumination of the concentrator and the collection of fluorescence objective lens. The optical path needs to be added to a two-colour beam separator, which is 45. angle with the light uranium, the excitation light is reflected into the objective lens, and gathered in the sample, the fluorescence produced by the sample and the objective lens lens surface, cover glass surface reflection of the excitation light into the objective lens at the same time, and back to the two-colour beam separator, so that the excitation light and fluorescence are separated from the residual excitation light and then be absorbed by the blocking filters. If you change the different excitation filter / dual-colour beam separator / blocking filter combination block, can meet the needs of different fluorescence reaction products. The advantages of this fluorescence microscope is the field of view of uniform illumination, clear imaging, the greater the magnification of fluorescence the stronger.
(B) fluorescence microscope method.
1. Open the light source, ultra-high-pressure mercury lamp to preheat for a few minutes to reach the brightest point.
2. Transmission fluorescence microscope needs to be installed between the light source and the concentrator required to stimulate the filter, in the back of the objective lens with the corresponding blocking filter. Falling fluorescence microscope needs to be inserted in the slot of the optical path of the required excitation filter / dual-colour beam separator / blocking filter inserts.
3. Observe with a low magnification lens and adjust the centre of the light source so that it is located in the centre of the whole illumination spot according to the adjusting device of different models of fluorescence microscopes.
4. Place the specimen slice, focusing can be observed. Use should pay attention to: the end of the installation of filters do not use the eye to observe directly, so as not to cause eye damage; with an oil mirror to observe the specimen, you must use a special non-fluorescent oil mirror; high-pressure mercury lamp can not be reopened immediately after the closure of the lamp, you need to be 5 minutes before you can start again, otherwise it will be unstable, affecting the life of the mercury lamp.
(C) observation Under the fluorescence microscope on the teaching stage with a blue-violet light filter, cells stained with o.01% of amidin orange fluorescent dye can be seen, and the nucleus and cytoplasm of the cells are excited to produce two different colours of fluorescence (dark green and orange-red).
