Fluorescence microscopes can be divided into two types according to the optical path principle:
1. Transmission fluorescence microscope
Older fluorescence microscopes use a spotlight to excite fluorescence by passing the excitation light source through the specimen material. Its advantage is strong fluorescence at low magnification, but its disadvantage is that its fluorescence decreases with increasing magnification. So it is only suitable for observing larger specimen materials.
2. Falling light fluorescence microscope
The excitation light falls down from the objective lens onto 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 splitter (dichroic mirror) needs to be added to the optical path, which forms a 45 degree angle with the optical axis. The excitation light is reflected into the objective lens and focused 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 at the same time and return to the dual color beam splitter to separate the excitation light and fluorescence. The residual excitation light is then blocked by the filter absorption. If different combinations of excitation filters, dual color beam separators, and blocking filters are used, they can meet the needs of different fluorescent reaction products.
The advantages of this fluorescence microscope are uniform field illumination, clear imaging, and stronger fluorescence with larger magnification.
3. Phase contrast microscope
Phase contrast microscope is a microscope that can convert the phase difference (or optical path difference) generated when light passes through an object into amplitude (light intensity) changes. Mainly used for observing live cells, unstained tissue sections, or stained specimens lacking contrast.
The human eye can only distinguish changes in the wavelength (color) and amplitude of visible light, but cannot distinguish changes in phase. Most biological specimens are highly transparent, and the amplitude of light waves remains basically unchanged after passing through, with only phase changes.
The phase contrast microscope basically converts the optical path difference of visible light passing through the specimen into amplitude difference, thereby improving the contrast between various structures and making them clear and visible. After passing through the specimen, the light undergoes refraction, deviating from the original optical path and being delayed by 1/4 λ (wavelength). If the optical path difference is increased or decreased by another 1/4 λ, the optical path difference becomes 1/2 λ, and the interference between the two light beams increases or decreases after the axis is combined, improving the contrast.
