Principle and Structural Features of Fluorescence Microscopes
The fluorescence light source generally uses an ultra-high-pressure mercury lamp (50–200 W), which emits light of various wavelengths. However, each fluorescent substance has a specific excitation wavelength that generates intense fluorescence. For this reason, an excitation filter (typically available in ultraviolet, violet, blue and green types) is required to allow only excitation light of a specific wavelength to pass through and irradiate the specimen, while absorbing all other wavelengths.
When irradiated by excitation light, each substance emits visible fluorescence with a longer wavelength than the excitation light within an extremely short time. Fluorescence is highly specific and generally weaker than the excitation light. To observe this specific fluorescence, a barrier (or suppression) filter must be installed behind the objective lens. It serves two purposes: first, it absorbs and blocks excitation light from entering the eyepiece to avoid interfering with fluorescence observation and damaging the eyes; second, it selectively transmits specific fluorescence to produce a distinct fluorescent color. The two types of filters must be selected and used in matching pairs.
Fluorescence microscopes are divided into two types based on their optical path:
1. Transmitted-light Fluorescence Microscope
In this design, the excitation light source passes through the condenser and the specimen material to excite fluorescence. A dark-field condenser is commonly used, though a standard condenser can also be applied; the reflecting mirror is adjusted to redirect and side-irradiate the excitation light onto the specimen. This is a relatively older type of fluorescence microscope. Its advantage is strong fluorescence at low magnification, while its disadvantage is that fluorescence intensity decreases as magnification increases. It is therefore better suited for observing larger specimen materials.
2. Epifluorescence Microscope
This is a modern, newly developed type of fluorescence microscope. Unlike the transmitted-light design, excitation light is projected downward through the objective lens onto the specimen surface - the same objective lens serves both as the illumination condenser and the fluorescence-collecting objective. A dichroic beamsplitter is installed in the optical path at a 45° angle to the optical axis. Excitation light is reflected into the objective lens and focused onto the sample. The fluorescence generated by the sample, along with excitation light reflected from the surfaces of the objective lens and coverslip, simultaneously enters the objective lens and returns to the dichroic beamsplitter, which separates excitation light from fluorescence. Remaining excitation light is then absorbed by the barrier filter.
By swapping filter blocks with different combinations of excitation filter, dichroic beamsplitter and barrier filter, the requirements for different fluorescent reaction products can be met. The advantages of this type of fluorescence microscope include uniform field illumination, sharp imaging, and increasing fluorescence intensity at higher magnifications.
