The special structures of the fluorescence microscope include:
(I) Filter System
The filter block is an important part of a fluorescence microscope. Its core components consist of an excitation filter (first barrier filter), an emission filter (second barrier filter) and a beam‑splitting mirror. The model designations and names of filters vary among manufacturers.
Excitation Filters and Emission Filters Based on the characteristics of the light source and fluorochromes, the following three matching combinations are commonly selected. They provide excitation light within a specific wavelength range and transmit fluorescence emitted by the sample to the eyepiece for imaging.
Ultraviolet excitation: The excitation filter transmits ultraviolet light and blocks visible light above 400 nm. The corresponding emission filter allows blue light to pass, producing a blue image in the field of view, e.g., for DAPI staining.
Blue light excitation: The excitation filter transmits blue light and blocks light of other wavelengths. The corresponding emission filter allows green light to pass, e.g., for GFP labelling.
Green light excitation: The excitation filter transmits green light and blocks light of other wavelengths. The corresponding emission filter usually allows red light to pass, e.g., for Rhodamine staining.
Beam‑splitting Mirror It completely blocks and reflects excitation light, while transmitting emission light of the corresponding wavelength range. Its model is matched with the excitation filter and emission filter.
(II) Objectives and Eyepieces
Most objectives can be used, but apochromatic, achromatic objectives are preferred. They have extremely low autofluorescence and suitable transmittance (wavelength range) for fluorescence applications. The fluorescence brightness of an image in the microscope field of view is proportional to the square of the objective numerical aperture and inversely proportional to its magnification. To improve the brightness of fluorescence images, objectives with a large numerical aperture should be used. Especially for weakly fluorescent specimens, select objectives with large numerical aperture and high light transmittance, paired with the lowest possible magnification eyepieces.
(III) Other Optical Components
Reflecting mirror: Its reflective layer is usually aluminium‑coated. Aluminium has low absorption for ultraviolet light and the blue‑violet band of visible light, with a reflectance of over 90% (silver only reaches about 70%). Plane mirrors are generally adopted. Condenser: Condensers specially designed for fluorescence microscopes are made of quartz glass or other UV‑transparent glass. Epi‑illumination unit: In addition to the function of a transmitted light source, it is more suitable for direct observation of opaque and semi‑transparent specimens such as thick slices, filter membranes, bacterial colonies and tissue cultures. Many newly developed fluorescence microscopes adopt epi‑illumination systems, known as epifluorescence microscopes.
