The visualization of fluorescence under the fluorescence microscope is ensured by special filters that allow the passage of individual wavelengths. Special filters of a fluorescence microscope include:
- Excitation Filter
- Emission Filter
- Dichroic Beam Splitter
Individual excitation filters allow the respective wavelength of light to pass which is necessary to excite a specific dye in the sample to be examined. The dichroic mirror reflects the stimulating wavelength to the objective, which concentrates the beam onto the specimen. The light reflected from the specimen is concentrated in the objective and in its excited state usually has a higher wavelength than the incident light. Passing through the dichroic mirror, the reflected light passes through the emission filter and is reduced to the wavelength of the emission. Residues of the stimulating light that have not yet been stopped at the dichroic mirror are filtered out at the emission filter. Ideally, only the emission light hits a detector built into the microscope and becomes visible in the respective color.
Optimum measurement results require uniform illumination, especially when a large field of view of several micrometers or millimeters is required. In the case of inhomogeneous illumination, for example, uneven activation of the molecules to be examined can occur. The result: The molecules in the center fluoresce more strongly than those in the periphery of the incident illumination beam. If the periphery is not illuminated equivalently to the center, shading continues to occur when the individually recorded image grids are later merged. Therefore, measurements such as cell and tissue samples cannot be used for reliable analysis. Problems like these can be overcome by using the a|TopShape and the a|BeamExpander. Elements like these are possible through the use of aspheres in the systems. Our systems convince with their compact design as well as their precision and highest optical quality. The use of the optical components a|TopShape and a|BeamExpander enables the transformation of the Gauss beam into a uniform Flat-Top profile and thus uniform illumination across the entire field of vision. The generated flat field illumination convinces by a high spatial coherence, unbeatable optical performance and a high homogeneity of > 95 %. The even excitation of the molecules and minimal image overlaps (5%) can be guaranteed to your complete satisfaction.
The following graphic shows the working principle and general structure of a fluorescence microscope.