Fluorescence Microscopy
Fluorescence microscopy is a special form of light microscopy, which has established itself worldwide and is often used in the field of life sciences thanks to its advantages in terms of contrast, selectivity and simplicity of staining.
by Dr. Jakob Bierwagen, AHF analysentechnik AG
Background
The human eye is an impressive organ whose resolution is about 2 angular minutes. As a result, one can achieve a spatial resolution of up to 15 microns – three times finer than a human hair. However, if you want to examine objects that have much smaller structures, a microscope is needed. The most commonly used type of microscope is the light microscope, where the user can deploy his eyes as detectors. In order to distinguish the very small objects from each other, sufficient contrast is required. Since organic samples are usually colorless or uniformly colored, contrast media such as fluorescent dyes are used for this purpose.
Incidentally, the fluorescence effect is an intramolecular effect in which a dye molecule absorbs a higher-energy photon (e.g. blue light), converts a part thereof into heat and then emits a lower-energy photon (e.g. green light). By this color change, it is possible to separate the light used for the excitation of the dye by dielectric filters. As a result, one sees only the green glowing structures which are stained with the dye.
It Depends on the Right Filter
It is crucial to use very good optical filters, since only in this way a sufficient suppression of 1: 10^6 of the excitation light is achieved in order to see the weak fluorescent light. To make sure that the microscope light source illuminates the sample at the correct wavelength, a dielectric filter is also used in the excitation path which suppresses all other wavelengths. Usually in a fluorescence microscope, both the excitation and the detection of the sample proceeds only via one objective lens. Due to this, one needs in addition to excitation and emission filter a so-called dichroic beam splitter. This is a mirror that is transparent or reflective, depending on the wavelength. In the case described above, it should reflect blue light from the excitation to the sample, while green light passes through the mirror and the emission filter to the viewer.
We Are Happy to Assist You
At AHF analysentechnik, you will find a wide selection of these optical filters, which have proven their worth in fluorescence microscopy. We are happy to assist you in choosing the right filter for your microscope and your specific application. You benefit from our long-term interdisciplinary expertise in microscopy.