In fluorescence microscopy, the specimens are treated with special reagents. Their individual molecules are able to absorb light for an extremely short time – usually billionths of a second – and then to emit it again. However, the emitted light features a wavelength which is slightly shifted “towards red“. If, for example, blue light is absorbed, green light will be emitted immediately afterwards. Green is changed to yellow, yellow to reddish orange and invisible UV light to visible light. This shift is termed Stokes shift after its discoverer. In fluorescence, the wavelength of the emitted light is about 20 to 50 nanometers longer than absorbed exciting light.|
Fluorescence molecules can only absorb light of a certain wavelength. Each of the various fluorochromes exhibits its own, very specific absorption spectrum, depending on the internal structure of the fluorescence molecules and sometimes also on their surroundings. Furthermore, not every photon is absorbed, but only a part of their radiating light. The absorbed photons are not emitted again in their entirety either. Good fluorescence markers feature a high “quantum yield” – a term describing the ratio of the emitted to the absorbed photons.
This effect is very useful for microscopy:
a specimen marked in this way is illuminated with pure, filtered blue light and viewed using a barrier filter which is completely opaque to blue light, but which transmits long-wave green, yellow and red light. The structures marked with fluorescence molecules – e.g. parts of a cytoskeleton – then light up green against a black background.
When microfluorescence was initially introduced, the specimens were usually dyed non-specifically with fluorochromes. This type of marking usually looks bright, since many fluorescence molecules are bonded everywhere. Nowadays, however, fluorescence methods are much more specific. This has been made possible in particular by the permanent coupling of the fluorescence molecules with biological substances, e.g. antibodies (in this case, it is no longer the dye which determines the bonding position, but the biologically active molecule). Normally, this results in weak fluorescence images in the microscope because much less dye is bonded. However, the information obtained, e.g. in the diagnosis of illnesses, is becoming much more exact.
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|A specimen marked with a fluorescence dye is illuminated in reflected light with intensive, blue light.||If the specimen is viewed through a barrier filter, the blue excitation light can no longer be seen. Instead, the yellow fluorescence light is clearly visible on the specimen.|