6. Problems and Limitations: Immunofluorescence is one of the main methods regarding imaging techniques due to its high specificity and it’s relatively simply implementation. Nevertheless there are also some problems when dealing with immunofluorescence. 6.1. Photobleaching Fluorochromes can lose the ability to fluoresce due to the photochemical destruction of a fluorophore. This phenomenon is called photobleaching [15]. Although this process is not fully understood yet, there are some hypotheses trying to explain photobleaching. One of the main theories is that molecular oxygen interacts with excited fluorophores. Dye molecules of the fluorophores can get from their original singlet excited state to the long-lived triplet excited state making it possible to interact with molecular oxygen. This allows the generation of singlet oxygen which has a longer lifetime and can react with the excited fluorophore, which can lead to irreversible reactions (bleaching of the fluorophore) [16]. Photobleaching is increased if the …show more content…
FACS measurements make it possible to detect target proteins via binding to a fluorescence labeled antibody. Therefore the flow cytometry instrument has different lasers which can excite the fluorophores. FACS can then measure the intensity of the emitted light. As well as the epi-fluorescence microscope also the FACS machine uses different filters in order to pass on only the light of a certain wavelength. Modern flow cytometry machines can have up to 10 lasers and even more detectors. An advantage of this method is that it is also possible to measure the size and the granularity of a cell. However, as in immunofluorescence microscopy, fluorescence compensation has to be taken into account when using FACS as well. The procedure for sample preparation is very similar to that in immunofluorescence experiments, which is an advantage when comparing the two