Limitations
Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Photobleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons excited during fluorescence. Photobleaching can severely limit the time over which a sample can be observed by fluorescent microscopy. Several techniques exist to reduce photobleaching such as the use of more robust fluorophores, by minimizing illumination, or by using photoprotective scavenger chemicals.
Fluorescence microscopy with fluorescent reporter proteins has enabled analysis of live cells by fluorescence microscopy, however cells are susceptible to phototoxicity, particularly with short wavelength light. Furthermore fluorescent molecules have a tendency to generate reactive chemical species when under illumination which enhances the phototoxic effect.
Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of the specific structures which have been fluorescently labeled. For example observing a tissue sample prepared with a fluorescent DNA stain by fluorescent microscopy only reveals the organisation of the DNA within the cells and reveals nothing else about the cell morphologies.
Read more about this topic: Fluorescence Microscope
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—James Thurber (1894–1961)
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