A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at ≈390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Förster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.
Fluoro-Jade B, like its predecessor Fluoro-Jade, is an anionic fluorescein derivative useful for the histological staining of neurons undergoing degeneration. However, Fluoro-Jade B has an even greater specific affinity for degenerating neurons. This notion is supported by the conspicuous staining of degenerating neuronal elements with minimal background staining. This improved signal-to-noise ratio means that fine neuronal processes including distal dendrites, axons and axon terminals can be more readily detected and documented. Although the staining time and dye concentration are reduced, the method is as rapid, simple and reliable as the original Fluoro-Jade technique. Like Fluoro-Jade, Fluoro-Jade B is compatible with a number of other labeling procedures including immunofluorescent and fluorescent Nissl techniques.