Minimizing photobleaching of Blue Fluorescent Protein (BFP) using the Agilent Cary Eclipse fluorescence spectrophotometer

Blue Fluorescent Protein (BFP) provides an example of a fluorophore that is susceptible to photobleaching: “…even with folding improvements, BFP still suffers from a relatively low fluorescence quantum yield [compared to green fluorescent protein] and relatively easy bleaching.” (Tsien, 1998)1. Despite the useful spectral properties of BFP for use in multi-labelling studies and techniques such as FRET (fluorescence resonance energy transfer),2 it is likely that its low quantum yield and susceptibility to photobleaching could limit ongoing detection in instruments that irradiate the sample continuously, or too intensely, or both. Accordingly, the present study aimed to determine whether using the Agilent Cary Eclipse fluorescencespectrophotometer, which utilizes a special xenon flash lamp, photobleaching of BFP could be eliminated, or at least reduced, compared to other commercially available instruments having continuous or pulsed light sources.