Blue fluorescent proteins excitation

Since GFP mutants emitting blue Ught have to be excited with near-UV lasers, they are less favourable dyes for sensitive intracellular applications due to the significant interference of UV radiation with cellular components. Nevertheless, advanced spectroscopic concepts such as three-photon excitation might shed new interest on blue fluorescent proteins, e.g. for combined two- and three-pho-... 

Cyan fluorescent proteins (CFPs) have blue-shifted excitation and emission spectra, because of the mutation Tyr66Trp inside the chromophore (Fig. 5.3C) [34], CFP fluorescence (Ex 435 nm/Em 474 nm) is less blue-shifted than for EBFP and CFP excitation is intermediate to the excitation of the neutral and anionic chromo-phores of avGFP [4], CFPs are widely used for dual-color imaging and FRET applications together with yellow fluorescent proteins (YFP, Section 3.6). 

Mitra, R. D., Silva, C. M. and Youvan, D. C. (1996). Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein. Gene 173, 13-7. 

A UV laser is needed for exciting the blue-fluorescing agents, 4, 6-diamidino-2-phenyhndole (DAPI) and Hoechst 33342, which are DNA-intercalating stains, and for indo-1, a fluorescent calcium chelator dye. Violet diode lasers that are offered in some newer instruments accommodate fluorochromes such as Cascade Blue, Pacific Blue, and cyan fluorescent protein, and are also capable of exciting DAPI (Shapiro and Perlmutter 2001 Telford et al., 2003). 

The first described member of the cyan fluorescent proteins (CFPs) resulted from a rationally designed chromophore mutation of Aequorea GFP. Heim and co-worker replaced Tyr66 with Trp and found the peak wavelength for excitation and emission of this GFP derivative (GFP-Y66W) to be shifted to 436 and 476 nm, respectively [52], Because of this blue-green/cyan light emission the protein was called cyan fluorescent protein or CFP. 

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