Two green fluorescent protein (GFP) chromophore analogs (4Z)-4-(N,N-dimethylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DMPI) and (4Z)-4-(N,N-diphenylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DPMPI) were investigated using femtosecond fluorescence up-conversion spectroscopy and quantum chemical calculations with the results being substantiated by HPLC and NMR measurements. The femtosecond fluorescence transients are found to be biexponential in nature and the time constants exhibit a significant dependence on solvent viscosity and polarity. A multicoordinate relaxation mechanism is proposed for the excited state relaxation behavior of the model GFP analogs. The first time component (τ(1)) was assigned to the formation of twisted intramolecular charge transfer (TICT) state along the rotational coordinate of N-substituted amine group. Time resolved intensity normalized and area normalized emission spectra (TRES and TRANES) were constructed to authenticate the occurrence of TICT state in subpicosecond time scale. Another picosecond time component (τ(2)) was attributed to internal conversion via large amplitude motion along the exomethylenic double bond which has been enunciated by quantum chemical calculations. Quantum chemical calculation also forbids the involvement of hula-twist because of high activation barrier of twisting. HPLC profiles and proton-NMR measurements of the irradiated analogs confirm the presence of Z and E isomers, whose possibility of formation can be accomplished only by the rotation along the exomethylenic double bond. The present observations can be extended to p-HBDI in order to understand the role of protein scaffold in reducing the nonradiative pathways, leading to highly luminescent nature of GFP.

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The good agreement of the graphite calorimeter and free-air chamber results indicates that both devices are performing as expected. Further investigations at higher dose rates than 50 Gy/s are planned. At higher dose rates, recombination effects for the free-air chamber are much higher and expected to lead to much larger uncertainties. Since the graphite calorimeter does not have problems associated with dose rate, it is an appropriate primary standard detector for the synchrotron IMBL x rays and is the more accurate dosimeter for the higher dose rates expected in radiotherapy applications.