14 June 2004 Fluorescence enhancement of asCP595 is due to consecutive absorbance of two photons
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Colored proteins are widely used as gene markers in biotechnology. Chromophores result from autocatalytic posttranslational reactions involving several amino acids. The protein asCP595 was isolated for the first time from the coral as a weakly fluorescent chromoprotein with a fluorescence maximum at 595 nm. Strong illumination in the blue wing of the low energy absorption band results in a superlinear increase of the fluorescence yield and shifts its fluorescence spectrum by about 10 nm to the red. Time resolved fluorescence measurements using excitation pulses with 10 ps duration revealed a multiexponential decay pattern with time constants in the range from 20 ps to 2.1 ns. The ratio of amplitudes related to the different time constants depends on the intensity of illumination favoring the ns component at high intensities. Transient absorption measurements using ultrashort excitation pulses (150 fs, 1 kHz repetition rate) did not reveal excited states with nanosecond lifetimes as observed in fluorescence upon excitation using 10 ps pulses. This observation leads to the notion that within 10 ps a second photon is absorbed by a state not yet populated within 150 fs. As a consequence we propose two different excited singlet states operative in asCP595, one with low fluorescence quantum yield peaking at 595 nm and one with high fluorescence quantum yield peaking at 605 nm which is populated via the consecutive absorption of two photons at high excitation intensities.
© (2004) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Alexander P. Savitsky, Alexander P. Savitsky, Michail B. Agranat, Michail B. Agranat, Konstantin A. Lukyanov, Konstantin A. Lukyanov, Tanja Schuttrigkeit, Tanja Schuttrigkeit, Till von Feilitzsch, Till von Feilitzsch, Christian Kompa, Christian Kompa, Maria-Elisabeth Michel-Beyerle, Maria-Elisabeth Michel-Beyerle, "Fluorescence enhancement of asCP595 is due to consecutive absorbance of two photons", Proc. SPIE 5329, Genetically Engineered and Optical Probes for Biomedical Applications II, (14 June 2004); doi: 10.1117/12.533013; https://doi.org/10.1117/12.533013

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