Absorption and fluorescence spectra of the probe Hoechst 33258
ABSTRACT The steric and electronic structure and absorption spectra of the fluorophore Hoechst 33258 (I) and a number of model compounds in various protonated forms were calculated by the semi-empirical quantum chemical methods PM3 and CNDO/S. The low-frequency transition was shown to provide an increase in electron affinity of the imidazole rings. The fluorescence quenching of I in neutral aqueous solutions was suggested to result from the keto form (or bipolar structure), derived via proton transfer in the excited state of the dimer associate of I. The polar (approximately 10 D) fluorescence state of I due to intramolecular charge transfer is markedly sensitive to the polarity of the environment.
- SourceAvailable from: Alexander Heisterkamp[Show abstract] [Hide abstract]
ABSTRACT: In two-photon laser-scanning microscopy using femtosecond laser pulses, the dependence of the photobleaching rate on excitation power may have a quadratic, cubic or even biquadratic order. To date, there are still many open questions concerning this so-called high-order photobleaching. We studied the photobleaching kinetics of an intrinsic (enhanced Green Fluorescent Protein (eGFP)) and an extrinsic (Hoechst 33342) fluorophore in a cellular environment in two-photon microscopy. Furthermore, we examined the correlation between bleaching and the formation of reactive oxygen species. We observed bleaching-orders of three and four for eGFP and two and three for Hoechst increasing step-wise at a certain wavelength. An increase of reactive oxygen species correlating with the bleaching over time was recognized. Comparing our results to the mechanisms involved in intracellular ablation with respect to the amount of interacting photons and involved energetic states, we found that a low-density plasma is formed in both cases with a smooth transition in between. Photobleaching, however, is mediated by sequential-absorption and multiphoton-ionization, while ablation is dominated by the latter and cascade-ionization processes.Biomedical Optics Express 01/2011; 2(4):805-16. · 3.18 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Exploration of environmental dynamics using intrinsic biological probe tryptophan is very important; however, it suffers from various difficulties. An alternative probe, kynurenine (KN), has been found to be an efficient probe for the ultrafast dynamics in the biological environment (Goswami et al.,  J. Phys. Chem. B., 114, 15236-15243). In the present study, we have investigated the efficacy of KN for the exploration of relatively slower dynamics of biologically relevant environments. A detailed investigation involving UV-Vis, steady-state/time-resolved fluorescence spectroscopy and Förster resonance energy transfer (FRET) studies on KN compared to a well-known solvation probe, H33258, a DNA-minor groove binder in a model nonionic reverse micelle reveals that ultrafast internal conversion associated with the hydrogen-bonding dynamics masks KN to become a dynamical reporter of the immediate environments of the probe.Photochemistry and Photobiology 09/2011; 88(1):38-45. · 2.29 Impact Factor