Absorption and fluorescence spectra of the probe Hoechst 33258

Institute of Macromolecular Compounds, Russian Academy of Sciences, St. Petersburg Russian Federation
Journal of Photochemistry and Photobiology A Chemistry (Impact Factor: 2.5). 09/1994; 83(1):39-47. DOI: 10.1016/1010-6030(94)03797-3


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.

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    • "We presume that our findings for the photobleaching-order of Hoechst (see Fig. 4) are closely related to a two-photon excitation in the first singlet followed by several sequential-absorption events in higher states. In contrast to eGFP, Hoechst has electronic states above the first singlet [32] in which another one-photon absorption is likely because of the very large photon density [8]. Bleaching is then evoked by one or more one-photon absorptions after excitation into the first singlet. "
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    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.
    Full-text · Article · Apr 2011 · Biomedical Optics Express
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    ABSTRACT: The groove binding dye Hoechst 33258 was investigated both in aqueous solution and in the adsorbed form on silver colloids and island films. Fourier transform Raman, resonance Raman and surface enhanced resonance Raman spectra were used together with the results of a normal coordinate analysis for a comprehensive assignment of the rich vibrational spectrum. Relative band intensities in the SERRS spectra indicate that the molecule is adsorbed with its long axis oriented along the silver surface and with the planes of the benzimidazole rings approximately parallel to the surface. The molecule is bound to the surface as a cation protonated at a piperazine nitrogen atom and is desorbed on complete deprotonation.
    No preview · Article · Jun 1995 · Journal of Raman Spectroscopy
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    ABSTRACT: The complexes of Hoechst 33258 with poly[d(A-T)2], poly[d(I-C)2], and poly[d(G-C)2], and poly[d(G-m5C)2] were studied using linear dichroism, CD, and fluorescence spectroscopies. The Hoechst-poly[d(I-C)2] complex, in which there is no guanine amino group protruding in the minor groove, exhibits spectroscopic properties that are very similar to those of the Hoechst-poly[d(A-T)2] complex. When bound to both of these polynucleotides, Hoechst exhibits an average orientation angle of near 45 degrees relative to the DNA helix axis for the long-axis polarized low-energy transition, a relatively strong positive induced CD, and a strong increase in fluorescence intensity--leading us to conclude that this molecule also binds in the minor groove of poly[d(I-C)2]. By contrast, when bound to poly[d(G-C)2] and poly[d(G-m5C)2], Hoechst shows a distinctively different behavior. The strongly negative reduced linear dichroism in the ligand absorption region is consistent with a model in which part of the Hoechst chromophore is intercalculated between DNA bases. From the low drug:base ratio onset of excitonic effects in the CD and fluorescence emission spectra, it is inferred that another part of the Hoechst molecule may sit in the major groove of poly[d(G-C)2] and poly[d(G-m5C)2] and preferentially stacks into dimers, though this tendency is strongly reduced for the latter polynucleotide. Based on these results, the importance of the interactions of Hoechst with the exocyclic amino group of guanine and the methyl group of cytosine in determining the binding modes are discussed.
    No preview · Article · May 1996 · Biopolymers
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