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Revealing Spectral Features in Two-Photon Absorption Spectrum of Hoechst 33342: A Combined Experimental and Quantum-Chemical Study Joanna Olesiak-Banska,*,† Katarzyna Matczyszyn,† Robert Zaleśny,*,†,‡ N. Arul Murugan,‡ Jacob Kongsted,§ Hans Ågren,‡ Wojciech Bartkowiak,† and Marek Samoc† †

Institute of Physical and Theoretical Chemistry, Wrocław University of Technology, Wyb. Wyspiańskiego 27, PL-50370 Wrocław, Poland ‡ Division of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute of Technology, SE-10691 Stockholm, Sweden § Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark ABSTRACT: We present the results of wide spectral range Z-scan measurements of the two-photon absorption (2PA) spectrum of the Hoechst 33342 dye. The strongest 2PA of the dye in aqueous solution is found at 575 nm, and the associated two-photon absorption cross section is 245 GM. A weak but clearly visible 2PA band at ∼850 nm is also observed, a feature that could not be anticipated from the one-photon absorption spectrum. On the basis of the results of hybrid quantum mechanics/molecular mechanics calculations, we put forward a notion that the long-wavelength feature observed in the two-photon absorption spectrum of Hoechst 33342 is due to the formation of dye aggregates.

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INTRODUCTION A requirement of most biological two-photon fluorescence microscopy (2PFM) investigations is the application of fluorescent markers, which selectively bind to specific biomolecules. Among the markers considered, organic and metal−organic dye molecules, fluorescent proteins and various nanoparticles have been utilized. Optimal fluorophores for 2PFM should have maximized 2PA cross sections, σ2PA, but should also exhibit features such as high water solubility, efficient fluorescence, low photodecomposition (bleaching) efficiency, and highly specific binding with a chosen biomolecule. Even though they may not be optimal in all respects, numerous fluorophores that are well-known from the one-photon imaging techniques are successfully applied in 2PFM, such as ethidium bromide, propidium iodide or DAPI for DNA staining.1−4 Hoechst dyes are one of the most widely used classes of DNA stains; they are low-molecular-weight compounds, capable of efficiently staining double-stranded DNA and emitting bright fluorescence upon DNA binding. They show strong one-photon absorption (a molar extinction coefficient of ∼104 M−1 cm−1) in the UV range and bright blue fluorescence. The Hoechst dyes display antitumor and antibiotic properties as well.5 Thus, they can serve as a model for the design of more advanced low-molecular-weight DNAbinding compounds: DNA stains, gene inhibitors, and drugs. A complication in the use of Hoechst dyes arises from the aromatic stacking interactions between the molecules. This © 2013 American Chemical Society

interaction is present in solutions at higher concentration of the dye, as confirmed in calorimetric and spectroscopic studies.6 Also upon noncovalent binding of Hoechst in the minor groove of DNA, when the dye-to-DNA ratio exceeds 1:1, several Hoechst molecules simultaneously enter the groove. Various models of multiple Hoechst molecules binding in DNA have been proposed, from the sequential binding7 up to sandwiches of dimers, trimers, and larger aggregates.8 Thus, the selfaggregation of Hoechst has an important impact on biophysical investigations as well as the imaging of Hoechst-stained DNA. The application of Hoechst 33342 (further referred to simply as Hoechst) in two-photon and three-photon microscopy was proposed by Lakowicz et al.9 They investigated the dependence of the Hoechst emission intensity on the incident power of multiphoton excitation, and up to ∼880 nm, they found a quadratic dependence, which confirms two-photon excitation.10,11 From two-photon excited fluorescence measurements, they estimated the 2PA cross section of Hoechst in the wavelength range from 560 to 760 nm to be ∼100 GM.11 However, the efficient excitation of biological markers and the optimization of microscope equipment requires full information about the 2PA spectrum of a fluorophore over a wide range of wavelengths. The simplest approximation that 2PA should Received: July 18, 2013 Revised: September 6, 2013 Published: September 9, 2013 12013

dx.doi.org/10.1021/jp407144k | J. Phys. Chem. B 2013, 117, 12013−12019

The Journal of Physical Chemistry B

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continuum model (default settings). At the same level of theory, we have computed the electrostatic-potential-derived (ESP) charges using the CHELPG procedure.16 These calculations have been performed using the Gaussian 09 suite of programs.17 The optimized geometry and the ESP charges for Hoechst were used in the molecular dynamics simulations. For Hoechst, we have used the general Amber force field (GAFF)18 in combination with the ESP charges as described above. For water, we have employed the TIP3P force field. The simulation box contained a single dye molecule and 12 665 water molecules. The box also contained a single chloride ion to neutralize the entire system. The simulations were carried out in an isothermal−isobaric ensemble in an orthorhombic box with dimensions of approximately 91, 73, and 69 Å. For the Hoechst dimer simulation, the force field parameters and the charges used for Hoechst dye were the same as those in the monomer simulation. However, the simulation box lengths were 93, 93, and 95 Å, which included the dimer, 2 chloride ions, and 23 051 water molecules. The initial geometry of the dimer has been taken from a simulation of Hoechst oligomer in water. An independent oligomer simulation starting with randomly placed dye molecules in water showed the formation of dimers and trimers during the simulation time scale. The configuration for the dimer used in the simulation is based on the coordinates picked up from the oligomer simulation of Hoechst. The interplanar distance between the two Hoechst dye molecules is approximately 3.5 Å in this initial configuration. Both monomer and dimer simulations employ a flexible molecular model for both dye and solvents, so the sampling over the intramolecular degrees of freedom is accounted for. The time step for the integration of the equation of motion was set to 1 fs. A cutoff of 10 Å has been used to calculate nonbonded interactions within the dye− solvent systems. The simulations were equilibrated until the density of the solute−solvent systems satisfied a convergence criterion. The simulations were carried out for 12 ns, and the trajectory from the last 4 ns was used to compute one- and twophoton properties. The dimer simulation was carried out for 4 ns, and the trajectory from the last 1 ns was used for the property calculations. All of the simulations described above were carried out using AMBER 11 software.19 Electronic structure calculations of Hoechst and its dimer have been performed using time-dependent density functional theory (TD-DFT) based on the Coulomb attenuating method with the B3LYP exchange-correlation functional (CAMB3LYP)20 and the TZVP basis set.21 The rationale behind this choice of functional is that intermolecular charge transfer states in the electronic structure of the Hoechst dimer are expected. Solvent effects have been taken into account using the polarizable embedding scheme where the polarization between the solute and solvent is taken into account using a self-consistent scheme linear in solvent polarization.22,23 The polarizable embedding approach is a variant belonging to the class of QM/MM models. By this we described the solute (dye) using DFT (as detailed above), and the water solvent was described on the basis of atomic point charges and atomic isotropic polarizabilities calculated using the LoProp24 approach as implemented in the MOLCAS25 software. The results presented in this work correspond to sampling over a set of 30 and 20 uncorrelated solute−solvent configurations in the case of the monomer and dimer of Hoechst, respectively. A satisfactory convergence of a cumulative average of the excitation energy was found for all

occur at the doubled wavelength of 1PA is often misleading. Although the Hoechst molecule is not centrosymmetric so that the mutual exclusion selection rule is not operative, additional factors concerning the 2PA transition probabilities may need to be taken into account. An important complication is that introduced by the possible self-aggregation of molecules at the concentrations of the dye used. In fact, as has been shown by some of us, organic dye aggregation may influence the twophoton absorption cross section to a large extent.12 In this article, we present an analysis of the 2PA spectrum of Hoechst, measured over a wide range of wavelengths from 500 to 1500 nm. The σ2PA value was calculated on the basis of the results of Z-scan measurements, where the nonlinear absorption cross section is determined directly by transmission measurements,13 thus it does not include possible uncertainties due to the varying quantum yield of luminescence, which may influence measurements based on two-photon fluorescence. Furthermore, we analyze the features of 1PA and 2PA absorption spectra as well as the effect of self-stacking based on the results of quantum-chemical calculations.

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MATERIALS AND EXPERIMENTAL METHODS 2′-[4-Ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1Hbenzimidazole trihydrochloride trihydrate, known as Hoechst 33342, was purchased from Sigma-Aldrich. Linear absorption spectra were measured for a series of concentrations of the dye in water with a Jasco V-670 UV−vis−NIR spectrophotometer. In the case of the Z-scan technique, nonlinear optical properties of dyes are conveniently measured in solutions placed in a 1 mm cuvette traveling along the focused Gaussian-shaped laser beam.14,15 A 10 mg mL−1 solution of Hoechst in water was prepared. Approximately 130 fs pulses were delivered by a laser system consisting of a Quantronix Integra-C regenerative amplifier operating as an 800 nm pump and a QuantronixPalitra-FS BIBO crystal-based optical parametric amplifier with a repetition rate of 1 kHz. The output from the Palitra amplifier was appropriately filtered using wavelength separators and colored-glass filters. It was then attenuated to the microjoule per pulse energy range. The beam was focused to a focal spot having a beam waist of w0 = 25−60 μm, which resulted in peak intensities in the range from 60 to 150 GW cm−2. Three InGaAs photodiodes (Thorlabs) collected the reference signal, the open-aperture (OA) signal, and the closed-aperture (CA) signal. The CA and OA Z-scan traces were recorded simultaneously in a single Z-scan run and used to determine the bulk nonlinear optical parameters of the solutions. Each wavelength data point also involved a measurement on a 4.66mm-thick fused silica plate, which provided the reference, a 1 mm glass cell filled with the solvent, and an identical cell with the solution.

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COMPUTATIONAL METHODS An integrated molecular dynamics and hybrid quantum mechanics/molecular mechanics (QM/MM) approach has been employed to study the structure and optical properties of Hoechst in water solvent. Two independent molecular dynamics simulations were carried out for the monomeric and dimeric forms of the dye. First, the geometry of the molecule was optimized using the Kohn−Sham (KS) formulation of density functional theory (DFT) using the B3LYP exchangecorrelation functional and the 6-311++G** basis set. The solvent effects were accounted for through the polarizable 12014



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analyzed transitions. For a set of one-photon excitation energies ℏωi, we have also determined the means of the excitation energy (separately for each excited state) n

ω̅ =

∑i = 1 ωifi n

∑i = 1 fi

(1)

where f i is the oscillator strength corresponding to excitation by a photon of angular frequency ωi . The two-photon absorption cross section was determined by assuming the Lorentzian function as the line-shape function σ2PA =

4π 2αa0 5ω 2 δ2PA 15c Γf

(2)

where α is the fine structure constant, a0 is the Bohr radius, c is the speed of light, and ω corresponds to the angular frequency of the incident light. The experimental value of the damping constant (Γf) equal to 0.55 eV was used to determine σ2PA for all transitions. δ2PA is the orientationally averaged two-photon absorption probability defined as δ2PA = FδF + GδG + HδH

where δF, δG, and δH are given by δF =

(3) 26

∑ SaaSbb* a,b

δG =

∑ SabSab* a,b

δH =

(4) Figure 1. (a) Molecular structure of Hoechst and (b) 1PA of aqueous solutions of high and low concentrations of Hoechst.

∑ SabSba* a,b

The second-order transition moment between states 0⟩ and |f ⟩ reads 0→f Sab

⎡ ⟨0|μ |n⟩⟨n|μ |f ⟩ ⟨0|μb |n⟩⟨n|μa |f ⟩ ⎤ a b ⎢ ⎥ =∑ + 1 ⎢ ωn − 1 ωf ⎥⎦ ω − ω n f n ⎣ 2 2

before and focusing after the focal plane of the Z-scan setup, which is a standard pattern in the case of positive nonlinear refractive index materials. Simultaneously, the transmittance in the OA trace decreases at the focus, z = 0, which is an indication of multiphoton absorption. The data was analyzed as described in ref 28 to obtain nonlinear optical microscopy parameters of the dye molecules. The experimental points are well reproduced by the theoretical curves, which justifies the assumption that the absorption is due to a two-photon process. The 2PA cross section spectrum of Hoechst is compared to the 1PA spectra in Figure 2c. It exhibits two maxima, around 575 and 850 nm, with σ2PA values equal to 245 and 35 GM, respectively. The former maximum overlaps with twice the wavelength of the one-photon absorption responsible for the transition to higher excited states (n1A ← 11A). Interestingly, a strong π−π* transition to the first excited state observed at 340 nm in the 1PA spectrum does not contribute significantly to the 2PA spectrum. This is in line with the results of quantum chemical calculations (see below). Additionally, a red-shifted band with its maximum at 850 nm is clearly visible. Although this maximum is located well above the doubled wavelength of the 1PA transition measured in a dilute solution of Hoechst (680 nm), it does fall within the range expected on the basis of the absorption of a more concentrated solution, thus it may be suspected that it may be related to the aggregation of the dye. To determine whether this is a plausible explanation of the origin of the long-wavelength maximum of 2PA, quantumchemical calculations have been performed on both monomers and dimers of Hoechst.

(5)

In the above equation, ℏωi is the energy difference between the excited state |i⟩ and the ground state |0⟩. In the case of the absorption of linearly polarized photons of equal energy, F = G = H = 2. All factors resulting from averaging are already included in eq 2. Electronic structure calculations have been performed using the locally modified version of the DALTON program.27

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RESULTS AND DISCUSSION Hoechst exhibits a structure typical of groove binders, with a crescent shape and flat hydrophobic aromatic rings (Figure 1 a). This set of structural properties together with a charge pattern of the molecule enables efficient binding of Hoechst in a DNA minor groove and gives rise to its strong fluorescence. However, such a molecular structure promotes the selfassociation of molecules in a solution of high concentration. It is a plausible explanation for a red shift in the absorption band, which we observe in the aqueous solution at cHoechst = 10 mg mL−1, as compared to the solution, where cHoechst = 10−4 mg mL−1 (Figure 1b). We investigated the NLO properties of an aqueous solution of Hoechst with the Z-scan technique. Figure 2a,b shows representative closed-aperture (CA) and open-aperture (OA) Z-scan traces (at 625 nm). The CA trace shows defocusing 12015



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Figure 2. Representative (a) closed-aperture and (b) open-aperture Z-scan traces of a Hoechst aqueous solution. (c) Comparison of one-photon (1PA) and two-photon absorption cross-section (σ2PA) spectra. Upper and lower scales of wavelengths correspond to 1PA and 2PA, respectively.

electron excitation corresponding to the 21A ← 11A transition is from the HOMO to the LUMO. There is also some admixture of the HOMO-1 → LUMO excitation, although it is accompanied by a roughly 10-fold decrease in probability. Figure 4 presents the frontier orbitals for a Hoechst monomer (in the very same conformation as that appearing in the dimer). It is seen that the lowest-energy π → π* transition does not lead to any substantial charge separation and is localized on the heterocyclic moieties. In the case of the dimer, the transition at λ1PA = 383 nm involves mainly the HOMO → LUMO oneelectron excitation (cf. Figure 3) that corresponds to an intermolecular charge transfer. As far as the transition in the dimer at λ1PA = 349 nm is concerned, there are two oneelectron excitations of equally large probability, namely, HOMO → LUMO+1 and HOMO−1 → LUMO+1. The former exhibits distinct intermolecular charge-transfer character. Surprisingly, this is not reflected in the value of the Λ diagnostic for the 31A ← 11A transition, which was found to be 0.49.29 In the orbital picture, transitions to higher excited states involve many one-electron excitations of equally moderate probability. The results of the transition analysis presented thus far correspond to a dimer geometry that is only an approximate representation of an aggregate. In fact, our results of molecular dynamics simulation indicate the formation of dimers and other oligomers such as trimer, tetramer, and pentamer. However, the largest population of oligomers is that of dimers, and these are expected to contribute to the optical properties of aggregates to a larger extent than other oligomers. We have not attempted to analyze the optical spectra of other oligomers because of their size (which amounts to more than 150 atoms) and the necessity of performing calculations for many such oligomers to account for the sampling effect. Because of the prohibitive computational expense, we limit the analysis of the electronic structure to dimers. As mentioned in the Computational Methods section, the initial configuration for the dimer was taken from the oligomer simulation. It follows from these simulations that once the dimer is formed it maintains its state of association. To put the analysis of the aggregation process on a quantitative basis, we have computed the center-of-mass distance distribution between the two monomer units in the dimer (cf. Figure 5) and also followed the evolution of this distance as a function of time (cf. Figure 5). These plots can easily be misinterpreted because the large distance between monomers can be attributed to the dissociated state. In fact, the large distances seen in these plots can be explained by the tendency of two monomers to slide over one another.

We determined the electronic structure of the monomer and dimer of Hoechst. The results of the excitation energy and oscillator strength calculations corresponding to the eight lowest electronic states for the monomer are presented in Table 1 and in Figure 3. Our theoretical estimate of the wavelength Table 1. Excitation Wavelengths (λ1PA, nm), Oscillator Strengths ( f), and Two-Photon Absorption Cross Sections (σ2PA, GM) Corresponding to the Lowest Electronic Transitions for a Set of 30 Uncorrelated Configurations of the Hoechst Monomer in a Water Solutiona transition

λ1PA

f

← ← ← ← ← ← ← ←

345 306 278 265 257 248 241 236

1.58 0.28 0.05 0.06 0.06 0.12 0.09 0.14

1

2A 31A 41A 51A 61A 71A 81A 91A

1

1A 11A 11A 11A 11A 11A 11A 11A

σ2PA 5 10 22 38 40 267 249 193

a

The experimental value of the spectral width equal to 0.55 eV was used together with eq 2 to determine σ2PA for all transitions.

corresponding to the first absorption band maximum for the monomer (345 nm) is in excellent agreement with the experimental value (342 nm). For a dimeric system, there is also a long-wavelength absorption band of substantial oscillator strength (0.33) shifted by 22 nm with respect to the absorption band maxima of the monomer. As seen in Figure 3, the first absorption band corresponding to the dimer of Hoechst at 367 nm is covered by the band with the maximum at 345 nm. We shall now focus on the nature of the electronic transitions observed in the spectra of monomeric and dimeric Hoechst. For this purpose, from the set of uncorrelated configurations of the dimer, we picked up the one corresponding to the largest oscillator strength of the long-wavelength transition (i.e., λ1PA = 383 nm and f = 0.95 for the 21A ← 11A transition). For comparison, the spectroscopic parameters characterizing the 31A ← 11A transition are λ1PA = 349 nm and f = 2.15. In what follows, we analyze the character of the 21A ← 11A and 31A ← 11A transitions for the dimer and the 21A ← 11A transition for a monomer (with the same geometry as that in the dimer) with solvents effects taken into account using the polarizable continuum model. This model allows us to treat the solute− solvent interactions for the monomer and dimer on an equal basis. In the case of the Hoechst monomer, the dominant one12016



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Figure 3. One-photon spectra simulated for the monomer (bottom) and the dimer (top) based on a set of statistically uncorrelated configurations extracted from the MD simulation. Dashed and dashed−dotted lines correspond to the 21A ← 11A and 31A ← 11A absorption bands, respectively. Solid lines correspond to the simulation of spectra including eight electronic transitions. The spectral width for each transition was estimated on the basis of the sampling of uncorrelated configurations.

Figure 5. Center-of-mass distance distribution between the two monomer units in the dimer (top) and its instantaneous value for different configurations in the trajectory (bottom).

Figure 4. Fronier orbitals involved in electronic transitions to the lowest excited states (i.e., 21A ← 11A and 31A ← 11A). See the text for details.

To gain more insight into this aspect of aggregation, we have also computed the minimum distance distribution function between the two monomers (only p-block atoms included), and the results are shown in Figure 6. This function is similar to the minimum distance radial distribution function calculated by Georg et al.,30 except that the latter one is normalized by a factor. As can be seen, the minimum distance between the components of the dimer does not exceed 3.6 Å, suggesting that the dimer is in the associated state throughout the simulation. The distance corresponding to the most probable value is 3.2 Å, and the minimum value observed for the distance is 2.8 Å. Such a small interplanar distance between the

Figure 6. Minimum distance distribution between two monomers. See the text for details.

monomers in a dimer suggests that the electronic structure of one monomer might be strongly perturbed by its neighbor. We 12017



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electronic excited states is characterized by a relatively low value of the 2PA cross section. As a result, none of these lowest-lying states correspond to the prominent feature at 575 nm in the measured two-photon absorption spectrum. The experimental 2PA spectrum also reveals the existence of a weak but clearly visible band at ∼850 nm of no obvious correspondence to any transition in the 1PA spectrum. We have shown, on the basis of the results of the QM/MM calculations, that the longwavelength feature might be directly linked to the selfassociation of Hoechst molecules in aqueous solution. Molecular dynamic simulations provided us with insight into the structure of aggregates and revealed the formation of dimers and other oligomers such as trimers, tetramers, and pentamers. The results of the electronic structure calculations show clear changes in the electronic structure of the dye upon dimerization in aqueous solution. In particular, a new lowenergy state involving intermolecular charge transfer is predicted, thus making the aggregation process a plausible origin of the feature at 850 nm in the 2PA spectrum.

have also computed the angle between the molecular axis of both monomers, and the results are shown in Figure 7.

Figure 7. Distribution of the angles shown in the inset. See the text for details.

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Interestingly, the zero degree dimer configuration is not the one largely populated but rather the one with the 10° angle. This can be easily explained by the fact that the bulky and flexible cyclohexane groups at the end do not allow the molecule to adopt a perfect stacking alignment. On the basis of what has been already discussed and considering the large oscillator strength corresponding to the 21A ← 11A transition in the dimeric system of Hoechst, one may formulate the hypothesis that the experimentally determined broadening and absorption maximum shift is indeed due to aggregation. This is supported by the results of the quantum-chemical calculations presented herein. We now turn to the discussion of two-photon absorption spectra. Two-photon absorption cross sections corresponding to transitions to the lowest eight states in the monomer of Hoechst are presented in Table 1. Note that the experimental value of the spectral width equal to 0.55 eV was used together with eq 2 to determine σ2PA for all transitions. The data reported in Table 1 clearly show that σ2PA for the 21A ← 11A transition is in fact rather small and the dominant contribution to the 2PA intensity is due to the two-photon 71A ← 11A and 81A ← 11A transitions. This is in good agreement with the experimental data presented in Figure 2. Considering the electronic structure of the Hoechst dimer, one might link the origin of the absorption band in the range of 700−850 nm in the experimental two-photon absorption spectrum to the intermolecular charge-transfer excitations in the aggregate. In fact, the values of σ2PA for many (out of 20) considered configurations of dimers were found to be in the range of 15− 20 GM. This is in good agreement with the experimental values of σ2PA, which in the range 700−800 nm do not exceed 30 GM.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], robert.zalesny@pwr. wroc.pl. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support from Iuventus Plus grant IP2012 004172 and the Foundation for Polish Science (under the “Welcome” program). R.Z. acknowledges financial support from the Wenner-Gren Foundations. This work was supported by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wroclaw University of Technology and by a grant from the Swedish Infrastructure Committee (SNIC) for the project “Multiphysics Modeling of Molecular Materials”, SNIC 023/ 07-18. J.K. thanks the Danish Center for Scientific Computing (DCSC), The Danish Councils for Independent Research (The Sapere Aude Programme), the Lundbeck Foundation, and the Villum Foundation for financial support.

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CONCLUSIONS We have performed one- and two-photon absorption measurements for the Hoechst 33342 dye in aqueous solution over a wide spectral range. The 2PA spectrum reveals the strongest absorption at 575 nm, with σ2PA = 245 GM. To gain insight into the electronic structure of the solvated dye, we have supported the experimental data with the results of quantum chemical calculations employing the polarizable embedding scheme combined with time-dependent density functional theory. It turns out, on the basis of the results of quantum-chemical calculations, that the two-photon transition to the lowest 12018



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