Chemical Physics Letters 583 (2013) 160–164

Contents lists available at ScienceDirect

Chemical Physics Letters

journal homepage: www.elsevier.com/locate/cplett

Polarization dependent two-photon absorption spectroscopy on a

naturally occurring biomarker (curcumin) in solution: A
theoretical–experimental study
Jose A. Tiburcio-Moreno a,b, J.J. Alvarado-Gil a, Carlos Diaz b, Lorenzo Echevarria b,c,⇑,
Florencio E. Hernández b,d,⇑
a
Departamento de Física Aplicada, Centro de Investigación y de Estudios Avanzados del IPN-Unidad Mérida, Antigua Carretera a Progreso km. 6, 97310 Mérida, YUC, Mexico
b
Department of Chemistry, University of Central Florida, P.O. Box 162366, Orlando, FL 32816-2366, USA
c
Departamento de Química, Universidad Simón Bolívar, Caracas 1020A, Venezuela
d
The College of Optics and Photonics, CREOL, University of Central Florida, P.O. Box 162366, Orlando, FL 32816-2366, USA

a r t i c l e i n f o a b s t r a c t

Article history: We report on the theoretical–experimental analysis of the two-photon absorption (TPA) and two-photon
Received 20 June 2013 circular-linear dichroism (TPCLD) spectra of (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadi-
In final form 30 July 2013 ene-3,5-dione (curcumin) in Tetrahydrofuran (THF) solution. The measurement of the full TPA spectrum
Available online 6 August 2013
of this molecule reveals a maximum TPA cross-section at 740 nm, i.e. more than 10 times larger than the
maximum reported in the literature at 800 nm for the application of curcumin in bioimaging. The TPCLD
spectrum exposes the symmetry of the main excited-states involved in the two-photon excitation pro-
cess. TD-DFT calculations support the experimental results. These outcomes are expected to expand
the application of natural-occurring dyes in bioimaging.
Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction photoexcitation of curcumin [10]. With a characteristic fluorescence

Turmeric is a precious substance present in the daily diet of mil-
lions of people in the world, predominantly in Middle Eastern and
Asian countries. This natural product has been extensively used as
a flavor enhancer in the Indian cuisine and for therapeutic pur-
poses in Asian and European medicine thanks to its anti-oxidant,
anti-inflammatory, anti-septic and anti-malarial activity [1].
Curcumin, a naturally occurring yellow-orange pigment found in
the rhizome of turmeric (Curcuma longa), is one of the main
components found in curry [2]. In addition to the already men-
tioned biological significance, [1] its anti-tumor properties [3]
and anti-amyloid activity, [4] curcumin presents a moderate-high
singlet oxygen quantum yield in nonpolar solvents [5] and a
relatively high fluorescence quantum yield (uF > 0.30) in polar sol-
vents [6]. The former has already resulted in the employment of
curcumin as a photodynamic therapy agent in skin cancer
treatment [7,8]. However, the application of the latter for marking
biological samples is still, to the best of our knowledge, scarce
in the literature [9]. Furthermore, intramolecular proton transfer
in the excited-state is a major photophysical process in the
⇑ Corresponding authors. Address: Department of Chemistry, University of
Central Florida, P.O. Box 162366, Orlando, FL 32816-2366, USA. Fax: +1
4078232252.
E-mail addresses: lorenzoer@usb.ve (L. Echevarria), florencio.hernandez@ucf.edu
(F.E. Hernández).
emission spectrum between 480 and 660 nm, noteworthy fluorescent
quantum yield and extraordinary biocompatibility, curcumin is an
excellent candidate for bioimaging that could surpass the expectation
of specifically engineered fluorescent dyes and nanoparticles with
intrinsic toxicity [11]. In addition, its extended p–electron delocaliza-
tion and donor–p–acceptor_acceptor–p–donor motif make of this
molecule a suitable marker for multiphoton bioimaging.
It is known that the electronic excitation via TPA using two pho-
tons of longer wavelengths than those required for linear absorption
yields a reduction of losses due to scattering and one-photon
absorption (OPA) [12]. The TPA quadratic dependence with the inci-
dent irradiance confers this excitation process more spatial resolu-
tion and penetration depth, as well as a better background
discrimination [13]. In addition, TPA reduces photobleaching of the
fluorophores and photodamage to living specimens [13]. These spe-
cific features have found enormous technological applications in the
fields of biology [13,14] and medicine, [15] among others [16–18]. In
this direction, Farkas et al. lately used curcumin for multimodal
wide-field two-photon excitation imaging of eyecup specimen from
AD transgenic mouse model [19]. More recently, Kumar and co-
workers [2] reported on the study of the two-photon fluorescence
of this naturally fluorescent dye in two different solvents (THF and
DMOS). These authors have shown the direct outstanding applica-
tion of curcumin in two-photon confocal microscopic studies of
HL-60 derived neutrophil cells and acknowledged the use of this
fluorophore in bioimaging due to its extraordinary biocompatibility

0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cplett.2013.07.080
 J.A. Tiburcio-Moreno et al. / Chemical Physics Letters 583 (2013) 160–164 161

and effective penetration into the cells. Although, curcumin has al- [23], employing the Becke’s three-parameter exchange, Lee, Yang
ready been tested as a multiphoton fluorophore, its maximum TPA and Parr correlation (B3LYP) hybrid functional [24–26] and the
cross-section (dTPA ðkÞ) between 780 and 900 nm, the investigated 6-31++G⁄⁄ basis set [27]. The TPA response (for the degenerate
spectral region, [2,19] is only few Goeppert–Mayer, i.e. <6 GM case) for the first 18 electronic excited states was computed with
(1 GM = 1  1050 cm4 s photons1) [20]. Consequently, the TPA ac- DALTON2011, [28] employing Time-Dependent DFT (TD-DFT) [29]
tion cross-section defined as dTPA ðkÞ ¼ dTPA ðkÞ  uF , which is the at the same level of theory as for the geometry optimization. All
parameter that serves to evaluate specific chromophores for two- theoretical calculations were performed in THF within the polariz-
photon fluorescence microscopy, is still relatively low due to the able continuum model (PCM) [30].
very modest dTPA ðkÞ. In order to unveil the full potential of this nat- The TPA spectra were obtained through, [31–33]
ural dye as a two-photon biomarker, a systematic characterization of
the two-photon absorption (TPA) of curcumin in solution is in great 4p3 aa50 X 2
dTPA
0f ðxÞ ¼ ðhxf Þ dTPA
0f ðx0f Þgð2x; x0f ; CÞ; ð1Þ
need. In light of these recent finding we have engaged in the study of c f
the TPA of this important molecule.
In this article, we report on the theoretical–experimental anal- 2 2
X
dTPA
0f ðxÞ  1:25273  10  ðhxÞ gð2x; x0f ; CÞ  dTPA
0f ðx0f Þ: ð2Þ
ysis of the TPA and two-photon circular-linear dichroism (TPCLD)
f
spectra of (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hept-
adiene-3,5-dione (curcumin) in Tetrahydrofuran (THF) solution In Eq. (1), c is the speed of light in vacuum, a0 is the Bohr’s ra-
(curcumin/THF). Using a tunable 90 fs amplified OPA-femtosec- dius, a is the fine structure constant, E ¼  hx is the photon energy
ond system working at 50 Hz repetition rate and over a broad spec- (half of the transition energy for the degenerate case) and dTPA
0f ðx0f Þ
tral range (600–900 nm), we measured the full TPA spectra of this is the orientational averaged two-photon probability for the
dye with linear and circular polarization employing the double L- degenerate case. A Lorentzian line-shape gð2x; x0f ; CÞ was used
scan technique [21]. Our results reveal a maximum TPA cross-sec- to broaden electronic transitions (Eq. (3)),
tion of at 740 nm and unveil the position and symmetry of the
main excited states involved in this process. Time-dependent den- 1 Cgf =2
gð2x; x0f ; CÞ ¼ ; ð3Þ
sity functional theory (TD-DFT) calculations, carried out using re- p ðxgf  2xÞ2 þ ðCgf =2Þ2
sponse theory at the B3LYP level with the 6-31++G⁄⁄ basis set,
solidly certify the experimental results. The outcomes of this Letter where C (in s1) is the full width at half-maximum (FHWM) line-
are expected to motivate further studies on this molecule and open width for all transitions. A value of C ¼ 0.15 eV was applied to better
a new road for the application of this bio-friendly natural dye in reproduce the experimental spectra. Partially solving Eq. (1) yields
the biomedical and pharmaceutical field. Eq. (2), which contemplates the necessary conversion factors to obtain
the TPA spectra in Göppert-Mayer units (GM), i.e. 1050 cm4  s 
1 1
2. Experimental methods mol  photon , when all the parameters are introduced in atomic
units.
Curcumin (98%) and THF (spectroscopic grade) were purchased The polarization dependent  dTPA
0f ðx0f Þ (in atomic units) is ob-

from Sigma–Aldrich and used without further purification. The lin- tained from [31].
ear absorption spectrum of curcumin in THF was taken using a sin- X       
dTPA ðx0f Þ ¼ 1 F  S0f 0f
þ ðG þ HÞ  S0f 0f
gle-beam spectrophotometer (Agilent 8453 Diode Array UV–Vis), 0f aa Sbb ab Sab ; ð4Þ
30 a;b
from 190 nm to 1100 nm, in a 1.0 cm quartz cuvette at a concen-
tration of 3  105 M. Contributions from the solvent and the x
here, S0f
ab is the transition matrix element which is a function of 2 ;
0f

quartz cuvette were subtracted from the spectrum. The fluores-

F, G and H are scalars used to define the polarization of the excita-
cence anisotropy was obtained with the PTI Quanta-Master spec-
tion source, all having a value of 2 for the linearly polarized light
trofluorimeter upon excitation at 350 nm with linearly polarized
(LPL) case and 2, 3, 3, respectively, for the circularly polarized light
light. The emission intensity was measured through a polarizer ori-
(CPL) case.
ented parallel (I//) and perpendicular (I\) to the direction of the
polarized excitation [22].
The TPA and polarization dependence TPA measurements were
performed in a homogeneous 2.3  102 M solution of curcumin/
THF, employing the double L-scan technique [21]. Two-photon
excitation was induced with a computer-controlled femtosecond
optical parametric amplifier (OPerA Solo) pumped by an amplified
laser system from COHERENT. The whole systems is capable of
generating 90 fs (FWHM) pulses over a wavelength range that
spans from 290 nm to 2.6 lm, with pulse energies of up to 4 mJ
and a variable repetition rate ranging from 1 Hz to 1 kHz. Experi-
ments were performed between 600 and 840 nm, with typical in-
put energies ranging from 7 to 45 lJ/pulse at a 50 Hz repetition
rate to avoid any contribution from accumulative effects. The pulse
width was determined using a single-shot autocorrelator from
Coherent Inc. and a frequency-resolved optical gaiting (FROG) from
Swamp Optics LLC. The excitation polarization state was adjusted
using broadband zero-order quarter-wave plates.

3. Computational and theoretical methods

Figure 1. Experimental absorption spectrum (blue dotted line) of curcumin in THF.
Experimental emission (red dashed line) and excitation anisotropy (black solid line)
The molecular structure of curcumin (see inset of Figure 1) was spectra for curcumin in ethylene glycol. (For interpretation of the references to
optimized using Density Functional Theory (DFT) in GAUSSIAN 09 color in this figure legend, the reader is referred to the web version of this article.)
162 J.A. Tiburcio-Moreno et al. / Chemical Physics Letters 583 (2013) 160–164

In order to recover the TPCLD spectrum of curcumin/THF we

used the definition of this property [34,35].
" #," #
TPCLD dTPA
CPL ðkÞ dTPA
CPL ðkÞ
Dd ðkÞ ¼ 1 þ1 ; ð5Þ
dTPA
LPL ðkÞ dTPA
LPL ðkÞ

where (dTPA TPA

CPL ðkÞ) and linearly (dLPL ðkÞ) are the TPA cross-sections ob-
tained using LPL and CPL, respectively. It is well known that TPCLD
is a property governed by the transition electric dipole moment
which provides information about the symmetry of excited states
of conjugated molecules [36,37].

4. Results and discussion

Figure 1 display the normalized absorption and emission spec-

tra of curcumin/THF at 3  105 M. The linear characterization of
this molecule in solution was performed to corroborate, through
its direct comparison with data reported in the literature, the opti-
cal properties of this compound. The obtained spectra match with
those previously reported by Kumar and co-workers [2]. It can also
be noticed, in the linear absorption spectrum, a resolved vibra-
tional structure with a total of two characteristic absorption peaks
(one centered at 422 nm and another at 438 nm), attributed to an
effective internal vibrational mode in that region.
In order to identify the most suitable region for TPA we mea-
sured the fluorescence anisotropy spectrum of curcumin in ethyl-
ene glycol at 3  105 M. This curve reveals three different
spectral regions 390–520 nm, 330–390 nm and 250–330 nm corre-
sponding to transitions S0 ? S1, S0 ? S2 and S0 ? Sn, respectively.
Although, curcumin is not a centrosymmetric molecule, parity
restriction, imposed by selection rules, are still very important
for this molecule [20]. Therefore, one should expect a strong TPA
in the central region (330–390 nm which correspond to a two-pho-
ton excitation range between 660–780 nm) where the two states
involve in the transition present equal-parity. Recent research
work on the study of the fluorescence anisotropy of curcumin done
by Gryczynski et al. [38] support our results.
In Figure 2 we show the experimental and theoretical TPA spec-
tra of a 2.3  102 M of this naturally occurring yellow-orange pig-
ment in a homogeneous THF solution. Measurements were
performed with LPL and CPL. First, it can be noticed an excellent
spectral match between theory and experiment. Among the calcu-
lated transitions, that to electronic state S2 (722 nm) presents the
highest TPA cross section within the measured region. In addition,
it can be observed a small peak at 842 nm corresponding to a
highly probable OPA transition to S1 (see inset Figure 3 –
420 nm). The amplitude and spectral position of these two bands
are in agreement with prediction based on differences in selection
rules for OPA and TPA. The analysis of the fluorescence anisotropy
spectrum (Figure 1) and that of TPCLD, which is presented later,
corroborate this outcome. Additionally, the preferential one- and
two-photon transitions between S0 ? S1 and S0 ? S2, respectively,
are validated by noticing in Figure 3 (molecular orbitals corre-
sponding to the two main transitions) the stronger intramolecular
charge transfer for the latter, which is typically associated with a
larger transition dipole moment [39]. It is important to highlight
that a spectral blue shift of approximately 80 nm (see Table 1)
was applied to the theoretical spectra (LPL and CPL) to reach the
spectral overlap. This procedure can be justified by recognizing
that theoretical calculations, using PCM to include solvent effects,
are performed under the Lorentz–Lorenz approximation which
does not consider specific solute–solvent interactions [40], thus a
theoretical spectral shift is not surprising. Though, transitions to
these two excited states are in agreement with those revealed by
the experiment, a strong overestimation of the TPA cross section,
correspondent to the lowest energy band can be remarked. This
Figure 2. Experimental TPA spectra of curcumin in THF (scattered squares) for LPL
(a) and CPL (b), with their corresponding theoretical TPA electronic transitions
(scattered spheres) and Lorentzian convolution [line width 0.15 eV FWHM] (dotted
lines). Solid black lines over the TPA data points are shown to guide the reader
through the TPA experimental spectra. Theoretical spectra are presented with an
80 nm blue shift.
outcome is due to imprecisions in the determination of the line
widths and transition energies [40]. Second, it can be observed that
the TPA cross-section using LPL is larger than CPL through almost
the whole spectrum. This fact strongly indicates that TPA transi-
tions are taking place between states of the same symmetry [36].
This point is corroborated below through TPCLD. Third, the notice-
able split on the most important TPA band, i.e. transition to the sec-
ond excited state, is attributed to a vibrational coupling. This effect
has already been associated with large Herzberg-Teller contribu-
tions in similar conjugated molecules [37,41]. Next, one should
highlight the fact that the TPA spectra exhibit two distinct peaks
(Figure 2a) with TPA cross sections of 80 GM and 90 GM at
700 nm and 740 nm, respectively. This represents more than 10
folds improvement in dTPA ðkÞ with respect to values reported by
Kumar et al. at 800 nm, i.e. 6 GM [2]. In fact, Kumar and co-workers
showed, for the first time, the important application of this natu-
rally occurring yellow-orange pigment for two-photon bioimaging
working, coincidentally, in one of the minimum of the TPA spec-
trum of this molecule (see Figure 2) [2]. Therefore, our results ex-
pose the full potential of this biocompatible two-photon
absorption marker for bioimaging, and unveil the optimum excita-
tion wavelength for the most efficient two-photon excitation in the
NIR. Because the fluorescence quantum yield of curcumin does not
vary within the two-photon excitation wavelength range under
study, the corresponding dTPA ðkÞ should also increase more than
one order of magnitude when exciting curcumin/THF via TPA at
740 nm. TPA measurements were not performed below 600 nm
to avoid stepping into the resonance enhanced TPA region were
OPA start becoming important [36]. Nevertheless, according to
 J.A. Tiburcio-Moreno et al. / Chemical Physics Letters 583 (2013) 160–164 163

Figure 3. Molecular orbitals of curcumin calculated using Gaussian 0920 at the B3LYP/6–31++G⁄⁄ level of theory in THF. The arrows represent one- and two-photon
transitions between S0 ? S1 (99%_HOMO ? LUMO) and S0 ? S2 (97%_HOMO-1 ? LUMO), respectively, at their corresponding maximum absorption. Inset – Normalized OPA
and TPA spectra.

Table 1
Results summary for the TPA theoretical calculation. n is the excited state number;

dTPA TPA
0f ;LP ðx0f ÞðGMÞ and d0f ;CP ðx0f ÞðGMÞ are the TPA cross-sections for LPL and CPL,
respectively and DdTPCLD is the TPCLD.

n 
dTPA 
dTPA DdTPCLD
0f ;LP ðx0f Þ 0f ;CP ðx0f Þ
k
(nm) (GM) (GM)
S1 921.816 9.66  1001 1.15  1002 0.087
S2 802.487 4.58  1003 3.02  1003 0.205
S3 692.649 1.73  1003 2.59  1003 0.200
S4 683.109 2.07  1001 2.88  1001 0.167
S5 654.270 8.66  1002 5.60  1002 0.212
S6 618.375 1.12  1003 7.59  1002 0.190
S7 610.760 1.83  1002 1.35  1002 0.156
S8 586.214 2.72  1001 1.85  1001 0.190
S9 536.728 1.79  1002 1.24  1002 0.183
S10 532.121 8.56  1001 6.16  1001 0.163
S11 509.176 6.48  1002 4.40  1002 0.190
S12 507.093 2.12  1002 1.52  1002 0.163
S13 506.058 7.37  1001 1.11  1000 0.200
S14 499.936 2.86  1001 4.30  1001 0.200
S15 492.001 6.18  1002 4.13  1002 0.198
S16 490.056 1.73  1000 2.59  1000 0.200
S17 489.090 9.52  1001 9.78  1001 0.015
Figure 4. Log–Log plot of the normalized transmittance change vs. input irradiance
S18 485.261 3.41  1002 5.11  1002 0.200
at 600 (black squares), 740 (blue spheres) and 840 nm (red triangles). (For
interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)

the theoretical spectra, dTPA ðk < 600 nmÞ of curcumin, in the visible
spectral region, remains below the maximum value obtained at
740 nm. In order to corroborate TPA within the studied spectral
range we measured the normalized transmittance change vs. input
at 600, 740 and 840 nm (see Figure 4). A slope equal to one, in the
log–log plot, evidences the typical quadratic law with respect to
the input irradiance for TPA processes [42].
Finally, we analyze the experimental and theoretical TPCLD
spectra of curcumin/THF (Figure 5). This novel spectroscopic tech-
nique can be utilized to determine the symmetry of the excited
states and the relative orientation between transition electric di-
pole moments [37]. First thing to notice in Figure 5 is the spectral
agreement between theory and experiment. The difference in mag-
nitude between the two spectra is attributed to the same factors
described above on the TPA spectra analysis. Second, according
to theoretical results reported by Nascimento [36] on the polariza-
tion dependence of TPA rates for randomly oriented molecules and
those by De Boni and co-workers on TPA polarization dependence
of perylene [37], a negative TPCLD value (DdTPCLD < 0) throughout
the whole spectral region indicates that the main TPA transitions
take place between states of the same symmetry. This can be cor-
roborated by carefully inspecting values of DdTPCLD in Table 1.
Although, excitations to excited states S1, S3 and S4 present
0 < DdTPCLD 6 0:2, and to S13, S14, S16, S17 and S18 DdTPCLD ¼ 0:2,
which correspond to transitions between states of different
164 J.A. Tiburcio-Moreno et al. / Chemical Physics Letters 583 (2013) 160–164
Figure 5. Theoretical (red dotted line) and experimental (black solid line) TPCLD
spectra of curcumin in THF. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
symmetry [36], the relative minuscule dTPA ðkÞ for all these particu-
lar transitions (see Table 1) present an insignificant contribution to
the overall shape and sign of the TPCLD spectrum of curcumin/THF.
This type of effect has already been observed by De Boni et al. in
perylene [37]. Third, the theory clearly reproduces the maximum
at 870 nm and the broad valley between approximately 630 nm
and 780 nm. These two regions, which correspond to S0?S1 and
S0 ? S2 transitions, are in perfect agreement with the fluorescence
anisotropy results (see Figure 1) [38]. Next, the small minimum
centered at 700 nm is attributed to the vibrational coupling men-
tioned above [37,41]. However, the origin of the deep minimum
observed at 810 nm, on the experimental TPCLD spectrum is un-
known. We believe this feature is caused by the overlap between
allowed TPA excited states of curcumin in solution due to all pos-
sible mechanisms of homogeneous and inhomogeneous broaden-
ing [43].
5. Conclusions
We have shown, for the very first time to our knowledge, the
full theoretical and experimental TPA spectra of curcumin in THF
solution. The TPA spectroscopy of this naturally occurring yel-
low-orange pigment, in the femtosecond regimen and over a broad
spectral range, revealed a maximum dTPA ffi 90 GM at 740 nm that is
more than 10-folds larger than that previously reported in the lit-
erature for the application of curcumin in bioimaging at 800 nm.
This important finding is expected to stimulate the use of this
non-toxic molecule for multiphoton bioimaging and photodynamic
therapy. Through the application of TPCLD, we were able to iden-
tify the symmetry and spectral position of the excited states on this
interesting molecule. Results reported herein are expected to
encourage the application of this and perhaps others natural occur-
ring biocompatible marker in the biomedical and pharmaceutical
fields.
Acknowledgements
This work was partially supported by the National Science
Foundation through Grants Number CHE-0832622 and
CHE-0840431. Tiburcio-Moreno would like to thanks CONACYT
Mexico for their financial support through the Ph.D. scholarship
program. F.E. Hernandez would like to thank Dr. Sergio Tafur
(STOKES ARCC) and Dr. Haining Wang (Chemistry Department),
for their assistance in implementing of Dalton 2011 at UCF.
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