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Cite This: J. Phys. Chem. B XXXX, XXX, XXX-XXX
pubs.acs.org/JPCB
Two-Photon Spectra of Chlorophylls and Carotenoid−Tetrapyrrole
Dyads
Daniel A. Gacek,† Ana L. Moore,‡ Thomas A. Moore,‡ and Peter Jomo Walla*,†
†
Technische Universität Braunschweig, Institute for Physical and Theoretical Chemistry, Department of Biophysical Chemistry,
Gaußstraße. 17, 38106 Braunschweig, Germany
‡
School of Molecular Sciences and Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, Arizona 85287-1604,
United States
S Supporting Information
*
ABSTRACT: We present a direct comparison of two-photon
spectra of various carotenoid−tetrapyrrole dyads and phthalocyanines (Pc) as well as chlorophylls (Chl) in the spectral range
between 950 and 1360 nm, corresponding to one-photon spectra
between 475 and 680 nm. For carotenoids (Car) with 8, 9, or 10
conjugated double bonds, the two-photon absorption cross section
of states below the optical allowed carotenoid S2 is at least about 3−
10 times higher than that of Pc or chlorophyll a and b at 550/1100
nm. A quantitative comparison of spectra from Pc with and without
carotenoids of eight and nine conjugated double bonds confirms
energy transfer from optically forbidden carotenoid states to Pc in
these dyads. When considering that less than 100% efficient energy
transfer reduces the two-photon contribution of the carotenoids in the spectra, the actual Car two-photon cross sections relative
to Chl/Pc are even higher than a factor of 3−10. In addition, strong spectroscopic two-photon signatures at energies below the
optical allowed carotenoid S2 state support the presence of additional optical forbidden carotenoid states such as S*, Sx, or,
alternatively, contributions from higher vibronic or hot S1 states dominating two-photon spectra or energy transfer from the
carotenoids. The onset of these states is shifted about 1500−3500 cm−1 to lower energies in comparison to the S2 states. Our
data provides evidence that two-photon excitation of the carotenoid S*, Sx, or hot S1 states results in energy transfer to
tetrapyrroles or chlorophylls similar to that observed with the Car S1 two-photon excitation.
■
explore states below Car S2, including fluorescence spectroscopy, Raman spectroscopy, and transient absorption via
femtosecond time-resolved spectroscopy and two-photon
spectroscopy.15 In 1972 Karplus and Schulten published a
theoretical study describing a low-lying optical forbidden state
S1 with identical symmetry to the ground state.16 Both were
assigned to have an Ag-symmetry which explains why a
transition between these two states is forbidden and neither a
corresponding absorption nor fluorescence can be readily
observed. However, two-photon spectroscopy can be applied to
directly excite these states due to the inversion of the Laporte
rule. While any transition between states having a g (gerade) or
u (ungerade) symmetry is one-photon forbidden, g ↔ g or u ↔
u transitions are two-photon allowed.
In addition, the understanding of the role of carotenoids
became more difficult as theoretical and experimental results
indicated that there are even more than a single forbidden state
below the Car S2. Andersson and Gillbro described in
carotenoids with N = 15 and N = 19 conjugated double
INTRODUCTION
Carotenoids play a crucial role in photosynthetic organisms, as
they contribute to the light-harvesting of photons in the blue
spectral range and are likely key players in the regulation of the
energy flow in the photosynthetic apparatus under strongly
varying light conditions.1 Excessive energy is a major problem
for the photosynthetic apparatus because it leads to photodamage and undesired side effects. Therefore, plants evolved
specific regulation mechanisms to protect the photosynthetic
apparatus under alternating light conditions.1−3 This process is
called nonphotochemical quenching (NPQ) and dissipates the
excessive energy by converting it into heat.4 Most models that
explain the photophysical mechanisms of this energy
dissipation involve chlorophyll−carotenoid (Chl−Car) interactions.5−14
However, carotenoids are difficult to understand theoretically
as well as to investigate experimentally because at least one
optically forbidden state exists below the Car S2 state, that is
actually responsible for their color and light absorption in the
blue spectral range. As the forbidden states are close to the Qstates of chlorophylls, they are important for both lightharvesting energy transfer as well as excess energy dissipation.
To date, various spectroscopic approaches have been applied to
© XXXX American Chemical Society
Received: August 25, 2017
Revised: October 4, 2017
A
DOI: 10.1021/acs.jpcb.7b08502
J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
Figure 1. (a) Absorption (solid) and fluorescence (dashed) of all samples: Pc1 (black), Pc1−9DB (red), Pc1−11DB (blue), Pc2 (gray), Pc2−8DB
(magenta), and Pc2−10DB (cyan). (b) Structure of the studied compounds. Fluorescence spectra are scaled relative to their different fluorescence
quantum yields with the spectrum of Pc1 normalized to 1.
bonds (DB) states of symmetries that could neither be affiliated
with Car S1 nor Car S2 state.17 Several Car S2−S1 intermediate
states (Sx) or discrete electronic states (S*) have been
proposed since then.18 In 2002, Cerullo et al. presented
evidence for a singlet excited intermediate state, denoted as Sx,
in β-carotene and lycopene.19 Further results were provided by
Wohlleben et al., that indicated that the new state could not be
populated by deactivation from the Car S2. They proposed a
hot ground state decaying by vibronic relaxation as suspected
earlier.17,20,21 Both hot ground states22 and Sx intermediate
states23 were further discussed by others, and even coherent
coupling to the Car S224 was proposed. More recently, Miki et
al. suggested that the Sx state undergoes a diabatic mixing of the
carotenoid Sx and S2, depending on the chain length of the
carotenoid. They considered that the Sx and S2 states are
energetically close in short-chain carotenoids (N ≤ 10).25
Already in 2001, Gradinaru et al. proposed a discrete electronic
singlet state that was named S* in the bacteria light harvesting
systems 1 (LH1). They even proposed that triplet states were
generated through fission of the electronic S* state, as the
triplet and S* spectra share many common features. On the
basis of further experimental results, the S* state was also
proposed to exist in LH2.26,27 Additionally, direct observation28
and communication between the Car S1 and S* was indicated
by results obtained with bacterial light harvesting systems,18,29−31 phthalocyanine−carotenoid systems,32−35 and
natural light harvesting pigments such as lutein and
zeaxanthin.36,37 It seems that the currently most widely
accepted hypotheses on the nature of S* are either structures
involving a twist of the carotenoid backbone or conformational
change in which the S1 state becomes the S* state37−40 or the
S* state is originated from a hot ground state.41 It has also been
proposed that both states coexist. For example, Sx (intermediate state) and S* (separate electronic state) could exist
depending on the structure of the carotenoid42 and the given
solvent.43 Still, others propose that neither is the case and that
all the reported features are explained by relaxation via
vibrationally hot S1 state44/S1 state45 or vibronic transitions
of the S0, S1, or both, due to their conjugated system.46
In summary, in the past decades, there has been a confusing
discussion not only about the existence of carotenoid optical
forbidden states but also about their role and involvement in
energy transfer and electronic interactions with tetrapyrroles
such as Pc or chlorophylls. First, there has been the abovementioned discussion on the existence of additional states,
often named S*, hot S1 or Sx states, between the optically
allowed Car S2 and optically forbidden Car S1 states. Second,
there has been a discussion as to what extent direct chlorophyll
or Pc two-photon excitation (TPE) contributes to two-photon
data from photosynthetic pigment−protein complexes or
artificial model systems. Third, there have been discussions
about whether excitonic or bidirectional energy transfer indeed
exists between forbidden carotenoid and tetrapyrrole states in
certain dyads or photosynthetic pigment−protein complexes
under certain conditions.7,10,12−14,35,47−50
To address these points, herein we present a systematic study
of the two-photon spectra of chlorophyll a, b as well as Pc and a
variety of carotenoid−Pc dyads in a wide spectral range along
with a direct quantitative comparison of the fluorescence
intensities observed after two-photon excitation. From these
data, we conclude the following: (1) The data support the
existence of additional states between Car S2 and Car S1. (2) In
certain spectral regions of optically forbidden carotenoid states,
direct two-photon excitation of chlorophylls and Pc in the same
spectral range has to be considered, as previously reported.50
However, there exist spectral ranges where two-photon
excitation of the carotenoids is much larger than that of
tetrapyrrole derivatives such as chlorophylls or Pc. (3)
Unusually high forbidden state energies and high two-photon
cross sections around 1100 nm in carotenoid−Pc difference
spectra in amine linked dyads with a carotenoid of 10
conjugated double bonds in toluene support special electronic
interactions. These observations could be related to excitonic or
bidirectional energy transfer, as has been suggested.12,32,33,35,50
■
MATERIALS AND METHODS
Sample Preparation. The synthesis of the samples (see
Figure 1) is described elsewhere.33,47,50 Chl a and Chl b were
obtained from Sigma-Aldrich and dissolved in acetone. All
B
DOI: 10.1021/acs.jpcb.7b08502
J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
electron multiplying charge coupled device (EMCCD) camera
(iXonEM + 897 back-illuminated, Andor Technology).
The two-photon emission spots observed with the camera
were integrated for each excitation wavelength and corrected
for camera background. To prevent camera overload when
using NIR excitation wavelengths, an OD 1 neutral density
filter was used in the detection path.
To assess the contribution of pure two-photon excitation to
the observed spectra, we also recorded dependences of the
signals on the excitation power (Figure 3). Albeit some
samples containing Pc1 (Pc1, Pc1−9DB, and Pc1−11DB) were
dissolved in THF and were adjusted to an optical density of
0.75 in the Pc Qy band in a 1.5 mm cuvette. All samples
containing Pc2 (Pc2, Pc2−8DB, and Pc2−10DB) were
dissolved in toluene. The chlorophylls as well as all samples
containing Pc2 were adjusted to an optical density of 0.5 in the
Chl Qy and Pc Qy bands. All samples and solvents were stored
at 2 °C and prepared at this temperature to ensure minimal
evaporation of the solvent. Cooled, cleaned single-indented
microscope slides were assembled with the sample solution and
covered with #1 coverslips. The outlining of the coverslip was
fixed and sealed with silicone and cellulose nitrate polymer.
The Chl a and Chl b samples were prepared in a Lab-Tek
chamber slide with a silicone seal, since it was not possible to
prepare the acetone solution in the same way as the other
samples due to the fast evaporation of the acetone. Therefore,
the intensities observed with Chl a and Chl b should be
regarded as an upper limit in comparison to the other samples,
as the concentration might be somewhat higher due to the fast
acetone evaporation.
Two-Photon Spectra Measurements. The two-photon
excitation range from 950 to 1400 nm was generated by two
different laser systems (Figure 2). For wavelengths in the range
Figure 3. Example of square dependency measurement.
formation of triplet states never can be excluded, here a
contribution to the observed fluorescence can be neglected, as
this would require processes like triplet−triplet annihilation
after diffusion controlled physical contact of two triplet
molecules in solution. This is very improbable, as the molecules
are quickly diffusing out of the excitation volume of 1 fL into a
much larger volume of many μL on a time scale of a few μs.
One-Photon Absorption and Fluorescence Data.
Absorption spectra and optical density adjustment were
measured by a PerkinElmer Lambda 25 UV−vis spectrometer.
The phthalocyanine and chlorophyll concentrations for all
experiments were adjusted by UV−vis spectra to yield an
optical density (OD) of 0.5 ± 5 or 0.75 ± 5% in the Chl a or Pc
Qy band in a 1.5 mm cuvette, respectively.
Fluorescence spectra were measured at ambient temperature
with a Varian Cary Eclipse fluorescence spectrometer using the
absorption maxima of the respective sample as the excitation
wavelength. All one-photon data was baseline corrected.
Two-Photon Data Processing. For every sample,
individual movies of 50 frames were recorded. The acquired
TPE data were integrated over a defined area and divided by
the number of pixels of this area to obtain an intensity of
arbitrary units. For background correction, an unilluminated
area of similar size was integrated and pixel corrected for every
movie. After background correction, the data were corrected for
variations in the concentration by dividing by the OD observed
in the Pc absorption in the Qy peak. To account for variations
in the readout using the Pc fluorescence caused by quenching,
the data were additionally divided by the Pc fluorescence
intensities observed after one-photon excitation. The twophoton difference spectra were obtained by subtraction of the
corresponding Pc spectra from the Pc−Car data.
Figure 2. Microscope laser setup for (a) the NIR (950−1060 nm) and
(b) the IR measurements (1050−1400 nm). The dichroic mirror is
reflecting all infrared two-photon excitation wavelengths while
transmitting the visible fluorescence light. F1: 900 nm long pass filter.
F2: 700/40 nm band-pass filter. F3, F4: Two IR-blocking 770 nm
short pass filters. All unmarked optical elements represent silver
mirrors.
from ∼1050 to 1400 nm, an optical parametric oscillator (IR
OPO) driven by a Chameleon Ultra II, 80 MHz laser system
was used (Figure 2b, all optical devices by APE Berlin and
Coherent Inc.). To ensure exclusive infrared (IR) light, the
excitation beam was cleaned by a 900 nm long pass filter
(FEL900, Thorlabs) and fed in a confocal microscope setup
(microscope body: IX71 by Olympus). The microscope utilized
a 1000 nm reflection/705 nm transition dichroic mirror (AHF
sp770rxc) and an IR microscope objective (UPlanApo/IR 60×
1.20 W). To achieve the near IR (NIR) range 950−1060 nm,
the Chameleon Ultra II laser light was directly fed in the
confocal microscope. For this range, all optics were retained
except for the 900 nm long pass filter (cf. Figure 2a).
For all wavelengths and measurements, identical two-photon
excitation powers corresponding to about 1 mW at a fixed point
of 5 cm over the microscope objective were adjusted by a linear
variable neutral density filter (NDL-10C-2, Thorlabs) before
the objective using a calibrated power meter (Coherent).
To ensure a Pc-/chlorophyll-fluorescence was only considered, the detection path was cleaned by two IR-block filters
(AHF T700spxr-1500) and one 700 nm band-pass filter
(FB700-40, Thorlabs). The fluorescence was detected by an
C
DOI: 10.1021/acs.jpcb.7b08502
J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
Figure 4. (a−h) Comparison of TPE data (black and blue dots) of all samples. TPE data measured at different days are presented by the blue and
black dots, respectively. Additionally, one-photon absorption (gray lines) of the samples and Gaussian peak fits to the spectral range of the
carotenoid S2 state are shown (black lines). (i−l) TPE difference spectra (black and blue dots). Absorption spectra of the dyads and Pc are indicated
by gray and green lines, respectively. Fluorescence spectra of the dyads and Pc are indicated by gray and green dashed lines, respectively. In addition,
Gaussian peak fits to the putative Car S*, Sx, or hot S1 as well as Car S1 are shown (red lines).
Gaussian Peak Fitting. The Car S2 and S1/S* data were
analyzed by multi Gaussian fit functions. First, multiple
Gaussian peaks with a fixed common peak width were fitted
to the data in the Car S2 spectral range of the one-photon
spectra in Figure 4e−h (black lines). Next, the relative
vibrational energies observed in this way for each carotenoid
were kept fixed and used to fit two peak series with variable 0−
0 positions and peak amplitudes to the two-photon difference
spectra representing the 9DB, 8DB, and 10DB carotenoids
shown in Figure 4i, j, and l, respectively. Again, common fixed
peak widths were used to fit the two-photon data. For the
spectral region of the putative S*, Sx, or hot S1 state, three to
four peaks were used, whereas only two peaks were used for S1
(red lines in Figure 4i, j, and l and Table 1). The peak positions
corresponding to the 0−0 transitions determined in that way
are indicated by vertical red lines in Figure 4e−l. All energies
D
DOI: 10.1021/acs.jpcb.7b08502
J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
Table 1. Fitting Parameters for Gaussian Peaks of the Two-Photon Spectra in Figure 4a
sample
Pc2−8DB (toluene)
Pc1−9DB (THF)
Pc2−10DB (toluene)
fit
peak
peak
peak
peak
peak
peak
peak
peak
peak
peak
peak
peak
peak
peak
peak
peak
peak
center (nm)
1
2
3
4
5
6
1
2
3
4
5
1
2
3
4
5
6
640/1280
590/1180
550/1100
510/1020
475/950
465/930
670/1340
610/1220
520/1040
485/970
455/910
640/1280
585/1170
540/1080
500/1000
470/940
440/880
center (cm−1)
amplitude (au)
fwhm (cm−1)
15,600/7800
17,000/8500
18,200/9100
19,600/9800
21,000/10,500
21,600/10,800
15,000/7500
16,400/8200
19,400/9700
20,800/10,400
22,000/11,000
15,600/7800
17,000/8500
18,600/9300
20,000/10,000
21,400/10,700
22,600/11,300
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
750
750
750
750
750
750
900
900
900
900
900
750
750
750
750
750
750
3.57
1.07
3.50
6.19
9.50
3.48
8.98
5.99
4.43
7.22
5.00
4.75
9.20
4.36
6.99
8.92
1.00
5
10
106
106
106
106
105
105
105
106
106
106
105
105
106
106
106
104
A sensitivity analysis based on varying the fitted parameters resulted in estimated errors for the center wavenumbers on the order of ±350 cm−1, for
the amplitudes on the order of ±10% and for the FWHM of about ±50 cm−1.
a
addition, a direct comparison with the carotenoid S2 onephoton absorption spectrum clearly demonstrates that the
onset of the two-photon excitable carotenoid states is shifted
more than 50/100 nm to the red, corresponding to more than
3500 cm−1 less energy. This provides evidence that indeed
other states below the optically allowed carotenoid S2 are twophoton excited. Figure 4j shows the difference spectrum of pure
Pc2 (Figure 4d) and Pc2−8DB (Figure 4f), thereby
representing a two-photon spectrum that can be attributed
exclusively to the carotenoid excitation. Again, the amplitudes
on the ordinate are in the same relative units as all other
spectra. These data confirm that selective two-photon
excitation of optical forbidden carotenoid states is possible in
the presence of tetrapyrrole/chlorophyll. Also, the difference
spectra of the other two samples, Pc1−9DB and Pc2−10DB,
that were known to exhibit energy transfer from optically
forbidden carotenoid states to the chlorophylls confirm
significant additional two-photon excitation of carotenoids at
state energies below the corresponding carotenoid S2 states
(Figure 4i and l).32,33 The only sample for which it was known
that no efficient energy transfer from optically forbidden states
occurs, Pc1−11DB, indeed displayed less significant twophoton excitation intensities in a spectral range below 550/
1100 nm (Figure 4g). We attribute these signals to direct twophoton excitation of the optically allowed Car S2 state that has
an identical onset at 550/1100 nm for the 11DB carotenoids.
The special role of the Pc2−10DB dyad in toluene will be
further discussed below.
The data shown in Figure 4e, f, and h also provide some
further insights into the question as to what extent optical
forbidden states exist below the optically allowed S2 state of
carotenoids. The energies previously reported in the literature
for the optically forbidden S1 state differ significantly. For
example, Sashima et al.51 reported a value for the carotenoid
spheroidene with 10 conjugated double bonds of 14,200 cm−1
based on Raman spectroscopy. On the other hand, Polivka et
al.52 reported a value of 13,400 cm−1 for the very same
carotenoid based on S1−S2 absorption. For the carotenoid
peridinin with seven conjugated double bonds, 16,200 cm−1
was reported on the basis of fluorescence techniques53 but
derived from the data in Figure 4 are summarized in the state
diagrams shown in Figure 5 as well as Tables 1 and 2. No fitting
was done for the two-photon data of the 11 DB carotenoid, as
here no contribution of forbidden states is expected due to the
lack of energy transfer to the Pc. Please note that it is
impossible to directly observe two-photon excitation spectra
from carotenoids, only, as they are nonfluorescent.
■
RESULTS AND DISCUSSION
Figure 4 shows a direct comparison of the absolute TPE spectra
of all Chl a, Chl b, Pc1, Pc2, Pc1−9DB, Pc2−8DB, Pc1−11DB,
and Pc2−10DB samples along with their corresponding
difference spectra and Gaussian peak fittings. In the spectral
range above 525/1050 nm, all spectra were measured twice on
different days with freshly prepared samples to ensure that the
amplitudes of the observed data are reproducible and therefore
allowed a quantitative comparison (the two data sets are
visualized by black and blue color in Figure 4). These data
demonstrate that the variation in the absolute intensities of the
spectra measured at different days was typically on the order of
about 20%. The data in the spectral range below 525/1050 nm
were measured only once, as indicated by the same black color,
but still connected to the two data sets measured in the spectral
range above 525/1050 nm. As outlined in the Materials and
Methods section, all samples were adjusted to have predefined
ODs in the Qy bands of Pc or Chl for a direct comparison.
The direct comparison of the absolute intensities of the twophoton excitation spectra of Chl a, b, Pc1, and Pc2 with the
Pc2−8DB dyad demonstrates that the presence of the 8DB
carotenoid (Figure 4f) results in significantly higher twophoton excitation in a spectral range energetically below the
optically allowed carotenoid S2 state (>500/1000 nm) than any
of the samples without carotenoid (Figure 4a−d). This was
expected, since it is known from many different studies that a
very effective carotenoid to tetrapyrrole energy transfer occurs
from the optically forbidden carotenoid states in this dyad.15,34
Our results provide direct evidence that significantly more
carotenoid two-photon excitation occurs in spectral ranges of
wavelengths around 575/1150 nm and below than that of
direct tetrapyrrole/chlorophyll two-photon excitation. In
E
DOI: 10.1021/acs.jpcb.7b08502
J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
Figure 5. Energy diagrams derived from the data shown in Figure 4.
18,500 cm−1 based on two-photon spectroscopy.15,54 However,
none of these experiments and predictions yielded values as
blue in the spectral range, as indicated by the dominant spectral
components in the study presented here. Even though peaks
with an onset in the spectral range of ∼650/1300 nm in Figure
4i, j, and l confirm states in a spectral range as predicted by the
previous studies for Car S1, there are even larger spectral
components having an onset in a range of ∼600/1200 nm and
below in these samples. As the onsets of these components are
all significantly red-shifted in comparison to the respective
onsets of the Car S2 states, we believe that these signatures
support the presence of an additional state between Car S1 and
Car S2, as suggested in the above-mentioned studies. The fact
that this state is strongly two-photon allowed indicates that it
might be the S* state related to the Car S1, as suggested by
groups of Frank, Kennis, and Polivka.37−40 If this was an Sx
intermediate state, it has a 1B−u symmetry, which should not be
strongly two-photon allowed. On the other hand, the high state
energies rather point to an Sx state, as S* have been assigned to
have significantly lower state energies. In any case, our data
demonstrate that this state ultimately transfers energy to Pc, as
otherwise no Pc fluorescence could be observed. Therefore, the
overall result of populating the additional state between Car S1
and Car S2 is very similar to directly populating the Car S1 state,
including potential subsequently ET to chlorophylls or
tetrapyrroles.
Of particular interest are the data of the Pc2−10DB samples
in toluene, as has been suggested by several authors that in this
system excitonic interactions or at least bidirectional energy
transfer might occur between optically forbidden carotenoid
states and optically allowed tetrapyrrole states.7,10,12−14,35,47−50
The proposal of such interactions has been rationalized by the
observation that effective energy transfer is possible involving
optically forbidden carotenoid states. As the same electronic
coupling that governs energy transfer can potentially also lead
to excitonic interactions, the latter should occur in cases when
the carotenoid states and the chlorophyll/tetrapyrrole states
have similar state energies. In addition, effective and almost
instantaneous population of optical forbidden carotenoid states
has been observed after excitation of the tetrapyrroles and vice
versa that further supports the presence of such interactions.12,13,35,47,50
F
DOI: 10.1021/acs.jpcb.7b08502
J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
Table 2. State Energies Derived from Figure 4a
sample
Chl a
Chl b
Pc1
Pc2
Pc1−9DB
Pc1−11DB
Pc2−8DB
Pc2−10DB
band
center (nm)
center (cm−1)
Qx
Qy
Qx
Qy
Qx
Qy
Qx
Qy
Qx
Qy
S2
S* ?
S1
Qx
Qy
S2
Qx
Qy
S2
S* ?
S1
Qx
Qy
S2
S* ?
S1
620/1230
660/1320
600/1190
650/1290
650/1290
680/1350
660/1310
690/1380
650/1300
680/1360
490/970
520/1040
670/1340
650/1300
680/1360
520/1040
660/1320
690/1380
490/970
550/1100
640/1280
660/1320
690/1380
520/1040
540/1080
640/1280
16,200/8100
15,100/7600
16,800/8400
15,500/7800
15,500/7800
14,800/7400
15,200/7600
14,500/7300
15,400/7700
14,700/7400
20,700/10,400
19,300/9700
14,900/7500
15,400/7700
14,700/7400
19,300/9700
15,200/7600
14,500/7300
20,600/10,300
18,200/9100
15,600/7800
15,200/7600
14,500/7300
19,300/9700
18,500/9300
15,600/7800
excitation of the very same state energies and from samples
under otherwise identical conditions, a change in their ratio
S1−Chl
TPE OPE
ΦCar
/F
still occurs only when there is a change
Coupling = F
in the energy transfer between optically forbidden carotenoid
states and chlorophylls/tetrapyrroles in these samples. Supplementary note 1 describes how even larger contributions of
direct chlorophyll two-photon excitation only leads to a
S1−Chl
but does not alter the general
constant offset in ΦCar
Coupling
S1−Chl
with
and direct correlation of increasing values in ΦCar
Coupling
increasing energy transfer between optical forbidden carotenoid
states and chlorophylls/tetrapyrroles and vice versa.
■
CONCLUSIONS
In summary, the data shown in Figure 4 provide a direct,
quantitative account of relative two-photon absorption of
carotenoids and subsequent energy transfer to Pc in
comparison to direct chlorophyll and tetrapyrrole two-photon
excitation. It also provides important insights into the question
of optically forbidden states that exist in carotenoids below the
optically allowed Car S2 state. Even though direct chlorophyll
and other tetrapyrrole two-photon excitation in spectral ranges
between the optically allowed Car S2 and chlorophyll/
tetrapyrrole Qy states is not negligible,50 wavelengths exist in
the spectral range between ∼550/1100 and 600/1200 nm in
which two-photon excitation of optically forbidden carotenoid
states clearly dominate. The difference spectra of the twophoton spectra of pure tetrapyrroles and their carotenoid
containing dyads confirm that effective energy transfer exists
from optically forbidden carotenoid states to the tetrapyrroles
in Pc1−9DB, Pc2−8DB, and Pc2−10DB. It has to be
considered that the intensity of Pc fluorescence observed
after carotenoid two-photon excitation depends also on the
quantum efficiency of the corresponding energy transfer. For
example, the quantum efficiency for the forbidden state to Pc
energy transfer has been estimated to be 0.3−0.6 in Pc1−
9DB.50 Therefore, for a relative comparison of the two-photon
cross sections of Pc1 and the 9DB carotenoid, the two-photon
spectrum of Pc1 (Figure 4c) must be compared with the
corresponding two-photon spectrum of the carotenoid (Figure
4i) corrected to even higher values by a factor 1/0.3 = 3.33−1/
0.6 = 1.67.
Dominant signatures in the observed spectra indicate the
existence of additional optically forbidden states between the
allowed state S2 and forbidden state S1 in the carotenoids. They
are strongly two-photon allowed, which points to S* (Ag−
symmetry) states and not Sx (1B−u symmetry) states, as only
the former should be two-photon excitable to such an extent.
On the other hand, the high state energies could also be a
signature for Sx states, as S* have been assigned to have
significantly lower state energies. Alternatively, these dominant
signatures might reflect higher vibronic or hot S1 states that
have just a more pronounced cross section or energy transfer
efficiency than the 0−0 transition of S1 in the two-photon
excitation spectra. Taking all vibrational state energies together
that are assigned to S1 and S* in Figure 4i, j, and l as well as
Table 1 (peaks 1−6) would also fit to a progression of
vibrational energies of just one electronic S1 state. In this
scenario, peak 1 would still be the 0−0 transition with a
relatively low two-photon cross section or energy transfer
efficiency but peaks 2−6 would correspond to higher vibronic
transitions with increasing two-photon cross sections or energy
transfer efficiencies.
a
A sensitivity analysis based on varying the fitted parameters resulted
in estimated error for the center wavenumbers on the order of ±350
cm−1.
Indeed, the largest intensities in the difference spectra around
550/1100 nm have been observed for the Pc2−10DB samples
in toluene (Figure 4h,l). In addition, while the forbidden (the
putative S*, Sx, or hot S1 state) and allowed states (Car S2) in
Pc1−9DB and Pc2−8DB (Figure 4i and j) are shifted to a
similar extent relative to each other, this forbidden state in
Pc2−10DB seems to be shifted less to the red relative to Car S2
even though it has the largest system of conjugated double
bonds (Figures 4 and 5). Also, the fitted Car S1 state is quite
high in energy. As to what extent this could be explained by
excitonic splitting of the state energies to higher and lower
energies or other special electronic properties in this particular
dyad shall be the subject of further theoretical studies.
However, the data provide further evidence that in this system
also effective energy transfer from optically forbidden
carotenoid states to tetrapyrole is possible even though this
probably would not have been expected from a carotenoid with
such a large system of conjugated double bonds.
Finally, the data shown in Figure 4a and b also confirm
results observed by Lokstein and co-workers that show that in a
similar spectral range as carotenoid two-photon excitation also
some two-photon excitation of Chl a and Chl b occurs and that
the chlorophyll Qy band is indeed less intense in two-photon
spectra, contrary to assumptions based on previously measured
Chl two-photon spectra.55 Thus, at wavelength ranges often
used for direct comparisons of one- and two-photon excitation
of photosynthetic pigment−protein complexes or synthetic
dyads, direct chlorophyll two-photon excitation also occurs to a
similar extent as previously reported.50 However, as always,
relative changes were compared for one- or two-photon
G
DOI: 10.1021/acs.jpcb.7b08502
J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
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Finally, higher two-photon cross sections of Pc2−10DB
around 550/1100 nm and blue-shifted spectral signatures of the
optically forbidden states in comparison to Pc1−9DB and
Pc2−8DB further support the presence of special electronic
circumstances in the former. This observation potentially
supports suggestions that in Pc2−10DB excitonic interactions
might be present and should be subject to further theoretical
studies.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jpcb.7b08502.
Calculation and description of the influence of
contributions of direct Chl or Pc two-photon excitation
S1−Chl
(PDF)
on the coupling parameter ΦCar
Coupling
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: p.walla@tu-braunschweig.de. Phone: +49-5313915328. Fax: +49-531-3915352.
ORCID
Peter Jomo Walla: 0000-0001-6956-5569
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the Office of Basic Energy
Sciences, Division of Chemical Sciences, Geosciences, and
Energy Biosciences, Department of Energy under contract DEFG02-03ER15393.
■
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J. Phys. Chem. B XXXX, XXX, XXX−XXX
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