Photosynth Res (2009) 101:105–118
DOI 10.1007/s11120-009-9454-y
REVIEW
Ultrafast transient absorption spectroscopy: principles
and application to photosynthetic systems
Rudi Berera Æ Rienk van Grondelle Æ John T. M. Kennis
Received: 18 February 2009 / Accepted: 5 June 2009 / Published online: 4 July 2009
Ó The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract The photophysical and photochemical reactions, after light absorption by a photosynthetic pigment–
protein complex, are among the fastest events in biology,
taking place on timescales ranging from tens of femtoseconds to a few nanoseconds. The advent of ultrafast laser
systems that produce pulses with femtosecond duration
opened up a new area of research and enabled investigation
of these photophysical and photochemical reactions in real
time. Here, we provide a basic description of the ultrafast
transient absorption technique, the laser and wavelengthconversion equipment, the transient absorption setup, and
the collection of transient absorption data. Recent applications of ultrafast transient absorption spectroscopy on
systems with increasing degree of complexity, from biomimetic light-harvesting systems to natural light-harvesting antennas, are presented. In particular, we will discuss,
in this educational review, how a molecular understanding
of the light-harvesting and photoprotective functions of
carotenoids in photosynthesis is accomplished through the
application of ultrafast transient absorption spectroscopy.
Keywords Ultrafast spectroscopy Photosynthesis
Light-harvesting antennas
R. Berera R. van Grondelle J. T. M. Kennis (&)
Department of Physics and Astronomy, Faculty of Sciences,
VU University Amsterdam, De Boelelaan 1081,
1081 HV Amsterdam, The Netherlands
e-mail:
[email protected]
Present Address:
R. Berera
Institute of Biology and Technology of Saclay, CEA
(Commissariat a l’Energie Atomique), URA 2096 CNRS
(Centre National de la Recherche Scientifique),
91191 Gif/Yvette, France
Abbreviations
(B)Chl (Bacterio)Chlorophyll
BPheo
Bacteriopheophtin
EADS
Evolution-associated difference spectra
ESA
Excited-state absorption
FWHM Full-width at half maximum
LHC
Light-harvesting complex
PSII
Photosystem II
RC
Reaction center
SADS
Species-associated difference spectra
SE
Stimulated emission
Introduction
The process of photosynthesis relies upon the efficient
absorption and conversion of the radiant energy from the
Sun. Chlorophylls and carotenoids are the main players in
the process. While the former are involved in light-harvesting and charge separation process, the latter also play
vital photoprotective roles. Photosynthetic pigments are
typically arranged in a highly organized fashion to constitute antennas and reaction centers, supramolecular
devices where light harvesting and charge separation take
place.
The very early steps in the photosynthetic process take
place after the absorption of a photon by an antenna system, which harvests light and eventually delivers it to the
reaction center (Van Grondelle et al. 1994). Despite the
enormous variety of photosynthetic organisms, the primary
events leading to photosynthetic energy storage are
remarkably similar (Sundström 2008). In order to compete
with internal conversion, intersystem crossing, and fluorescence, which inevitably lead to energy loss, the energy
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and electron transfer processes that fix the excited-state
energy in photosynthesis must be extremely fast. In order
to investigate these events, ultrafast techniques down to a
sub-100 fs resolution must be used. In this way, energy
migration within the system as well as the formation of
new chemical species such as charge-separated states can
be tracked in real time. This can be achieved by making use
of ultrafast transient absorption spectroscopy. The basic
principles of this technique, instrumentation, and some
recent applications to photosynthetic systems that involve
the light-harvesting and photoprotective functions of
carotenoids are described in this educational review. For
earlier reviews on ultrafast spectroscopy, see e.g., Jimenez
and Fleming (1996), Groot and Van Grondelle (2008), and
Zigmantas et al. (2008).
Ultrafast transient absorption spectroscopy
The principle of ultrafast transient absorption
spectroscopy
The process of energy transfer in a photosynthetic membrane typically takes place on a time scale from less than
100 fs to hundreds of ps (Sundström et al. 1999; Van
Amerongen and Van Grondelle 2001; Van Grondelle et al.
1994). The advent of ultrashort tunable laser systems in the
early 1990s has opened up a new and extremely fascinating
area of research. Nowadays, the high (sub 50 fs) time
resolution has made it possible to investigate the very early
events taking place within a light-harvesting antenna in real
time (Sundström 2008). In transient absorption spectroscopy, a fraction of the molecules is promoted to an electronically excited state by means of an excitation (or pump)
pulse. Depending on the type of experiment, this fraction
typically ranges from 0.1% to tens of percents. A weak
Photosynth Res (2009) 101:105–118
probe pulse (i.e., a pulse that has such a low intensity that
multiphoton/multistep processes are avoided during probing) is sent through the sample with a delay s with respect
to the pump pulse (Fig. 1). A difference absorption spectrum is then calculated, i.e., the absorption spectrum of the
excited sample minus the absorption spectrum of the
sample in the ground state (DA). By changing the time
delay s between the pump and the probe and recording a
DA spectrum at each time delay, a DA profile as a function
of s and wavelength k, i.e., a DA(k,s) is obtained. DA(k,s)
contains information on the dynamic processes that occur
in the photosynthetic system under study, such as excitedstate energy migration, electron and/or proton transfer
processes, isomerization, and intersystem crossing. In order
to extract this information, global analysis procedures may
be applied (see below). One advantage of time-resolved
absorption spectroscopy over time-resolved fluorescence is
that with the former, the evolution of non-emissive states
and dark states can be investigated. This is of particular
importance in photosynthesis where carotenoid dark (nonemissive) states play a number of vital roles.
In general, a DA spectrum contains contributions from
various processes:
(1)
(2)
The first contribution is by ground-state bleach. As a
fraction of the molecules has been promoted to the
excited state through the action of the pump pulse, the
number of molecules in the ground state has been
decreased. Hence, the ground-state absorption in the
excited sample is less than that in the non-excited
sample. Consequently, a negative signal in the DA
spectrum is observed in the wavelength region of
ground state absorption, as schematically indicated in
Fig. 1 (dashed line).
The second contribution is by stimulated emission.
For a two-level system, the Einstein coefficients for
absorption from the ground to the excited state (A12)
Fig. 1 Left panel: Schematic depiction of the transient absorption spectroscopy principle. Right panel: Contributions to a DA spectrum: groundstate bleach (dashed line), stimulated emission (dotted line), excited-state absorption (solid line), sum of these contributions (gray line)
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Photosynth Res (2009) 101:105–118
(3)
(4)
and stimulated emission from the excited to the
ground state (A21) are identical. Thus, upon population of the excited state, stimulated emission to the
ground state will occur when the probe pulse passes
through the excited volume. Stimulated emission will
occur only for optically allowed transitions and will
have a spectral profile that (broadly speaking) follows
the fluorescence spectrum of the excited chromophore, i.e., it is Stokes shifted with respect to the
ground-state bleach. During the physical process of
stimulated emission, a photon from the probe pulse
induces emission of another photon from the excited
molecule, which returns to the ground state. The
photon produced by stimulated emission is emitted in
the exact same direction as the probe photon, and
hence both will be detected. Note that the intensity of
the probe pulse is so weak that the excited-state
population is not affected appreciably by this process.
Stimulated emission results in an increase of light
intensity on the detector, corresponding to a negative
DA signal, as schematically indicated in Fig. 1 (dotted
line). In many chromophores including bacteriochlorophyll (BChl), the Stokes shift may be so small that
the stimulated emission band spectrally overlaps with
ground-state bleach and merges into one band.
The third contribution is provided by excited-state
absorption. Upon excitation with the pump beam,
optically allowed transitions from the excited (populated) states of a chromophore to higher excited
states may exist in certain wavelength regions, and
absorption of the probe pulse at these wavelengths
will occur. Consequently, a positive signal in the DA
spectrum is observed in the wavelength region of
excited-state absorption (Fig. 1, solid line). Again, the
intensity of the probe pulse is so weak that the
excited-state population is not affected appreciably by
the excited-state absorption process.
A fourth possible contribution to the DA spectrum is
given by product absorption. After excitation of the
photosynthetic, or more generally photobiological or
photochemical system, reactions may occur that result
in a transient or a long-lived molecular state, such as
triplet states, charge-separated states, and isomerized
states. The absorption of such a (transient) product
will appear as a positive signal in the DA spectrum. A
ground-state bleach will be observed at the wavelengths where the chromophore on which the product
state resides has a ground-state absorption. A wellknown example of such a transient product state is the
accessory bacteriochlorophyll (BChl) anion in the
bacterial reaction center (RC), which acts as a
transient intermediate in the electron transfer process
from the primary donor P to the bacteriopheophytin
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(BPheo). The rise and decay of this species can be
monitored through its specific product absorption at
1,020 nm (Arlt et al. 1993; Kennis et al. 1997a).
Pulse duration, time resolution, and spectral selectivity
Laser pulses as short as 5 fs are now available for transient
absorption spectroscopy (see, e.g., Cerullo et al. (2002);
and Nishimura et al. (2004)). A short pulse duration Dt
implies a large spectral bandwidth Dv according to relation
DtDv = 0.44 for Gaussian-shaped pulses. This relation is
known as the time–bandwidth product. For instance, a 10fs pulse with a center wavelength of 800 nm has a spectral
bandwidth of 4.4 9 1013 Hz at full-width at half maximum
(FWHM), which corresponds to about 100 nm in this
wavelength region. Thus, one has to make a trade-off
between time resolution and spectral selectivity. Consider
the example of the bacterial RC, which has the primary
donor absorbing at 860 nm, the accessory BChls at
800 nm, and the BPheos at 760 nm. With a 10-fs pulse at
800 nm, one would simultaneously excite all the cofactors.
In order to selectively excite one of the cofactor pairs to
study its excited-state dynamics, spectral narrowing to
*30 nm is required, which implies a longer excitation
pulse of *30 fs (Streltsov et al. 1998; Vos et al. 1997). For
the photosystem II (PSII) RC, where the energy gaps
between the pigments are significantly smaller, the excitation bandwidth has to be narrowed even more to \10 nm
for selective excitation, with corresponding pulse durations
of *100 fs (Durrant et al. 1992; Groot et al. 1997).
On very fast timescales, transient absorption signals have
contributions from processes additional to those described
in the previous section. These non-resonant contributions
are often lumped together under the terms ‘‘coherent artifact’’ and ‘‘cross-phase modulation.’’ As transient absorption signals result from light–matter interaction through the
third-order non-linear susceptibility v(3) (Mukamel 1995),
non-sequential light interactions that do not represent population dynamics of electronic states will contribute to the
signals. Such undesired signals can be ignored by excluding
the initial phases of the femtosecond dynamics from the data
interpretation and analysis. On the other hand, they may be
explicitly included in the analysis by considering their
physical origin. In such a case, assumptions need to be made
about the lineshapes and dephasing times of the chromophore in question (Novoderezhkin et al. 2004). Cross-phase
modulation effects are due to a change in the index of
refraction of solvent and cuvette induced by the pump beam
and give rise to oscillatory patterns around zero delay
(Kovalenko et al. 1999). These artifacts can in principle be
subtracted from the data by recording an experiment in a
cuvette with the solvent.
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Equipment: amplified Ti:sapphire laser systems
and optical parametric amplifiers
Generally speaking, two types of ultrafast transient
absorption spectroscopy setups are widely used today for
photosynthesis research, distinguished by the repetition
rate and pulse energies at which they operate: the first type
involves systems with a repetition rate of 1–5 kHz with a
relatively high pulse energy. The second type involves
systems with a repetition rate in the range 40–250 kHz
with a relatively low pulse energy. In addition, the direct or
cavity-dumped output from a Ti:sapphire oscillator has
frequently been employed for transient absorption spectroscopy, but will not be discussed here (Arnett et al. 1999;
Kennis et al. 1997b; Nagarajan et al. 1996; Streltsov et al.
1998; Vulto et al. 1999).
The first type of spectroscopy typically provides the
experimenter with excitation energies of 5–100 nJ, which
when focused on 150–200 lm diameter (the regular focusing conditions in our laboratory) typically results in 2–20%
of the molecules being promoted to the excited state. This
value is only approximate, since the accurate estimate of the
excitation density depends on several factors, namely, the
exact size of the focus, the concentration of the chromophores, and their extinction coefficient. The relatively high
excitation densities achieved with these systems make them
suitable to study complexes with a relatively small number
of connected pigments such as pigments in solution (Billsten
et al. 2002; Cong et al. 2008; De Weerd et al. 2003; Niedzwiedzki et al. 2007; Polivka et al. 1999), isolated reaction
centers (De Weerd et al. 2002; Holzwarth et al. 2006a,
2006b; Wang et al. 2007), isolated light-harvesting antenna
complexes (Croce et al. 2001; Gradinaru et al. 2000, 2001;
Ilagan et al. 2006; Krueger et al. 2001; Papagiannakis et al.
2002, 2003; Polı́vka et al. 2002; Polivka and Sundström
2004; Zigmantas et al. 2002), artificial antenna systems
(Berera et al. 2006, 2007; Kodis et al. 2004; Pan et al. 2002),
and photoreceptor proteins that bind only a single chromophore (Kennis and Groot 2007; Wilson et al. 2008). With
appropriate detection schemes that involve multichannel
detection on a shot-to-shot basis, signal detection sensitivities of *10-5 units of absorbance over a broad wavelength
range can be achieved, implying that molecular species with
a small extinction coefficient or that accumulate in very low
(transient) concentrations can be detected (Berera et al.
2006; Wilson et al. 2008). A drawback of a 1–5-kHz system
is that with its relatively high excitation densities, multiple
excited states may appear in a single multichromophoric
complex, resulting in singlet–singlet annihilation processes
among (B)Chls (Van Grondelle 1985).
With the laser systems that operate at 40–250 kHz, a
lower pulse energy can be used for excitation with respect
to the kHz systems owing to their higher repetition rate,
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Photosynth Res (2009) 101:105–118
which allows more laser shots to be averaged per unit time.
Typically, pulse energies of 0.5–10 nJ are used, roughly
corresponding to excited-state populations of \1–10%.
Under the right circumstances, detection sensitivities of
*10-6 units of absorbance can be achieved. Accordingly,
this kind of system has been used to study exciton migration in large systems with many connected pigments such
as chloroplasts and light-harvesting complex (LHC) II
aggregates (Holt et al. 2005; Ma et al. 2003; Ruban et al.
2007). In addition, it has been used to examine exciton
migration in isolated LH complexes under annihilation-free
conditions (Monshouwer et al. 1998; Novoderezhkin et al.
2004; Palacios et al. 2006; Papagiannakis et al. 2002).
Drawbacks of this type of systems involve the shorter time
between pulses (4–20 ls), which may lead to the build-up
of relatively long-lived species such as triplet or chargeseparated states. In addition, multichannel detection on a
shot-to-shot basis has been limited to 14 channels at such
high repetition rates (Ruban et al. 2007), although significant strides are currently being made in our laboratory to
resolve this limitation.
Figure 2 shows a scheme of an ultrafast transient
absorption setup, as it exists today in the Biophysics Laboratory of the Laser Center at the Vrije Universiteit (LCVU)
in Amsterdam, The Netherlands. A broadband oscillator
(Coherent Vitesse) generates pulses of *30 fs duration with
a wavelength of 800 nm, a bandwidth of *35 nm at a
repetition rate of 80 MHz. The pulses from the oscillator are
too weak to perform any meaningful spectroscopy and
therefore have to be amplified. Femtosecond pulse amplification is not a trivial matter because at high energies, the
peak power in a femtosecond pulse becomes so high that
amplification and pulse-switching media such as crystals
and Pockels cells easily get damaged. A Pockels cell is an
electro-optical device containing a crystal, such as potassium dihydrogenphosphate (KH2PO4), capable of switching
the polarization of light when an electrical potential difference is applied to it. In this way, the amount of stimulated
emission from the laser cavity can be controlled. For this
reason, femtosecond pulse amplification is carried out
through the chirped-pulse amplification principle: the pulse
from the oscillator (hereafter, referred to as ‘‘seed pulse’’) is
first stretched to *200 ps by a stretcher, which temporally
delays the ‘‘blue’’ wavelengths within the pulse bandwidth
of *35 nm with respect to the ‘‘red’’ wavelengths by means
of a grating pair. Then, the seed pulse is coupled into a
regenerative amplifier (Coherent Legend-UltraShort Pulse
(USP)). There, the seed pulse travels through a Pockels cell
which sets its polarization in such a way that it becomes
trapped within the amplifier’s cavity. On traveling back and
forth in the cavity, it passes through a Ti:sapphire crystal
that is pumped at 1-kHz repetition rate by a diode-pumped
Nd:YLF pump laser at 527 nm (Coherent Evolution, 30 W).
Photosynth Res (2009) 101:105–118
109
Fig. 2 Schematic
representation of an
experimental ultrafast transient
absorption setup
At each passage through the crystal, the trapped seed pulse is
amplified until saturation is reached. Then, the Pockels cell
switches the polarization of the amplified pulse which
results in its ejection from the amplifier. The amplified pulse
is compressed to *45 fs by temporally synchronizing the
‘‘blue’’ and ‘‘red’’ wavelengths within the pulse bandwidth,
essentially the reverse of the ‘‘stretching’’ procedure. At this
point, the output from the laser system is a 40-fs pulse at an
energy of 2.5 mJ, a center wavelength of 800 nm, a bandwidth of 30 nm, and a repetition rate of 1 kHz.
In order to perform transient absorption spectroscopy
with a Ti:sapphire laser alone, one is restricted to a
wavelength region for the excitation pulse around 800 nm,
allowing only the study of some BChl a-containing systems
(Arnett et al. 1999; Kennis et al. 1997b; Nagarajan et al.
1996; Novoderezhkin et al. 1999; Streltsov et al. 1998;
Vulto et al. 1999). In order to shift the wavelength to other
parts of the visible and near-IR spectra, optical parametric
amplifiers (OPAs) or optical parametric generators (OPGs)
are typically used. In an OPA, non-linear birefringent
crystals such as beta barium borate (BBO) are pumped by
the direct output of the amplified laser system at 800 nm or
frequency-doubled pulses at 400 nm. The pump is temporally and spatially overlapped with a white-light continuum
in the crystal, and depending on the angle between the laser
beam and the symmetry axis of the crystal, two particular
wavelengths of the white-light continuum called ‘‘signal’’
and ‘‘idler’’ are amplified through the second-order nonlinear polarizability of the crystal, of which the signal has
the shortest wavelength and is routinely selected for further
use. Since pump, signal, and idler beams have different
polarizations, the group velocity of pump, signal, and idler
beams can be made equal by varying the angle between the
laser beam and the symmetry axis of the birefringent
crystal. This allows energy from the pump beam to be
converted to the signal and idler beams over a large
propagation length up to millimeters. This is the so-called
phase-matching condition. Conservation of energy requires
that the sum of the frequencies of signal and idler add up to
the frequency of the pump beam. Thus, 800-nm-pumped
OPAs operate in the near-InfraRed (IR) (1,100–1,600 nm
for the signal) while 400-nm-pumped OPAs operate in the
visible (475–750 nm for the signal) spectrum. Using the
output of an OPA as a basis, essentially all wavelengths
from the UltraViolet (UV) to mid-IR can be generated at
relatively high pulse energies by using non-linear mixing
processes such as frequency-doubling, sum-frequency
generation, and difference-frequency generation in suitable
non-linear crystals. Obviously, visible and near-IR light are
the most useful wavelengths for the study of photosynthetic
systems. In addition, mid-IR wavelengths are very useful
for probing molecular vibrations of chlorophylls and
carotenoids (Groot et al. 2005, 2007). The pulse duration
out of the OPA roughly corresponds to that of the amplified
Ti:sapphire laser system. The pulse energy from our
regenerative laser amplifier of 2.5 mJ allows simultaneous
pumping of several OPAs. The latter option is important
for experiments that require multiple pump pulses, such as
pump–dump or pump–repump experiments (Kennis et al.
2004; Larsen et al. 2003; Papagiannakis et al. 2004).
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The transient absorption setup
In order to vary the time delay between the excitation and
probe pulses, the excitation pulse generated by the OPA is
sent through an optical delay line, which consists of a
retroreflector mounted on a high-precision motorized
computer-controlled translation stage. The translation stage
employed in our experiments has an accuracy and reproducibility of 0.1 lm, which corresponds to a timing accuracy of 0.5 fs. The delay line can be moved over 80 cm,
implying that time delays up to 5 ns can be generated
between excitation and probe beams. The excitation beam
is focused in the sample to a diameter of 130–200 lm and
blocked after the sample. In most cases, the polarization of
the pump beam is set at the magic angle (54.7°) with
respect to that of the probe to eliminate polarization and
photoselection effects (Lakowicz 2006).
For the detection of the pump-induced absorbance
changes, a part of the amplified 800-nm light is focused on
a sapphire or calcium fluoride plate (though other materials
such as quartz, MgF2, water, and ethylene glycol can also
be used) to generate a white-light continuum. In the
absence of special precautions, the white-light continuum
may range from *400 to *1,100 nm (depending on the
material) and be used as a broadband probe; its intensity is
so weak that it does not transfer an appreciable population
from the ground to the excited state (or vice versa). It is
focused on the sample to a diameter slightly smaller than
the pump, spatially overlapped with the pump, collimated,
and sent into a spectrograph. There, it is spectrally dispersed and projected on a silicon diode array that consists
of tens to hundreds of elements. The diode array is read out
by a computer on a shot-to-shot basis, in effect measuring
an absorption spectrum with each shot.
Under some experimental conditions, detection with a
diode array is not possible or appropriate. For instance, for
many experiments in the near-IR and the UV, other
detector types need to be employed that, in combination
with the white-light continuum intensities at those wavelengths, lack the sensitivity required for array detection. In
these cases, single wavelength detection is often employed.
In the mid-IR (*3–10 lm), mercury cadmium telluride
(MCT) arrays that consist of 32 or 64 elements are available (Groot et al. 2007). Another detection method in the
visible spectrum employs a charge-coupled device (CCD)
detector. Frequently, a reference beam is used to account
for shot-to-shot intensity fluctuations in the white-light
continuum. In such a case, the white-light continuum beam
is split in two beams, the probe and the reference. The
probe is overlapped with the pump beam in the sample,
while the reference beam is led past the sample (or through
the sample past the excited volume). The probe and reference beams are then projected on separate diode arrays.
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During data collection, the probe beam is divided by the
reference beam, which may lead to improved signal to
noise because the intensity fluctuations of the white-light
continuum are eliminated.
By the nature of the white-light generation process, the
white light is ‘‘chirped’’ on generation, i.e., the ‘‘blue’’
wavelengths are generated later in time than the ‘‘red’’
wavelengths. The exact temporal properties depend on the
specific generation conditions. Hence, the white-light
continuum has an ‘‘intrinsic’’ group-velocity dispersion.
When traveling through optically dense materials such as
lenses and cuvettes, the group velocity dispersion in the
white light readily increases to picoseconds. This effect can
be minimized by using parabolic mirrors for collimation
and focusing of the white-light beam between its point of
generation and the sample. The group velocity dispersion
may be accounted for in the data analysis and described by
a polynomial function. Alternatively, the white-light continuum can be compressed by means of a grating pair or
prism pair in such a way that the ‘‘red’’ and ‘‘blue’’
wavelengths in the probe beam coincide in time.
The instrument response function of this particular transient absorption apparatus, which can be measured by frequency mixing in a non-linear crystal placed at the sample
spot or by the transient birefringence in CS2 or water, can
usually be modeled with a Gaussian with a FWHM of
120 fs. If required, the white-light continuum can be compressed down to *10 fs by means of a grating pair or prism
pair; in such a case, the instrument response function is
generally limited by the duration of the pump pulse.
For measurements at room temperature, the sample is
placed in a 1–2-mm quartz cuvette which is either connected to a flow system or mounted on a shaker to prevent
exposure of the same excited volume to multiple laser shots
and to prevent sample degradation.
Collection of transient absorption spectra
A transient absorption experiment proceeds as follows: the
time delay between excitation and probe beams is fixed.
Before reaching the sample, the excitation beam (that
delivers a pulse every 1 ms) passes through a mechanical
chopper that is synchronized to the amplifier in such a way
that every other excitation pulse is blocked. Thus, alternately the sample is being excited and not excited. Consequently, the white-light continuum that is incident on the
detector diode array alternately corresponds to a ‘‘pumped’’
and ‘‘unpumped’’ sample, and the detector alternately
measures the intensity of the probe beam of a ‘‘pumped’’
and ‘‘unpumped’’ sample, I(k)pumped and I(k)unpumped.
I(k)pumped and I(k)unpumped are stored in separate buffers
(while keeping the time delay between pump and probe
fixed), and a number of shots that is sufficient for an
Photosynth Res (2009) 101:105–118
acceptable signal-to-noise ratio is measured, usually 103–
104. With the shot-to-shot detection capability of the
multichannel detection system, particular spectra that
deviate from the average (‘‘outliers’’) can in real time be
rejected during data collection, significantly improving
signal-to-noise ratio. A second white-light beam (the reference beam) not overlapping with the pump pulse can also
be used to further increase the signal-to-noise ratio. From
the averaged values of I(k)pumped and I(k)unpumped, an
absorbance difference spectrum DA(k) is constructed
according to
DAðkÞ ¼ logðIðkÞpumped =IðkÞunpumped Þ:
Then, the delay line is moved to another time delay between
pump and probe, and the above procedure is repeated. In
total, absorbance difference spectra at approximately 100–
200 time points between 0 fs and *5 ns are collected,
along with absorbance difference spectra before time zero
to determine the baseline. In addition, many spectra are
collected around the time that pump and probe pulse
overlap in time (‘‘zero delay’’) to enable accurate recording
of the instrument response function. This whole procedure
is repeated several times to test reproducibility, sample
stability, and long-term fluctuations of the laser system. In
this way, an entire dataset DA(k,s) is collected.
Anisotropy experiments in transient absorption
spectroscopy
In photosynthetic antennae and reaction centers, the pigments are bound in a well-defined way. Energy and electron transfer processes and pathways can be specifically
assessed through the use of polarized excitation and probe
beams. The time-dependent anisotropy is defined as
rðtÞ ¼ ðDAk ðtÞ DA? ðtÞÞ=ðDAk ðtÞ þ 2DA? ðtÞÞ:
With DAk(t), the time-dependent absorbance difference
signal with pump and probe beams is polarized parallel,
and with DA\(t), the time-dependent absorbance difference
signal with pump and probe beams is polarized perpendicular. In light-harvesting antennae, the decay of r(t)
indicates the elementary timescales of exciton migration,
be it through incoherent hopping or exciton relaxation
(Kennis et al. 1997b; Nagarajan et al. 1996; Novoderezhkin
et al. 1998; Savikhin et al. 1994, 1998, 1999; Vulto et al.
1999; Vulto et al. 1997). Energy transfer or exciton
relaxation processes often occur among (pools of) Chls
that have their absorption maxima at similar wavelengths.
Consequently, these processes are associated with
small spectral shifts of the DA spectra and are therefore difficult to observe under magic angle detection conditions. Through time-resolved anisotropy experiments, the
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timescales of such fast exciton migration events can
accurately be determined.
Data analysis
In time-resolved spectroscopic experiments, the very large
amounts of data collected can be analyzed by global and
target analysis techniques (Van Stokkum et al. 2004). A
typical time-resolved experiment DA(k,s) in fact consists of
a collection of thousands of data points, i.e., tens to hundreds wavelengths times one to two hundred data points. In
order to extract valuable information, one could simply
take slices of the data; for instance, one could take one
wavelength and look at its evolution in time (a so-called
kinetic trace), or one could plot the signal at different
wavelengths for a given time point (a DA spectrum). This is
normally the first stage of the data analysis where the
experimentalist has a glimpse of an expected (or unexpected) process. The next step in the data analysis is to
apply the so-called global analysis techniques, in an
attempt to distill the overwhelming amount of data into a
relatively small number of components and spectra. In the
most basic model, the femtosecond transient absorption
data are globally analyzed using a kinetic model consisting
of sequentially interconverting evolution-associated difference spectra (EADS), i.e., 1?2?3? in which the
arrows indicate successive monoexponential decays of
increasing time constants, which can be regarded as the
lifetime of each EADS. The first EADS correspond to the
time-zero difference spectrum. This procedure enables a
clear visualization of the evolution of the (excited) states of
the system. Based on the insight obtained from this model
and from the raw data, one can then take a further step in
the analysis and apply a so-called target kinetic scheme.
The EADS that follow from the sequential analysis are
generally made up from a mixture of various molecular
species. In general, the EADS may well reflect mixtures of
molecular states. In order to disentangle the contributions
from these molecular species and obtain the spectrum
signature of the ‘‘pure’’ excited- and product state intermediates (the so-called species-associated difference
spectra, SADS), a specific kinetic model must be applied in
a so-called target analysis procedure. In this way, the
energy and electron transfer mechanisms can be assessed in
terms of a number of discrete reaction intermediates. A
comprehensive review of global and target analysis techniques has been published (Van Stokkum et al. 2004). In
the next section, we illustrate a few examples of timeresolved experiments and data analysis. We will start with
the description of elementary energy transfer processes in
artificial systems followed by more complex examples in
natural light-harvesting compounds.
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112
Example 1: the light-harvesting function of carotenoids
Carotenoids play an important role in light-harvesting
antennae, not only in photoprotection but also by harvesting blue and green light and transferring the excited-state
energy to nearby (B)Chls (Frank et al. 1999; Polivka and
Sundström 2004; Ritz et al. 2000). Carotenoids have a
complicated excited-state manifold: they have a strongly
allowed transition from the ground state (which has Agsymmetry in ideal polyenes) to a state with Bu? symmetry
called S2. This transition is responsible for their strong
absorption of blue-green light. Below the S2 state lies the
optically forbidden S1 state that has Ag- symmetry, along
with a number of additional optically forbidden states, the
physical nature of which remains unclear (Polivka and
Sundström 2004).
Ultrafast spectroscopy has proven to be a valuable tool
to map out the energy transfer pathways from carotenoid to
(B)Chl and understand these processes at the molecular
level. In particular, simple artificial photosynthetic lightharvesting systems have given important insights into the
physical mechanisms that underlie the various energy
transfer and relaxation processes (Berera et al. 2007; Kodis
et al. 2004; Marino-Ochoa et al. 2002). Figure 3a shows a
minimal artificial light-harvesting mimic suitable for the
study of the light-harvesting role of carotenoids. The model
Fig. 3 a Molecular structure of
a carotenophthalocyanine lightharvesting dyad 1. b Evolutionassociated difference spectra
(EADS) that result from a
global analysis on transient
absorption experiments on dyad
1. The excitation wavelength
was 475 nm. c Kinetic traces at
560 nm (upper panel) and
680 nm (lower panel). d Kinetic
scheme that describes the
excited-state processes in dyad
1 upon carotenoid excitation.
Solid lines denote energy
transfer, dotted denote internal
conversion, dashed denotes
intersystem crossing processes.
Source: Berera et al. (2007)
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Photosynth Res (2009) 101:105–118
system, referred to as dyad 1, is made up of two moieties: a
carotenoid with nine conjugated double bonds in its pelectron system and a phthalocyanine (Pc) molecule. The
Pc molecule has a maximal absorption at 680 nm (called
the Q band), and it acts as a Chl a mimic. The carotenoid to
Pc energy transfer efficiency is very high in this particular
dyad, *90% (Berera et al. 2007).
In order to map out the energy transfer pathways from
carotenoid to Pc, the carotenoid moiety of dyad 1 was
excited at 475 nm for ultrafast time-resolved spectroscopy
(Berera et al. 2007). Figure 3b shows the results of a
global analysis of the time-resolved data. Figure 3c shows
kinetic traces at selected wavelengths for dyad 1. Six time
constants were needed for a satisfactory fit of the data.
The first EADS (Fig. 3b, dotted line) is formed instantaneously at time zero and represents population of the
optically allowed S2 state of the carotenoid. It presents a
region of negative signal below 570 nm originating from
the carotenoid ground-state bleach and from stimulated
emission (SE). In addition, the Pc Q region around
680 nm shows a band shift-like signal. The latter is due
the response of the Pc molecule to the charge redistribution on the nearby carotenoid upon excitation to the S2
state.
The first EADS evolve in 40 fs into the second EADS
(Fig. 3b, dashed line), which is characterized by a strong
Photosynth Res (2009) 101:105–118
bleach/SE signal at 680 nm. This corresponds to a population of the Pc excited state (the Q state) indicating that
the carotenoid S2 state is active in transferring energy to
Pc. The dip at 610 nm originates from a vibronic band of
the Pc Q state. In addition, excited-state absorption is
observed in the 480–600 nm region, which can be assigned
to the optically forbidden S1 state and the so-called S* state
(Gradinaru et al. 2001). This observation indicates that
internal conversion from the carotenoid S2 state to the
lower-lying states has taken place in competition with
energy transfer to Pc. The S1 excited-state absorption has a
maximum around 560 nm while that of the S* state is
around 525 nm.
The evolution to the third EADS (Fig. 3b, dash-dotted
line) takes place in 500 fs. It corresponds to a decrease of
excited state absorption (ESA) at the red wing of the S1
absorption, which may be assigned to vibrational cooling
of the S1 state (Polivka and Sundström 2004). Moreover, an
increase of the Pc Q bleach at 680 nm is observed which is
likely to originate from the energy transfer from the S1 and
possibly the S* state to Pc. Note that the third EADS
overlap with the fourth EADS (Fig. 3b, solid line) in the Pc
Q region and is not visible.
The fourth EADS (Fig. 3b, solid line) appear after
900 fs and has a lifetime of 7.8 ps. The signal at 525 nm,
where the main contribution to the spectrum is given by S*,
has decreased, whereas the signal in the 540–620 nm
region, where the absorption is mainly due to S1, has
slightly increased, indicating the decay of S* in about
0.9 ps, partly by internal conversion to S1.
The evolution to the fifth EADS (Fig. 3b, dash–dot–dot
line) takes place in 8 ps. At this stage, the carotenoid ESA
has decayed, and the fifth EADS correspond very well to
that of the excited Pc Q state with a flat ESA in the 450–
600 nm region. Around 680 nm, the bleach increases with
respect to the previous EADS, which implies that the
carotenoid S1 state has transferred energy to Pc.
The final EADS (Fig. 3b, short-dotted line) is formed in
2 ns and represents the component that does not decay on
the time scale of the experiment. It features the typical
carotenoid triplet ESA in the 475–550 nm region as well as
a bleach/band shift-like signal in the Pc Q region. Thus, the
carotenoid triplet state rises directly upon decay of the
singlet excited state of Pc. This observation implies that
triplet–triplet energy transfer from Pc to the carotenoid
occurs much faster than the inter system crossing (ISC)
process in Pc, which effectively occurs in 2 ns.
Figure 3c shows the kinetic trace recorded at 680 nm
(lower panel) and at 560 nm (upper panel), corresponding
to the maximum of the Pc Q absorption and the maximum
of carotenoid S1 excited state absorption. At 680 nm, the
ultrafast rise of the bleach corresponding to the carotenoid
S2 ? Pc energy transfer (40 fs) is followed by two slower
113
rise corresponding to hot S1 and/or S* ? Pc (500–900 fs)
and S1 ? Pc energy transfer (8 ps). At 560 nm, the
carotenoid S1 signal decays in 8 ps and matches the 8 ps
rise of the Pc bleach. The energy transfer pathways in dyad
1 are summarized with the kinetic scheme in Fig. 3d. Note
that this scheme is simplified; a full account of the kinetic
modeling of energy transfer pathways in dyad 1 along with
the SADS of the involved molecular species is given in
Berera et al. (2007). The carotenoid to Pc energy transfer
dynamics in dyad 1 is reminiscent of several natural lightharvesting antennas where high energy transfer efficiency
from carotenoids to chlorophylls is obtained; this occurs by
transfer of energy to Chl from multiple excited states of the
carotenoid (Holt et al. 2004; Kennis et al. 2001; Papagiannakis et al. 2002; Polivka and Sundström 2004; Ritz
et al. 2000; Walla et al. 2000, 2002; Wehling and Walla
2005; Zhang et al. 2000; Zigmantas et al. 2002).
Example 2: carotenoids in non-photochemical
quenching in photosystem II and artificial systems
When exposed to high light illumination, oxygenic photosynthetic organisms protect themselves by switching to a
protective mode where the excess energy in photosystem II
(PSII) is dissipated as heat through a mechanism known as
non-photochemical quenching (NPQ) (Demmig-Adams
et al. 2006; Horton et al. 1996; Müller et al. 2001). The
mechanism of energy dissipation in the PSII antenna has
long remained elusive but over the last years, significant
progress has been made in resolving its molecular basis. In
particular, the involvement of carotenoids in the quenching
of Chl singlet excited states has clearly been demonstrated.
Yet, controversy persists on whether the quenching process(es) involve energy or electron transfer processes
among Chls and carotenoids, and which particular Chl and
carotenoid pigments constitute the quenching site (Ahn
et al. 2008; Berera et al. 2006; Holt et al. 2005; Ma et al.
2003; Ruban et al. 2007).
On the basis of energetic considerations, it was speculated that the low-lying optically forbidden S1 state of
carotenoids could act as an energy sink to Chl singlet
excited states (Frank et al. 1994). In order to address this
issue, ultrafast transient absorption spectroscopy was
applied on the same artificial light-harvesting dyad as
discussed previously, but with extended conjugated pelectron system of the carotenoid moiety with 10 or 11
C=C double bonds, implying lower excited-state energies
(Fig. 4a). Strikingly, the Pc lifetime is reduced from its
natural lifetime of 3 ns to 15–300 ps, depending on the
length of the carotenoid’s conjugated p-electron system
(Fig. 4b) and the solvent polarity. Furthermore, Berera
et al. (2006) have demonstrated that the carotenoid S1
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114
Photosynth Res (2009) 101:105–118
Fig. 4 a Molecular structure of
a carotenophthalocyanine lightharvesting dyad 1, 2, and 3. The
carotenoids of dyad 1, 2 and 3
contain 9, 10 and 11 conjugated
C=C double bonds,
respectively. b Upper panel:
kinetic traces at 680 nm of dyad
1, 2, and 3 and a model Pc in
tetrahydrofuran (THF). Lower
panel: kinetic traces of dyad 3
dissolved in acetone detected at
480 nm (solid line) and 576 nm
(dashed line). Excitation
wavelength for b and d was
680 nm. c Kinetic scheme that
describes the excited-state
decay processes In dyad 2 and 3
upon Pc excitation. Solid line
denotes energy transfer, dotted
line denotes internal conversion
process. d Evolution-associated
difference spectra (EADS) that
result from a global analysis on
transient absorption
experiments on dyad 3
dissolved in acetone. Source:
Berera et al. (2006)
excited state acts as the acceptor of excited-state energy
from the covalently linked Pc, as schematically shown in
Fig. 4c, thereby providing an efficient channel for energy
dissipation.
A crucial aspect of Pc and Chl excited-state quenching
by the carotenoid S1 state is the notion that such processes
occur through a so-called inverted kinetic scheme, i.e., the
quenching state S1 is slowly populated by rate constant kslow
(in 15–300 ps) and quickly depopulated with rate constant
kfast (in *5 ps). The latter time constant is inherent to the
photophysics of the carotenoid S1 state, i.e., internal conversion to the ground state occurs on this timescale through
efficient vibronic coupling between the ground and S1 states
(Chynwat and Frank 1995). In such an inverted kinetic
scheme, the donor (Pc) decays with a single rate constant
kslow. The acceptor (carotenoid S1) will rise with rate constant kfast and decay in parallel with the donor with rate
constant kslow, and reach a maximum transient concentration that remains low, and with sufficiently separated rate
constants, it is approximately equal to kslow/kfast. Thus, in
the specific case of the artificial light-harvesting dyads, the
carotenoid S1 signal is expected to rise with a rate constant
that corresponds to the internal conversion rate of S1 to the
ground state and to have a low amplitude throughout the Pc
excited-state lifetime. This is exactly what is observed
experimentally: Figure 4d shows the EADS for dyad 3,
whose carotenoid has 11 conjugated double bonds, dissolved in the very polar solvent acetone. Under these
123
circumstances, the Pc is strongly quenched to a main lifetime of 15 ps and the quenching state is expected to accumulate to a readily observable transient concentration of
approximately a third of the Pc population at time zero. The
Pc moiety of dyad 3 was excited at 680 nm. Four components are needed to obtain a satisfactory fit of the data, with
lifetimes of 4.9, 15, and 89 ps and a non-decaying component. A closer examination of the EADS reveals the
nature of the quenching process: the first component
(Fig. 4d, solid line), appearing at time zero, shows bleach of
the Pc Q state in the 680 nm region—an almost flat excited
state absorption region that represents the excited Pc molecule. The first EADS evolve in 4.9 ps to the second EADS
(Fig. 4d, dashed line), characterized by an increase of the
amplitude in the 530–600 nm region and a decrease below
530 nm. The Pc bleach at 680 nm remains the same. This
change indicates that another species is populated in 4.9 ps.
In fact, the positive signal in the 530–600 nm region is due
to the carotenoid S1 ESA, while the region below 530 nm
corresponds to the carotenoid ground-state bleach. Thus, the
second EADS is a superposition of Pc singlet excited state
and a contribution from the carotenoid S1 state. The second
EADS evolve to the third EADS (Fig. 4d, dotted line) in
15.6 ps. The third EADS is characterized by an overall
decrease of the Pc and carotenoid S1 signal with respect to
the second EADS, indicating that these molecular species
have decayed together. The third EADS has a lifetime of
89 ps and represents a fraction of dyad 3 that decays more
Photosynth Res (2009) 101:105–118
115
slowly, presumably as a result of conformational heterogeneity (Berera et al. 2006). A target analysis that fully
accounts for the spectral evolution in terms of distinct
SADS for the Pc and carotenoid S1 excited states is given in
Berera et al. (2006).
The inverted kinetics of the carotenoid S1 state are
illustrated in the lower panel of Fig. 4b, where kinetic
traces at 480 and 576 nm are shown upon excitation of Pc
at 680 nm. The 576 nm trace represents the carotenoid S1
excited state absorption region and shows a rise with a time
constant of 4.9 ps that mainly decays in 15 ps. Thus,
population of the carotenoid S1 state rises in 4.9 ps and
then decays in parallel with excited Pc. Likewise, the
480 nm trace first gets a positive amplitude that originates
from Pc ESA. Then, the signal apparently decays in 4.9 ps.
The latter is interpreted as a growing in of the carotenoid
ground-state bleach that results from a population of the
carotenoid S1 state. Thus, the 480 and 576 nm traces show
the rise in 4.9 ps and decay in 15 ps of the quenching state,
i.e., the carotenoid S1 state.
Time-resolved experiments in LH complexes of oxygenic photosynthesis have revealed kinetic patterns similar
to those observed for the artificial dyads, in particular the
major light harvesting antenna of plants, LHCII (Ruban
et al. 2007) and the aggregated IsiA antenna complexes
from cyanobacteria (Berera et al. 2009). Figure 5 shows
selected kinetic traces for LHCII in the unquenched,
trimeric state (panel a) and in a quenched aggregated state
(panel b), following a 100 fs, 10 nJ laser pulse at 675 nm.
In the quenched state, the trace at 537 nm not only represents the carotenoid S1 ESA, but it also has a positive
amplitude coming from Chl ESA. It clearly shows a slower
decay in the first *10 ps compared to the decay of the Chl
Qy state at 679 nm. The opposite trend is seen at 489 nm
(carotenoid ground state absorption region), where the
trace shows a faster decay in the first *10 ps. If only Chl
signals were to contribute to the kinetics, one would expect
homogeneous decay. Thus, in analogy with the dyad case
(vide supra), the observed DA signals show that concomitantly with the decay of the Chl excited state, a carotenoid
excited state is populated. Application of a target analysis
with a kinetic model that incorporates quenching and singlet–singlet annihilation (Fig. 5, panel c) revealed the
SADS of the quenching state, which correspond to the
carotenoid S1 state. On the basis of the wavelength of its
maximum ground-state bleach, Ruban et al. (2007) concluded that Lutein 1 likely acts as a quencher of Chl
excited states in this isolated system.
In conclusion, carotenoids can accept energy from a
neighboring tetrapyrrole thereby acting as strong quenchers
(Berera et al. 2006, 2009; Ruban et al. 2007). The carotenoid S1 state acts as a quencher and effective energy
dissipator since its lifetime is 100–1,000 times shorter
compared to the lifetime of the Pc or Chl excited state. By
Fig. 5 Selected kinetic traces for unquenched LHCII trimers (a) and
quenched LHCII aggregates (b) at 677 nm (top), 489 nm (middle)
and 537 nm (bottom), following a 100 fs, 10 nJ laser pulse at 675 nm.
The vertical axis shows the measured change in absorption, the
horizontal axis is linear up to 1 ps and logarithmic thereafter. The
long short-dashed line represents the 1 ps phase due to chlorophyll
excited state relaxation, the dotted line the excited state decay of
chlorophyll, the dashed line the absorption changes due to the
quencher Q, and the dash-dotted line the build-up of the triplet state.
The kinetic model is shown in (c) and the corresponding speciesassociated difference spectra (SADS) in (d). Source: Ruban et al.
(2007)
123
116
making use of ultrafast spectroscopy, we have been able to
follow the process of energy dissipation in real time and to
determine the underlying physical mechanism. In particular, it is important to note that the quenching phenomena in
the artificial dyads, PSII, and IsiA antenna systems occur
through inverted kinetic schemes where the lifetime of the
quencher is inherently shorter lived than the Chl excited
state. This results in low transient concentrations of the
quenching states, which requires transient absorption data
at high-signal to noise. Further, application of target analysis techniques utilizing specific kinetic models is required
to extract the spectroscopic signature of the quenching
states and to identify the molecular mechanism of nonphotochemical quenching.
Acknowledgments J.T.M.K. and R.B. were supported by the Earth
and Life Sciences council of the Netherlands Foundation for Scientific Research (NWO-ALW) through a VIDI and a Rubicon grant,
respectively. The authors thank Cosimo Bonetti for providing Fig. 2.
This manuscript was edited by Govindjee.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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