Coherent Oscillations in the PC577 Cryptophyte Antenna Occur in the

Article pubs.acs.org/JPCB

Coherent Oscillations in the PC577 Cryptophyte Antenna Occur in the Excited Electronic State Scott D. McClure, Daniel B. Turner,# Paul C. Arpin, Tihana Mirkovic, and Gregory D. Scholes* Department of Chemistry and Centre for Quantum Information and Quantum Control, 80 Saint George Street, University of Toronto, Toronto, Ontario M5S 3H6, Canada ABSTRACT: Transient absorption spectroscopy is a useful measurement for investigating ultrafast dynamics in molecules. We have developed a transient absorption spectrometer that utilizes balanced and fast detection methods to suppress noise and maintain high temporal and spectral resolution. We use the spectrometer to investigate the ultrafast dynamics in a photosynthetic pigment− protein complex, the phycobiliprotein PC577 isolated from the cryptophyte alga Hemiselmis pacifica CCMP706. We analyze coherent oscillations in the transient absorption data and attribute them to vibrational coherences. Analysis of the dynamic Stokes shift and motion of the wave packet on the potential-energy surface indicate that the coherences arise from vibrational wave packets in the excited electronic state of the protein.

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INTRODUCTION

complexes. There is presently evidence for both vibrational and electronic coherences.33−36 A current question is how to assign the origin of coherent oscillations when electronic delocalization (electronic coherence) influences the excited state. A difficulty facing researchers is that clear solutions to this problem have so far been attained only in limiting cases or special cases of electronic coupling. Nevertheless, several groups have developed or modified techniques to help unravel differences between the various types of coherences and determine signatures that could be evident in spectroscopic data.4−6 A particular question of recent interest is whether electronic coherence generates oscillating cross peaks in 2D ES that actually represent ground-state vibrations but are positioned in the 2D map as if they were excited-state features.7 Here, we seek to find distinguishing features between excited-state and ground-state coherences in measurements of a representative cryptophyte antenna complex. We report that the oscillations observed in broadband ultrafast measurements of PC577 are primarily excited-state features. We investigate the coherences generated by ultrafast broadband electronic excitation of a photosynthetic protein, PC577, isolated from the cryptophyte algae Hemiselmis pacifica (CCMP 706). This PC577 light-harvesting complex contains eight bilin chromophores (two dihydrobiliverdins (DBVs) and six phycocyanobilins (PCBs)) covalently bonded to a protein scaffold (Figure 1). PC577 acts as the main peripheral antenna for cryptophyte algae. Owing to the high concentration of the complex associated with the thylakoid membrane and its

Transient absorption spectroscopy is widely used to study the photophysics of molecules and chemical reactions by photoinitiating dynamics with a strong laser pump pulse and then monitoring the time evolution with a weaker probe pulse. The development of nearly transform-limited laser pulses within the realm of tens of femtoseconds1,2 allowed researchers to investigatein addition to ultrafast decay of populations coherent oscillations in the signal amplitude. Often these oscillations indicate wave packet dynamics evolving from a coherent superposition of vibrational states.3 The dynamics of a wave packet on the excited-state potentialenergy surface are typically classified as either incoherent or coherent. In the incoherent regime, the wave packet thermalizes, meaning its energy is dissipated to other degrees of freedom. In the coherent regime, the damping of the wave packet is slow compared to its periodic motion about the potential well.8,9 In the former, we expect only population decay in the emission spectrum of the wave packet as it decays to the ground state, and in the latter, we expect coherent oscillations superimposed on the decay. Selected examples include organic molecules in solution,10−16 polymers and crystals,17−19 and even elementary chemical reactions.20,21 Femtosecond transient absorption studies of photosynthetic proteins and other light-activated biological systems continue to be of interest. Previous reports include studies of isolated bacterial reaction centers,22−24 the Fenna−Matthews−Olson complex,25−27 bacteriorhodopsin,28−30 and light-harvesting complexes.31,32 Each of these studies found evidence for coherence within biological systems. Recent experiments using two-dimensional electronic spectroscopy (2D ES) have provided further insights into coherences in photosynthetic © 2014 American Chemical Society

Received: December 5, 2013 Revised: January 7, 2014 Published: January 15, 2014 1296

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RESULTS

We developed a broadband transient absorption spectrometer that combines fast acquisition and balanced detection with the spectral resolution and sensitivity of a charge-coupled device (CCD) camera. Our apparatus is similar to one demonstrated previously;42 however, we used a CCD instead of a photodiode array, and we balanced the laser fluctuations with a reference beam. The instrument suppresses noise and extracts spectral information. In the Experimental Methods section, we describe the spectrometer in detail, including the optics and electronics behind its operation. We also report quantitatively on its suppression of noise. We performed transient absorption measurements on the photosynthetic light-harvesting complex PC577 isolated from the cryptophyte algae H. pacifica. The absorption spectrum of PC577 spans over one hundred nanometers centered at 577 nm. The complex contains two kinds of chromophores: the two DBV molecules absorb at the high-energy side of the absorption spectrum, and the six PCBs molecules absorb at the low-energy side. The linear absorption and fluorescence spectra of the complex are shown in Figure 2a,d, along with the spectrum of the laser pulse used for the transient absorption measurements. The same pulse was used to both excite and probe the molecule. In Figure 2b, we show representative transient absorption data recorded over the range of pump−probe delay times from 0−2 ps for PC577. The measurements were performed five times during a 90 min period. We see a dominant negativeamplitude (excited-state absorption) feature for wavelengths longer than approximately 660 nm. A clear node at about 650 nm separates the excited-state absorption from the positiveamplitude (ground-state bleach and stimulated emission) features dominating the lower wavelengths of the transient absorption spectrum. We see that the positive-amplitude features at about 580 nm decay more quickly than the feature centered at about 620 nm, typical of downhill energy transfer. We fit the traces at 577 and 620 nm separately to biexponential decay functions to estimate the approximate population relaxation time scales for the DBVs and the PCBs, respectively. We repeated this procedure for five independent measurements and display the results in Table 1. Oscillatory features are evident in the transient signal traces throughout the probe spectral window. The most prominent oscillations are superimposed on the ground-state bleach centered at probe wavelength λprobe ∼ 635 nm and superimposed on the excited-state absorption at λprobe ∼ 680 nm. We analyzed transients extracted from the data at λprobe = 616 nm from each of the measurements. We plot the average and standard deviation of these five transients for the first 1.5 ps after excitation in Figure 3. The transients from different data sets were linearly shifted, because a small error in the delay stage caused time zero to drift slightly between measurements. The compensation shifts ranged from 4 to 16 fs. We did not use any other normalization, interpolation, or scaling techniques. The coherent oscillatory features in the probe signal are clear amidst the population decay for the first 1.5 ps. These oscillatory features are evidently composed of several different frequencies, each with comparable amplitude. Even the smallamplitude structure in the oscillations is reproducible. To analyze the frequency components of the oscillations, we subtracted the biexponential fit to the amplitude decay at each probe wavelength and plot the residuals of the data in Figure 2c

Figure 1. Structural model of PC577 light-harvesting complex from Hemiselmis pacif ica (CCMP 706) from X-ray diffraction.41 The complex is composed of eight bilin chromophores: two dihydrobiliverdins (purple) and six phycocyanobilins (cyan) covalently bound to a protein scaffold (green). The protein has an open structure that separates the chromophores at its center.

complementary absorption spectrum to chlorophyll, PC577 helps the organism to capture a greater spectral cross section of sunlight than would be possible with solely the membranebound complexes that include photosystem I and photosystem II.37 Cryptophyte light harvesting involves both intraprotein energy transfermostly from the two high-energy DBVs to the six low-energy PCBsand interprotein energy transfer (that takes place only in vivo) between the PCBs to other pigment− protein complexes and ultimately the photosystems. Previously, we have reported studies of cryptophyte antenna complexes such as PE545 from Rhodomonas sp. and PC645 from Chroomonas sp. where the chromophores located in the center of the complex are closely associated and tend to coherently share electronic excitation.34,38−40 After broadband excitation, we see a combination of electronic and vibrational coherences.5 Recently, a cryptophyte genus was discovered where the antenna complex has an “open” structure, so that the central chromophores are separated41 (Figure 1). In this type of antenna complex, excitation is anticipated to not be delocalized. PC577 is one antenna with such an open structure. The weak electronic delocalization in PC577 means that the coherent oscillations observed after broadband photoexcitation are likely due predominantly to vibrational coherence. This weak electronic delocalization provides an opportunity to examine coherences in a limit closer to Förster theory. We studied PC577 at ambient temperature, dispersed in aqueous buffer at a dilute concentration. This paper focuses on the results of broadband transient absorption spectroscopy. This spectroscopic method tends to be more intuitive than 2D ES and, moreover, can be helpful in ascertaining the origin of coherent oscillations in the signal amplitude, as we describe in this report. We observe features in the transient spectra both due to population decay and coherent oscillations. Our measurements lead us to discuss how the vibrational wave packet explores the potential-energy surfaces of PC577 during the first few hundreds of femtoseconds after excitation. 1297

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Figure 3. Coherent oscillations at λprobe = 616 nm. We display the mean (black line) and standard deviation (blue shaded area) of transients from five independent measurements. We applied a linear temporal shift to each transient to correct for errors in the delay stage but otherwise did not apply any normalization or scaling techniques.

In Figure 4a, we plot the result of Fourier transformation of the transient absorption data at each probe wavelength. As suggested by the time-domain traces, there are multiple highamplitude oscillations. The strongest oscillations appear at 190 and 270 cm−1, and the oscillations at 470, 500, 670, and 800 cm−1 are also all reproducibly discerned. The intensity of any oscillatory component decreases to the noise threshold at around λprobe ∼ 640 nm. The intensities of the strongest modes are about 2.5 times less on the red side of λprobe = 640 nm. There is another spectral region, λprobe = 660 to 690 nm, where the intensities of the strongest modes (190 and 270 cm−1) are seen to decrease to the noise threshold before becoming observable again at about λprobe = 700 nm. We focus on the 270 cm−1 oscillation mode, because it is the highest in amplitude. The complex data from the Fourier transform contains amplitude and phase information, and in Figure 4c we plot this information for the 270 cm−1 mode. The plot shows that the amplitude and the phase of this oscillation as a function of λ probe change dramatically around λprobe = 640 nm. The amplitude diminishes into the noise threshold, and the relative phase changes by about 2.5 radians. In other words, the red and blue sides of λprobe = 640 nm are about 80% out-of-phase. The other modes show similar characteristics. The oscillatory features in the time domain are strong enough relative to the noise to apply a fitting technique. We first filter the time-trace data shown in Figure 3 in the frequency domain by applying a Heaviside filter from 130 to 1000 cm−1. This filter eliminates the constant background and the noise above 1000 cm−1 where we do not have the time resolution to observe oscillatory modes. We fit the filtered data in the time domain to a linear combination of eight damped cosine functions using a nonlinear least-squares algorithm. We chose eight functions because this was the number of clearly resolved peaks in the frequency domain. We maximize the robustness of the fit by minimizing the least absolute value residuals. The independent fits to each of the five transients as a function of pump−probe time delay all achieved a coefficient of determination value greater than 0.95. We seeded the fit algorithm with an estimate of the frequency of the eight peaks, but we did not specify bounds. All other parameters (the amplitude, phase, and decay constants) of the fit function were seeded randomly and without bounds (Table 2).

Figure 2. Steady-state and dynamic spectroscopy of PC577. (a) The linear absorption spectrum of PC577 (blue) and the laser pulse spectrum (orange) used for the transient absorption measurements. The dashed line (black) highlights the peak of the linear absorption spectrum. (b) A representative transient absorption spectrum of PC577 measured from 0−5 ps in 5 fs steps. We show the first 2 ps after excitation. The amplitude of the data corresponds to the change in the intensity of the probe light after interacting with the pumped sample. Positive (red) features represent increased signal emission when pumping, whereas negative (blue) features represent enhanced sample absorption when pumping. Positive times signify that the pump pulse arrived before the probe pulse. (c) The transient absorption signal of PC577 showing the coherent oscillations for the first picosecond after excitation. The background decay features were independently removed at each probe wavelength. A sharp node is seen around 640 nm. The oscillations on either side of this node are nearly out-of-phase relative to each other. The dashed line (orange) indicates a biexponential fit to the node in the oscillations. (d) The steady-state fluorescence spectrum of PC577 after excitation at 550 nm. The dashed line (black) highlights the peak of the fluorescence spectrum.

Table 1. Coefficients of the Biexponential Fits ΔI/I(t) = A1exp(−t/τ1) + A2exp(−t/τ2) of the Signal Amplitude at Probe Wavelengths 577 and 620 nm probe wavelength (nm)

A1 (%)

τ1 (fs)

A2 (%)

τ2 (ps)

577 620

1.9 ± 0.1 0.7 ± 0.1

640 ± 40 550 ± 70

2.5 ± 0.1 5.3 ± 0.1

15.16 ± 1.37 29.63 ± 1.80

for the first picosecond of the measurement. The highestintensity oscillations start out with a peak-to-peak amplitude of approximately 2% differential signal. There is a distinct region around λprobe = 640 nm where the amplitude of the oscillations goes to zero. The amplitude of the oscillations at λprobe < 640 nm has the opposite sign as the oscillations in the region λprobe > 640 nm. These features become clearer when we present the data in the frequency domain.

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DISCUSSION Population Dynamics. Transient absorption data contain ground-state bleach, stimulated emission, and excited-state 1298

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emission can reveal information about ground and excited potential-energy surfaces, respectively. The transient absorption spectrum of PC577 (Figure 2b) consists of a strong positiveamplitude feature in the center of the spectral probing window surrounded by negative-amplitude features on either side. The positive-amplitude feature is composed of ground-state bleach and stimulated emission components. In PC577, it is not possible to directly differentiate between the ground-state bleach and excited-state stimulated emission, because the Stokes shift is small.43 The negative-amplitude features are due to absorption of the probe light by the electronically excited chromophores in PC577. The density of states increases dramatically after the first excited state in large molecules like the chromophores in PC577, and it is therefore expected that the transient absorption spectrum would reveal some of the electronic states near twice the energy of the first excited state. The ground-state bleach and stimulated emission features become comparable in magnitude to the underlying excited-state absorption features near the edges of the linear absorption spectrum (540 and 660 nm). These effects then mutually cancel, and we observe no differential intensity in the transient absorption spectrum. A fit to each probe wavelength of the transient absorption spectra provides insight into the ultrafast dynamics of PC577. The biexponential fit hints at the multiple processes of population decaymainly by energy transferoccurring within the PC577 protein, as is known from studies of other cryptophyte antenna complexes.39,40,44 The transient absorption spectra show a fast decay of the positive-amplitude features around λprobe = 570 nm during the first few hundred femtoseconds after excitation. This spectral region corresponds to the maximum of the ground-state absorption by the highestenergy chromophores in PC577the DBV chromophores and the decay indicates the transfer of the excitation energy from the DBVs to the lower-energy PCB chromophores. The fit in this region gives two time constants of 640 ± 40 fs and 15.16 ± 1.37 ps of comparable magnitude from five independent measurements. These decay times represent average time scales of intracomplex energy transfer. The bulk of the ground-state absorption around 620 nm is due to the PCB chromophores. The biexponential fit to the transient absorption data in this spectral region gives a 550 ± 70 fs time constant and a 29.63 ± 1.80 ps time constant. In contrast to the transients in the DBV window, the femtosecond component of the PCBs is very small in amplitude relative to the picosecond component. The femtosecond component may be due to the energy transfer from the DBVs. Energy transfer within cryptophyte light-harvesting complexes appears to be governed by a fast (hundreds of femtoseconds) decay from the initially excited high-energy pigments and subsequent relaxation (tens of picoseconds) within the lower-energy pigments of the complex. For example, the population decay times for PC577 are similar to the pigment−protein complex PC645, which also contains DBV and PCB pigments.45 Previous studies have shown that in PC645, the energy transfer from DBVs to PCBs is on a time scale of hundreds of femtoseconds, and the energy transfer from one PCB pigment to another is on a time scale of tens of picoseconds.40,44,46 In vivo, one PC577 phycobiliprotein would transfer its energy to another pigment−protein complex on a time scale that would be comparable to but faster than the recovery of the bleach features. In intact cryptophytes

Figure 4. Analysis of oscillatory modes in the transient absorption data. (a) The power spectrum of PC577 from the backgroundsubtracted transient absorption data. Bright features correspond to high-amplitude oscillations at that particular probe wavelength. Shown is one of five measurements. (b) A representative power spectrum for the oscillations at λprobe = 616 nm. (c) The amplitude and phase of the 270 cm−1 oscillations present in the PC577 data are plotted as a function of the probe wavelength. Both the amplitude and phase exhibit a sharp change around λprobe = 640 nm.

Table 2. Eight Oscillatory Modes Obtained from a Fit with a Sum of Damped Cosines at λprobe = 616 nma oscillatory mode (cm−1) 190 270 370 450 470 510 660 810

± ± ± ± ± ± ± ±

10 10 40 110 10 10 10 10

damping time (ps) 0.4 0.44 0.7 0.21 0.4 0.3 0.6 0.11

± ± ± ± ± ± ± ±

0.1 0.08 0.1 0.03 0.2 0.2 0.1 0.01

a Average and standard deviation of the values obtained from five independent measurements.

absorption components. The sign of the amplitudes of groundstate bleach and stimulated emission is the same but opposite to excited-state absorption. Ground-state bleach and stimulated 1299

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PC577, we observe a damping time constant for this oscillation of 330 fs. None of the higher-wavenumber modes (1100, 1300, and 1700 cm−1) that were observed in the data of PC645 were observed in the transient absorption data of PC577. The absence of these modes is likely, because the pulse used in the transient absorption measurements is temporally longer than the pulse used in the 2D ES measurements. Vibrational coherences are only generated and measured when the pump and probe pulses are shorter in time than the period of each vibrational mode, and we were not able to resolve any oscillatory modes greater than 1000 cm−1. Determining the nature of the vibrational coherences is a challenging task with transient absorption data. Transient absorption measurements can probe vibrational coherences on both the ground-state as well as the excited-state potentialenergy surfaces. It is important to distinguish between the two possibilities to help better understand superpositions of excitedstate vibronic levels (involving electronic as well as vibrational coherences).49 Measurement of an excited-state vibrational coherence provides information on its damping time, which is often assumed to be much shorter than our results suggest. With a temporal δ-function pulse (meaning infinitely short), and making typical assumptions such as harmonic modes, the contribution of ground-state coherences to the broadband transient absorption signal should be negligible.50 One can envision that the instantaneous pump impulsively prepares a wave packet on the excited-state potential and transfers that wave packet back to the ground-state potential before it has time to explore the phase space of the excited state.51,52 Thus, the wave packet is returned to stationary equilibrium conditions, not leaving a “hole” in the ground-state density that can be probed. For a short but finite-duration pulse, however, it might be possible to see ground-state contributions to the coherences by means of resonant impulsive stimulated Raman scattering.29 This happens when the pump pulse is long enough to allow the wave packet to evolve on the excited-state potential before a second interaction with the pump projects the wave packet back to the ground state.28,53 Minimum of Potential-Energy Surface. One method for unambiguously assigning excited-state coherences is fluorescence upconversion, as the oscillatory signals can only occur from the excited state that emits fluorescence.54 However, this method does not allow the broad spectral probing window that is useful for observing wave packet dynamics. A method for application in transient absorption measurements involves intentionally adding dispersion to the excitation pulse. Depending on the sign of the dispersion, wave packet motion on a particular potential-energy surface can be enhanced.55 Our transient absorption measurements do not rely on this method, however. Instead, using the formalism developed for wave packets, we provide evidence for the vibrational coherences being predominantly in the excited state of the PC577 chromophores. The formalism56 developed for coherent superpositions of vibrational modes (wave packets) is useful for interpreting how the amplitude and phase of the oscillations vary with λprobe in the transient absorption data. Direct Franck−Condon excitation of the ground-state equilibrium population with coherent, broadband light will generate a population on multiple, closely spaced vibrational levels along different vibrational coordinates in the excited state of the molecule. This superposition of vibrational levels is nonstationary under the Hamiltonian of the system and will lead to a wave packet

containing a pigment−protein complex similar to PC577, for example, the energy transfer from the DBVs to the photosystems was found to be on the order of tens of picoseconds.47 Coherent Dynamics. Subtracting the biexponential fits from each λprobe of the transient absorption data removes the population dynamics and reveals the coherent dynamics (Figure 2c) caused by impulsive excitation of intramolecular vibrations. The oscillations are present over much of the spectral probing window albeit with interesting amplitude and phase behavior, to be discussed later. The maximum amplitude of the oscillations is only about 1% (ΔI/I), and so the high signal-to-noise of the spectrometer aids in observing the oscillations above the background. Figure 4a shows the power spectrum as a function of probe wavelength. As expected from the oscillations in the time domain, there are many different modes of comparable amplitude present in the frequency domain. Unlike vibrational coherences observed previously in studies of organic dye molecules, there are many oscillations in photosynthetic complexes at low frequencies, around 50 and 100−250 cm−1.25 Although the coherent oscillations in the experiments on PC577 could, in principle, be due to electronic coherences (superpositions of electronic states), the open structure of PC577 means that all the electronic couplings are relatively weak, and thus the coherences are most likely to be vibrational. We also find the spectral dependence of the oscillations consistent with vibrational coherences (vide infra). All Franck− Condon active vibrations can be observed in the transient absorption spectra, with the number of modes determined by the pulse bandwidth overlap with the absorption spectrum.12,48 In large organic molecules, the displacements of most of the vibrational modes are within the small-displacement regime, under the harmonic-potential approximation. This principle limits the number of frequencies that we observe in the spectra. We fitted the coherences to damped cosines. At λprobe = 616 nm (where the oscillations are the strongest), we observe eight reproducible modes (Table 1). The strongest-amplitude modes at 190 and 270 cm−1 are damped with a time constant of about 400 fs, which is about the median of the other modes. Notably, the 810 cm−1 mode dephases fastest in about 100 fs, whereas the 370 and 660 cm−1 modes last for over 600 fs. The overall vibrational dephasing results both from pure dephasing and population decay (relaxation) of the vibrational levels involved in the coherence.15,22 Damping of the oscillations on the blue edge of the signal during the first few hundred femtoseconds is most likely due to population relaxation of the vibrational modes accompanying the energy transfer. Many of the underlying oscillatory modes in the data of PC577 corroborate with the 2D ES data of another phycobiliprotein, PC645 from the cryptophyte Chroomonas mesostigmatica CCMP269.5 PC645 contains eight chromophores: two dihydrobiliverdins, two mesobiliverdins, and four phycocyanobilins. Because each protein contains similar chromophores, we expect PC577 and PC645 to show similar coherent oscillations. (We note, however, that the two techniques are not identical, because 2D ES measurements separate excitation frequencies, whereas transient absorption measurements integrate all excitation frequencies.) We find common oscillatory modes (within the experimental uncertainty) of 190, 270, and 470 cm−1 in the PC645 and PC577 data. In the 2D ES data recorded for PC645, the 270 cm−1 oscillation did not appear to dephase during the 400 fs observation window. In the transient absorption data for 1300

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2a)). Thus, in PC577, the ground-state oscillations are weak relative to the excited-state oscillations. In the PC577 data, the absolute amplitude of the oscillations decreases sharply at λprobe approaching 577 nm. This makes sense, because the pump pulse is strongly pumping in the blue and thus mainly exciting the DBV molecules around 577 nm. We can only stimulate emission from this spectral region in the first few hundred femtoseconds, because after that time, the DBVs have been depopulated by energy transfer to the PCBs. Therefore, oscillations associated with the PCB excited state are the strongest and also represent the high-energy turning point. The oscillations extend to λprobe = 650 nm even though the ground-state absorption spectrum does not. This observation provides further evidence of stimulated emission from the excited-state potential-energy surface.62,63 The above picture is one-dimensional in coordinate space. To be observed experimentally, one vibrational mode has to dominate.64 The minimum of the potential-energy surface would not necessarily have to be the same for each vibrational mode. The signature of this possibility would be a frequencydependent nodal structure. Because all of the modes in the spectrum go to zero at approximately the same probe frequency, this is the global minimum of the multidimensional, excited-state potential-energy surface.65 We conclude from analysis of the broadband transient absorption data that vibrational coherences in PC577 are predominantly in the excited state of the constituent chromophores. This analysis implies that ultrafast intramolecular and intermolecular energy transfer occurs before vibrational modes are damped. This conclusion is important for determining the relevance of the various models of electronic energy transfer for phycobiliproteins like PC577 and highlights the necessity to include vibrational motion in any such model.66,67 The vibrations also provide opportunities for controlling energy transfer.65 Because the strongest modes dephase within a few hundred femtoseconds, they are only active during the PC577 intraprotein energy transfer. The longer-lasting modes like the 660 cm−1 mode are active during the time scales of interprotein energy transfer. It is interesting that there appears to be a lack of vibrational coherences around the DBV molecules, or perhaps the excited-state potentialenergy surface of these molecules are more red shifted than the PCB molecules relative to the common ground state of the PC577 complex. The 660 cm−1 mode, which dephases on about the same time scale as the DBV-to-PCB energy transfer, could be a signature of this mode belonging to DBV. At the very least, it is active when the DBV molecules are excited. This hypothesis would imply that at least the minimum (perhaps not the overall shape) of the potential-energy surface of the DBV molecules is shifted to the same phase-space coordinates as the PCB molecules. Dynamic Stokes Shift. The spectral and temporal resolution of the spectrometer enables us to characterize the solvation dynamics of chromophores in the PC577 lightharvesting complex through the dynamic Stokes shift.68−70 The Stokes shift is defined as the energy difference between the peak of the fluorescence spectrum and the peak of the absorption spectrum. It yields twice the reorganization energy associated with relaxation of the environment around the change in electrostatic properties (for example the transitiondipole moment) between ground and electronic excited states. The dynamic Stokes shift is the time-resolved environment relaxation, or solvent polarization, after photoexcitation. It is of

that explores the multidimensional phase space of the excited vibrational coordinates. The wave packet will oscillate about the minimum of the multidimensional potential-energy surface, and on the basis of the energy spacing between the ground-state and excited-state surfaces, the position of the wave packet as a function of pump−probe time delay will be evident in the transient absorption spectra.55 A key feature is that the phase of the oscillations undergoes a π radians shift at λ probe corresponding to the global minimum of the potential-energy surface.43 The oscillations in the PC577 transient absorption data are prevalent across much of the spectral probing window but show interesting features at certain probe wavelengths. Notably, the oscillations decrease in amplitude and abruptly change phase around λprobe = 640 nm (Figure 2c). This node is also visible in the frequency domain, where we see the amplitude of the various modes decrease around λprobe = 640 nm (Figure 4a). The node is clearly resolved for a few hundred femtoseconds after the impulsive excitation. We are thus observing the wave packet moving about the excited-state minimum at 640 nm. The minimum of the excited-state potential-energy surface at the free energy minimum of the chromophore−solvent system is where steady-state fluorescence peaks. Indeed, we see that the peak of the steady-state fluorescence is close to the minimum of the oscillations.57 As discussed in the next section, the slight blue shift of the node relative to the steady-state fluorescence peak at early pump−probe time delays is due to the dynamic Stokes shift. The phase change in the oscillations at λprobe = 640 nm is also consistent with the interpretation described above. In Figure 4c, we show the phase profile of the strongest mode at 270 cm−1. There is an abrupt change of about 2.5 radians at λprobe = 640 nm. This phase change corresponds to about 80% of the full π radians shift that we expect from the wave packet formalism. Because of this dramatic shift, the oscillations on the blue side of λprobe = 640 nm are observed to be almost out of phase with the same oscillations on the red side of λprobe = 640 nm (Figure 4c). Closer to the edges of the spectral probing window, the phase of the oscillations is less characteristic, probably because of the effects of residual fourth-order chirp on the pulse that was not corrected during pulse compression. The oscillations centered at 680 nm are observed to have an artificial slope because of these effects. We see that the amplitude of the wave packet is stronger to the blue side of the node. This observation might be attributed to anharmonicity causing the well to have a sharper rise on the blue side. The initial width of the wave packet on the excitedstate potential-energy surface is given by the spectral width of the laser pulse used to generate the wave packet, and through vibrational dephasing, relaxation, and anharmonicity over the energy surface, the wave packet broadens over time.43 Also, the excitation of more vibrational levels leads to greater dispersion and loss of coherence.22 Ground-state oscillations would show similar spectroscopic signatures as excited-state oscillations; however, the abrupt amplitude dip and phase shift would be centered at λprobe, corresponding to the peak of the ground-state absorption spectrum.30,58−61 At λprobe < 577 nm in the transient absorption data, there are some weak oscillations that may correspond to ground state-oscillations. However, there is neither a clear amplitude dip nor an abrupt phase change at 577 nm (the maximum of the ground-state absorption spectrum (Figure 1301

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probing window, these oscillations are damped within a few hundred femtoseconds. Interestingly, the oscillations are not simply a continuation of the modes present in the middle of the probing window, but rather they decrease in amplitude and reach a mutual minimum at about λprobe = 675 nm. This spectral region coincides with the peak amplitude of the excited-state absorption feature observed in the transient absorption measurement. We note that we cannot generate or observe coherences on a higherlying excited state within the formalism of third-order spectroscopy (we need a fifth-order technique). Certainly, the decrease in the amplitude of the modes deserves more investigation. At the very least, the presence of these oscillations superimposed on the excited-state absorption feature provides additional evidence that the coherences are coupled to the excited state of the chromophores in the protein, and we are indeed observing a wave packet on the excited-state potentialenergy surface. As shown in Figure 5, the wave packet that is

fundamental interest, because it provides the correlation function for the energy gap fluctuations that cause line broadening.71 Impulsive excitation of the chromophores leads to an excited-state configuration where the solvent and intramolecular nuclear degrees of freedom have not relaxed in response to the new dipole configuration of the chromophore. To compensate, the environment (water and protein) reorganizes its charge distribution to minimize the energy of solvation.72 This reorganization is evident in the way that the node that indicates the global minimum in the excited-state potential shifts with time (Figure 2c). There are many different dimensions involved with the excited-state potential-energy surface; we have eight resolvable superpositions of vibrational coordinates, as well as one solvation reaction coordinate.73 The solvent reorganization is represented by a Gaussian statistical distribution of chromophores with slightly different energies on the excited-state solvation reaction coordinate. Upon initial excitation from the ground state, this distribution is still centered on the minimum of the ground-state configuration. The solvent reorganization changes this distribution over time and is represented by the dynamic Stokes shift. Time-resolved fluorescence would reveal a red shift in the fluorescence over time.54 In transient absorption measurements, the wave packet oscillations are a window into the changing equilibrium point. The phase change of the wave packet occurs at the mean of the distribution, which is red shifting in time as the distribution becomes more statistically likely to be nearer the bottom of the excited-state potential-energy surface, representing the degree of solvation. The process of solvation occurs with a range of characteristic time scales, from hundreds of femtoseconds to picoseconds in polar solvents like water.68,69,74 The fast component is related to the solvent reorganization near the solute, and the slow component is due to the solvent away from the solute.68,74,75 Nearly 80% of the dynamic Stokes shift occurs within the first 100 fs, whereas the remainder takes places over hundreds of picoseconds. We observe these time scales by following the window of the wave packets. We extracted the time scales of the dynamics by fitting the node of the oscillations at each time delay value for five independent measurements. We chose a fit function of two decaying exponentials plus a constant. The average fit function had two exponential decay time scales of 62 ± 5 fs and 46 ± 12 ps. The fast component is about 75% of the total dynamic Stokes shift observed in our data and is likely predominantly the inertial solvation response69,70 (Figure 2c). The fit function can be used to determine the normalized correlation function of transition-energy gap fluctuations of chromophores in PC577, which can be Fourier transformed to yield a spectral density.76 Broadband transient absorption provides a direct way to obtain this information. Higher-Lying State. We observe additional features in the far-red regions of the probing window. In the transient absorption data, the region λprobe > 660 nm is dominated by excited-state absorption. The residuals of biexponential fits in this λprobe window show coherent oscillations centered at about 705 nm. Residual fourth-order chirp in the probe pulse contributes to the wavelength-dependent phase of the oscillations. The fit in the time domain at λprobe = 680 nm shows that several of the same modes that were present in the midspectral range are also present in the excited-state absorption region. The 190, 270, and 810 cm−1 oscillations are all reproducible within the error of the measurements. Similar to the same oscillations in the middle of the spectral

Figure 5. Transitions of the wave packet. The two light-matter interactions of the pump pulse in the transient absorption measurement generate a wave packet on the excited-state potential surface. The wave packet oscillates about the surface until a third light-matter interaction with the probe pulse. The probe pulse can stimulate the transition of the wave packet to either the ground-state potential or a higher-lying potential.

generated on the first excited-state potential surface by the pump pulse can transition to either the ground-state potential or a higher-lying potential upon interaction with the probe pulse. The strength and spectral dependence of either transition is given by the location of the oscillating wave packet on the lowest-energy excited potential at the time of interaction.

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CONCLUSIONS We have described in detail a transient absorption spectrometer capable of suppressing noise present in ultrafast laser systems. Through a combination of fast acquisition rates and balanced detection, we have developed an instrument with high temporal resolution and broad spectral resolution. The instrument is critical for investigating the spectral dependence of transiently emitted nonlinear optical signals. We have described the 1302

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probe and reference pulses, respectively. The transmitted portion is used as the pump pulse. The collimation of the beams is adequate to scan the pump delay relative to the probe over a range of 3800 ps with negligible changes in the pump divergence and with a resolution of 0.5 fs. In all places before the sample, we focus and collimate the beams with protectedsilver, spherical mirrors. We keep the angle of incidence onto the spherical mirrors below 4° to minimize the astigmatism caused by using them in an off-axis configuration. We independently control the power of the pump and probe beams with an achromatic λ/2 waveplate and a 0.7 mm thick wire-grid polarizer pair in each arm (Figure 6). We set the pump-beam power to about 60 μW, which means that the pump pulse energy was about 12 nJ/pulse. This power typically corresponded to a maximum bleach signal (ΔI/I) that was less than 0.05. This power level is low enough that effects such as

application of the setup to the study of a photosynthetic pigment−protein complex, PC577. The complex exhibits vibrational coherence. We analyzed the wave packet dynamics on the potential-energy surfaces. We have documented and described the evidence for the coherent oscillations being present on the excited-state potential-energy surface of the protein. The oscillations are coupled to the viewing window of the dynamic Stokes shift that occurs on a time scale of 62 fs and 46 ps. The oscillations are also found in the spectral region of excited-state absorption to a higher-lying state of the protein. There is a need to continue to investigate not just the presence of coherent oscillations but also their spectral dependence, amplitude, and phase. These properties will give insight into the nature of the potential-energy surfaces of the sample being studied. Broadband transient absorption data complements two-dimensional electronic spectroscopy, and using both techniques in unison may be useful for differentiating between electronic and vibrational coherences in future studies.

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EXPERIMENTAL METHODS We desired a spectrometer with high temporal resolution (an instrument response of tens of femtoseconds), with a viewing bandwidth across the visible spectrum, and with a high signalto-noise ratio. The transient absorption spectrometer that we constructed satisfies these three criteria. Transient absorption spectrometers require two pulsed beams. One beam of pulses perturbs the sample, and the other beam monitors the transient changes in the absorption of the sample. Dynamics of the system are then observed by incrementally delaying one beam of pulses relative to the other. After interacting with the sample, each probe pulse is imaged onto a CCD using a diffraction grating and appropriate focusing optics. A dispersed probe pulse shows the wavelength dependence of the dynamics in the emitted third-order signal. Our spectrometer also utilizes a third pulse as a reference for correcting the intensity fluctuations in the probe beam. Optical Apparatus. The optics upstream of the transient absorption spectrometer are similar to a design described previously.35 Briefly, a Ti/sapphire oscillator seeds a regenerative amplifier. The amplifier outputs 150 fs pulses centered at 800 nm and with 0.6 mJ of energy. These pulses seed a homebuilt noncollinear optical parametric amplifier (NOPA).77,78 We tune the NOPA to produce pulses with an intensity maximum at 580 nm and with minimal intensity structure from 600 to 700 nm (see Figure 2a). The profile of these pulses is ideal for pumping the sample predominantly at high energies and then probing the dynamics across its energetic landscape. Thus, we can use the output of the NOPA for both the pump and probe pulses in the transient absorption spectrometer. Next, we compress the output of the NOPA temporally using a 4-f grating compressor and a single-prism compressor.51,79,80 This procedure minimizes second-order and third-order dispersion in the pulses that could cause distortions of the spectral features.81 The compressed pulse gives an instrument response function (nonresonant response of the solvent, as measured at the sample position) of less than 30 fs. With this compressed pulse, we can impulsively excite and observe any active modes in the sample of approximately 1000 cm−1 or less. The compressed, broadband pulses are then input to the main part of the transient absorption spectrometer. We obtain three copies of the incident beam with a wedged beam splitter; the front reflection and back reflection (each less than 1% of the energy of the incident polarized beam) are used as the

Figure 6. Schematic of the experimental apparatus for measuring transient absorption spectra. For clarity, irises used to align and isolate beams and some turning mirrors are not shown. A beam splitter (BS) generates three beams from the incident beam. The two reflected portions are used as the probe and diagnostic beams while the transmitted portion is used as the pump beam. The diagnostic beam, which is reflected off the beam splitter at a slightly different angle than the probe beam, is directed into a photodiode (PD) by a pick-off mirror (M1). The probe beam propagates through a UV-fused-silica window (W) to compensate for extra glass in the pump arm. Both the probe and the pump propagate through separate half-wave plate (λ/2) and polarizer (P) pairs for independent power adjustment. A chopper (C) blocks the pulses at a rate of 625 Hz. The path length of the pump beam is adjusted using a retroreflector (R) mounted on a delay stage (D). A zero-degree spherical mirror (CM1) with focal length of 250 mm focuses the pump beam onto the sample, whereas a separate mirror (CM2) with focal length of 100 mm focuses the probe onto the sample. The angle of incidence of the pump beam onto the sample is 4° from being parallel to the probe beam and is blocked after traversing the sample with a beam block (B). The probe beam is recollimated by an achromatic lens (L1) placed after the sample. A second achromatic lens (L2) focuses the collimated beam into the spectrometer (G) which images the wavelength-dependent signal onto a CCD detector. 1303

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Figure 7. Overview of the electronics used for fast, balanced detection. The setup is designed to record transient absorption spectra and balance the laser intensity every eight laser shots. The output of the laser is set at 5 kHz, and the electronics downcount the pulse train to sets of eight pulses. A data acquisition (DAQ) card serves as the central hub for controlling and monitoring the measurements. A photodiode reads the intensity of each laser pulse and relays the measurement to the DAQ card after being enhanced by a preamplifier. Two TTL (CH7 and CH8) signals coming from the timing delay generator (TDG) of the laser are used to control the synchronization and delay of the electronics. CH8 is used to clock the timing of the DAQ card relative to the analog signals being received from the photodiode. CH7 drives the chopper that periodically blocks the pump pulses of the laser. A copy of CH7 is selectively controlled by a delay generator before triggering the camera to measure spectra. Labview software acts as the user interface.

section. Noise suppression requires fast acquisition methods, which are somewhat encumbered when spectrally resolving the probe with a CCD. An alternative method is to use a monochromator and a photodiode and scan the measured wavelength of the probe beam. However, this approach is limited by the linear correlation between the spectral resolution and the acquisition time.82 Our spectrometer suppresses noisewithout foregoing temporal and spectral resolution by using a CCD camera tuned for speed and electronics to precisely coordinate the timing of measurement events (Figure 7). The fundamental concept is to measure the intensity of the reference beam with a photodiode and to correlate that measurement with the concurrent signal at the CCD. We use all of the other electronics (preamplifiers, delay generators) to accomplish this task accurately, as described below. We operate the CCD camera (Andor DU971N-FI Newton) at a detection rate of 1250 frames per second, whereby the CCD chip is cropped, and images are acquired in a successive sequence. The camera readout and cleaning events take a combined time of less than 200 μsthus our camera does not miss laser pulses even though the repetition rate of the laser is 5 kHz. The detection procedure involves measuring quartets of consecutive laser pulses. The laser is chopped at 625 Hz so that the camera measures four consecutive laser signal pulses in one exposure and then measures four consecutive signal laser pulses in the next exposure. The cycle repeats 350 times for each timedelay value. The fast exposure of the camera integrates the intensity fluctuations of four laser shots so that noise below 625 Hz is suppressed.81 Noise with higher frequencies is then suppressed by averaging over hundreds of independent pulse quartets. We use the TTL signals of the laser to synchronize the timing of measurements with the pulse quartets. For the camera exposure to overlap with the arrival of the pulses, we use a frequency-downcounted TTL signal from the laser to both

photobleaching and multiphoton absorption are negligible (as verified by performing control experiments at twice the power). The probe beam passes through the sample perpendicular to the surface while the pump beam passes through at a slight angle. This configuration enabled us to isolate the probe beam and yet minimize the time-smearing of the pump beam. We focused the probe beam onto the sample using a spherical mirror with a shorter focal length ( f = 10 cm) than the spherical mirror (f = 25 cm) used to focus the pump beam. The focused spot size of the probe beam (16 μm beam waist) was therefore smaller than the pump spot size (41 μm beam waist), and thus the excitation density in the sample is fairly constant over the spatial cross section of the probe beam. The pump fluence was typically about 5 mJ/pulse/cm2. Our determination of time zero (the moment when the pump and probe pulses arrive at the sample concurrently) involves the measurement of the autocorrelation of the nonresonant response of the solvent (a buffer solution that is primarily water) of our sample. The precise measurement of time zero is important, because the time scale of the sample dynamics is on the order of femtoseconds. During consecutive measurements, the time zero drifts typically a few femtoseconds due to the inaccuracy of the delay stage (Newport IMS600LM). This drift is linear across the entire scan time window, and thus we correct the data by adjusting time zero. We scan typically for a few picoseconds before time zero (the probe pulse arrives at the sample before the pump pulse) to obtain an accurate measurement of the background noise such as scatter of the pump and fluorescence of the sample. These background spectra are averaged and automatically subtracted from the data. Methods of Fast, Balanced Detection. The preceding section details how the spectrometer meets the constraints of high temporal resolution and broad spectral detection. We describe the third constraintsuppressing noisein this 1304

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drive the chopping of the laser pulses and trigger the camera. We use a digital delay generator (Stanford Research Systems DG645) to precisely adjust the triggering of the camera to coincide with the exposure of groups of pulse quartets. The DAQ card (National Instruments PCI-6221) is triggered by the fire signal of the CCD and clocked by the TTL output of the laser.83 The spectra from the CCD and the voltage of each laser pulse are relayed into a Labview code via the USB and the DAQ card, respectively. We first amplify the photodiode output using a current preamplifier (Stanford Research Systems Model SR570) before relaying it into the DAQ card. The current preamplifier is beneficial, because it both increases the output voltage amplitude and lengthens the decay time of the signal. The Labview code normalizes the spectra to the laser intensity fluctuations, using the formula Ip

Ibalanced =

Sp

− Iu Su

Iu Su

(1)

where Ip and Iu are the respective intensities of the signal with pumped and unpumped excitation and Sp and Su are the respective intensities of the photodiode signal. The intensities of the pumped and unpumped spectra are each the sum of four consecutive signals (four laser pulses), and the photodiode voltages are the sum of the corresponding signals of the reference pulses on the photodiode. This procedure is averaged 350 times for every time-delay point. The output of this procedure is a balanced ΔI/I signal with spectral resolution across 800 pixels of the CCD. This procedure is fully automated and takes place in real time during the experiment. This balanced ΔI/I output is a quantitative measurement of pump-induced changes in the absorption of our samples, and its changes in time yield dynamic information about the samples. Unfortunately the chopper and the camera clock themselves independently; a small frequency mismatch between the two internal clocks causes them to slowly drift out of phase. This puts an upper limit on the amount of averaging that the system can perform before requiring retriggering. The digital delay generator (Stanford Research Systems DG645) enables us to maximize the amount of averaging by allowing us to precisely adjust the timing of the camera trigger relative to the incoming pulses. In practice, we achieve an upper limit of 3500 cycles, but for the current experiments, we limit the measurements to 350 cycles to avoid damage to the protein samples over prolonged time periods. In general, the procedure of taking the average of the individual intensities rather than the average of the quotient achieves a greater suppression of the noise.84 However, for the noise in our laser, the two procedures only begin to diverge when averaging more than 500 cycles. Thus, for our current measurements at 350 cycles, the two noise suppression procedures are indistinguishable. Laser Noise Analysis. In this section, we quantify the intensity fluctuations in the laser, and then we show how the spectrometer suppresses this noise. Figure 8a shows the energy output of the laser, as measured on a photodiode. The data represent typical fluctuations of the laser for time periods relevant to transient absorption experiments. The NOPA fluctuations correspond to an almost 2% change in intensity which is comparable to, or larger than, the signals emitted from PC577. Moreover, the fluctuations are oscillatory, and a Fourier transform shows that there are indeed discrete frequencies in the noise (Figure 8b). Discrete modes are often observed in the

Figure 8. Analysis of laser noise. (a) Histograms showing the deviation of the pulse energy from the mean, over 500 000 pulses. The left panel corresponds to the output of the regenerative amplifier. The right panel corresponds to the amplified output from the NOPA. Superimposed on both distributions is a Gaussian fit. (b) A Fourier transform of the NOPA intensity over time, representing noise under typical operating conditions. In addition to white noise, several discrete modes are noticeable over 2 orders of magnitude. (c) The correlation between four consecutive laser pulses. Uncorrelated data would be circular in the x−y plane, whereas these data roughly follow the diagonal. This result indicates high correlation and the capability to filter low-frequency noise.

noise of ultrafast laser systems85,86 and will complicate the identification and analysis of quantitative information in the true signal. Low-frequency oscillations could be incorrectly interpreted as sample population dynamics; high-frequency oscillations would appear to be very similar to coherences. The spectrometer uses two methods to suppress noise: balanced detection and fast acquisition. Figure 9 shows the results of balanced detection. Balanced detection normalizes the intensity of the signals to the intensity of the laser pulses used to probe the signals. The figure shows that balanced detection suppresses the laser intensity fluctuation noise in the probe beam and thus the overall signal relative to the unbalanced measurements. However, because the signal also depends on the intensity of the pump pulses and the pointing of the probe beam, the greater reduction of noise is obtained from the differential (between pumped and unpumped data) itself.84 This result is the outcome of the fast acquisition of the spectrometer. Acquiring data quickly has two advantages for noise suppression in the spectrometer: filtering low-frequency noise and enabling significant averaging. Low-frequency noise will be apparent as an intensity drift within consecutive pulses. Because 1305

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minimize protein aggregation on the flow cell walls and to minimize the chance of photobleaching the protein, we flowed the sample using a peristaltic pump (Cole-Parmer) at a rate of 1.05 mL/minute. The protein solution flowed through an ice bath during the scans to prevent temperature-activated denaturation of the protein. We translated the flow cell between successive transient absorption measurements to prevent light-catalyzed aggregation of the protein on the walls. We measured the linear absorption spectra (Varian Cary 6000i UV−vis spectrometer with a resolution of 1 nm) of the proteins before and after transient absorption measurements to ensure that the protein did not denature. We measured the steady-state fluorescence spectra with a fluorescence spectrometer (Varian Eclipse). All steady-state and time-resolved measurements were performed at room temperature.

Figure 9. Noise suppression results. The two red lines correspond to the integrated noise intensity of all pixels of the CCD. The two blue lines correspond to the noise at the single pixel of the CCD at the peak of the laser intensity. The lighter traces of each pair are unbalanced, and the darker traces are balanced to the noise present in the photodiode. Each trace is a function of the number of kinetic cycle pairs. We label 350 kinetic cycle pairs, because the data were measured at this value. The noise of the integrated spectrum decreases as the square root of the number of averages. Shown are the data of one measurement under typical conditions.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address #

Department of Chemistry, New York University, New York, NY 10003.

the spectrometer is integrating over four consecutive pulses for each data point, the intensity drift within those four pulses will be evident in the data. However, Figure 8c shows that there is a strong correlation between four consecutive pulses.84 The correlation implies that low-frequency noise is only significant at time scales longer than four pulses and is thus filtered by the fast acquisition methods of the spectrometer. Therefore, there is only high-frequency noise after filtering, some of which is attributable to noise in the camera. High-frequency noise can be suppressed by taking more averages. This procedure takes more time, but because the spectrometer acquires data quickly, we can take hundreds of averages at each time delay without the total measurement time becoming too long. A long measurement time is both tedious and detrimental to the sensitive biological samples. The current measurements are averaged for 350 cycles, which gives a noise threshold of about 10−4. Figure 9 shows that the noise suppression follows a square-root law with the number of averages; there are diminishing returns as we increase the averages to more than 350. Sample Preparation. We isolated the PC577 phycobiliproteins from the cryptophyte algae Hemiselmis pacifica (strain CCMP 706). We grew cells of H. pacifica in an artificial medium (Prov 50 from NCMA) on a 12/12 h dark-light cycle 18 mmol m−2 s−1. We harvested the cells from the growth medium by centrifugation and resuspended them in a 0.1 M sodium phosphate buffer. We extracted the proteins through freezing and thawing (−20 and 4 °C) in the dark. We achieved further purification through centrifugation and successive ammonium sulfate precipitation (40, 55, and 80%) and then stored the samples at −20 °C. Before transient absorption measurements, we thawed the proteins and dialyzed them against a 0.025 M phosphate buffer to remove excess ammonium sulfate. The optical density of the samples was adjusted with the phosphate buffer. We aimed for a sample optical density of about 0.15 to reduce the possibility of reabsorption effects when interacting with the laser pulses.87 We filtered both the protein and the buffer using 0.2 μm spaced nylon filters (VWR 28145−487) to remove any contaminants. We placed the sample in a homemade flow cell. The front window of the cell was a 0.5 mm piece of UVfused silica. The cell had a sample path length of 0.5 mm. To

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada, DARPA (QuBE) and the United States Air Force Office of Scientific Research (FA9550-13-1-0005).

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REFERENCES

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