www.sciencemag.org/cgi/content/full/340/6128/52/DC1

Supplementary Materials for

Broadband 2D Electronic Spectroscopy Reveals a Carotenoid Dark State in Purple Bacteria

Evgeny E. Ostroumov, Rachel M. Mulvaney, Richard J. Cogdell, Gregory D. Scholes*

*Corresponding author. E-mail: greg.scholes@utoronto.ca

Published 5 April 2013, Science 340, 52 (2013) DOI: 10.1126/science.1230106

This PDF file includes:

Materials and Methods Assignment of 2DEAS Peaks Figs. S1 to S13 Captions for Movies S1 to S4 References (39–47)

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/340/6128/52/DC1)

Movies S1 to S4

2

Materials and Methods

Sample preparations

The LH2 complexes from Rhodopseudomonas (Rps.) acidophia 10050 and

Rhodobacter (Rba.) sphaeroides 2.4.1 were prepared as previously described (39, 40).

The rhodopin glucoside and spheroidene carotenoids were isolated from anaerobically

grown cells of wild type strains of Rps. acidophila 10050 and Rba. sphaeroides 2.4.1,

respectively. The carotenoids were isolated using acetone extraction and petroleum ether

partitioning. The isolated spheroidene was further purified by alumina column

chromatography using 1% – 15% diethyl ether in petroleum ether. The purified samples

were stored in acetone at 4oC. For 2D measurements the LH2 complexes from Rps.

acidophila and Rba. sphaeroides were dissolved to a final concentration of 0.3 OD/mm,

contained 0.1% LDAO to prevent aggregation. Measurements were taken in a 1 mm flow

cell. The spheroidene and rhodopin glucoside carotenoids were measured in acetone

solution at 1 OD/mm final concentration in the maximum of absorption. The higher

concentration was used since the excitation pulses covered only the far red shoulder of

the main absorption band and absorption within the excitation spectral region did not

exceed 0.2 OD. In order to confirm that no photo-degradation had occurred during the

measurements, the absorption spectra before and after the 2D experiments were

compared.

2D electronic spectroscopy

The 2DES setup is described in detail elsewhere (41). Briefly, a regenerative amplifier

(Spitfire, Spectra-Physics) seeded by Ti:sapphire oscillator generates 150 fs pulses at 800

nm. A home-built NOPA converts the 10 mJ pump pulses into 10 mW broad-bandwidth

pulses (<70 nm FWHM) which are than compressed to 13 fs. The typical frequency-

resolved optical gating (FROG) map of the excitation pulse is shown in Fig. S1. A

diffractive optic splits the compressed pulse into four beams arranged in a box geometry

so that the signal is emitted in the background free direction. The emitted signal is

heterodyne detected by the spectrograph (Shamrock, Andor). The time axis t1 (coherence

time) is scanned from -60 to 60 fs (negative time range corresponds to non-rephasing

signal, positive to the rephasing signal), a Fourier transform along t1 yields the excitation

frequency axis (reported in wavelength). The dynamics of the absorptive 2D signal are

monitored as a function of t2 delays (waiting time). Several different t2 time ranges and

steps have been used in measurements of each sample in order to exclude artifact signals

and confirm repeatability of the results. Following t2 times were used in presented data:

0–400 fs with 5 fs step in Fig. 2A, 3C-D, S5-8, S11, Movie S1-2 and S4; 0–200 fs with 1

fs step in Fig. 2B, 3, S9A; 0–400 fs with 2 fs step in Fig. 3A-B and Movie S3. The

phasing of the 2D spectra has been performed using the projection-slice theorem (42, 43),

with the pump-probe spectra measured under the same experimental conditions right after

the 2D experiment. In presented figures the 2D spectra were generated in Matlab using

specially generated color map in order to emphasize signals of interest and diminish

noise. In the movies the 2D spectra were generated in Matlab using a default color map

with no noise filtering.

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Fig. S1. FROG map of excitation pulses used to collect data shown in Fig. 2B, S7–8 and

Movie S2. Spectral and temporal profiles are shown to the right and below the plot. Fit of

the profiles to a single Gaussian function is shown by red line.

Global analysis

Global analysis was performed using home-written Matlab code (MATLAB R2011a,

The Mathworks). The procedure of the non-linear fitting algorithm was based on the

Runge-Kutta method and included a separate fit of the oscillatory contribution and kinetic

relaxation decay. For fitting of the oscillatory contribution, the frequency spectrum was

generated by simulating the Fourier spectrum (Fig. S9B) with Gaussian functions and

wavelength-dependent phase factors. This simulated spectrum was transformed into the

time-domain and used within the fitting algorithm to extract pure kinetic contributions.

The principles of the global analysis applied kinetic data without oscillations can be

found in the following reviews (24, 25). In the present work several kinetic schemes were

tested: the lifetime scheme, the evolutionary scheme and the branching scheme. Each of

these schemes resulted in a good fit quality, however only one kinetic scheme describes

the actual dynamics, while other schemes are merely an alternative mathematical

solution. In order to find the kinetic scheme that provides the best overall description of

the data, first some preliminary information has to be obtained using the lifetime analysis.

By increasing the number of lifetimes and evaluating the quality of the fit, the number of

interconverting species can be estimated. The decay-associated spectra (DAS, which are

pre-exponential amplitudes at different wavelengths) can often give a good first estimate

for energy transfer processes (25). In order to find the actual SAS (species-associated

spectra) and excited state dynamics, different kinetic schemes have to be tested using the

preliminary information from lifetime analysis as well as auxiliary information from

other resources (transient absorption/fluorescence studies etc.). Since the quality of the fit

appears the same for all schemes (they are linear combination of each other and,

therefore, mathematically indistinguishable), the judgment of the optimal kinetic scheme

4

has to be made by the consistency of SAS spectral features, rate values and the scheme

pathways with complementary information about the studied system (25). This

information includes time constants obtained by alternative time-resolved methods,

spectra of isolated chromophores, electronic structure estimated from theoretical

calculations etc. Of particular importance are the SAS, which should show clear well-

resolved peaks consistent with the spectral properties of the chromophores (see (44)).

During analysis, first the time evolution of species populations is calculated using a

kinetic scheme. During the second step, the calculated population evolution is convoluted

with the instrument response function, estimated from the cross-correlation profile of the

laser pulses (Fig. S1). Data points over the first 15 fs of population time were excluded

from the fitting process because during the pulse overlap multiple pathways are allowed

and simulation of the resulting signal in 2D spectroscopy is not possible. In order to avoid

local minima in the solution space, the fitting procedure was repeated several times

(N>30) with various starting values of the system parameters. Different numbers of

species were tested for the fitting model. The scheme with four species resulted in both

meaningful spectra and good fit quality.

Assignment of spectral features

In Fig. S2-S4 all identified features are marked in accordance with assigned state(s).

Each diagonal feature (GSB) is designated by a single symbol, while off-diagonal

features are marked by two symbols. The first symbol denotes the locally excited state,

and the second symbol denotes the acceptor state. The signals in the 2DEAS can be

separated into several contributions: (i) Positive diagonal peaks, which represent GSB

signals (Fig. S13, R1 pathway) and stimulated emission (SE) from the locally excited

states (Fig. S13, R3 pathway), (ii) Positive cross-peaks, which represent GSB via

common ground state (Fig. S13, R2 pathway) and SE following the energy transfer from

the locally excited states (Fig. S13, R4 pathway), (iii) Negative peaks, which are due to

ESA signals, originated from the locally excited states (Fig. S13, R5 pathway), or from S1

state, which is populated from the locally excited states (Fig. S13, R6 pathway).

Lifetime analysis (parallel kinetic scheme)

The 2DDAS (2D decay-associated spectra), obtained in this work for LH2

complexes of Rps. acidophila, are shown in Fig. S2. Four lifetime components were

necessary to obtain a good fit, while larger number of lifetimes did not improve the fit

significantly. The values of the 66 fs and 398 fs lifetimes are in good agreement with the

values reported from transient absorption studies of LH2 complexes from Rps. acidophila

(11, 28, 29). The other two lifetimes have not been reported previously. The 2DDAS-1

reveals two weak diagonal signals at 530 nm and 560 nm, which are attributed to

GSB/SE from S2 and X states respectively. The strong off-diagonal positive peak at

530/560 nm excitation/detection wavelengths is due to S2-to-X energy transfer and/or

GSB via common ground state. The negative signal at 585 nm detection wavelength

might indicate the rise of the signal from Qx state populated from both S2 and X states, as

follows from elongation of the signal between 530-560 nm excitation wavelengths. The

5

2DDAS-2 and 3 have features similar to each other, but have opposite sign. This effect is

indicative of strong mixing among the spectral components in 2DDAS-2 and 3 and, as a

result, their spectral features do not correspond to any particular physical state. For that

reason interpretation of 2DDAS-2 and 3 is not possible. The 2DDAS-4 shows a positive

diagonal signal at 540 nm, caused by GSB from S2 state, and negative signal arising from

S1 ESA, with typical elongation along the detection wavelength scale. The ESA signal is

observed at both 530 nm and 560 nm excitation wavelengths due to S2-to-S1 and X-to-S1

energy transfer, respectively. In conclusion, the lifetime analysis revealed four species

with relaxation time constants of approximately 30 fs, 70 fs, 150 fs and 400 fs.

Fig. S2. 2D decay-associated spectra (2DDAS) of Rps. acidophila and corresponding

population dynamics. The parallel kinetic scheme and lifetimes are shown to the right of

the population dynamics plot.

6

Evolutionary analysis (sequential kinetic scheme)

Using the time constants from lifetime analysis we applied an evolutionary kinetic

scheme to reveal spectral profiles of the sequential species. The 2DEAS (2D

evolutionary-associated spectra) are shown in Fig. S3. The first spectrum (2DEAS-1, Fig.

S3) reveals three diagonal contributions: a strong peak at 530 nm, and two weak peaks at

560 nm and 585 nm. These diagonal features are due to GSB/SE signals from the Car S2,

X and BChl Qx states, respectively. Since GSB represents depopulation of the ground

state, these diagonal features are present in all 2DEAS-1-4, but are not visible in 2DEAS-

2 and 4 because of overlap with negative ESA signal. The strong S2/X cross-peak results

from ultrafast S2 X energy transfer and consists of three contributions: (i) X SE, and (ii)

GSB signal arising from S2/X pathway via common ground state. The Qx/X cross-peak is

unlikely to originate from up-hill energy transfer and is more likely due to the GSB signal

arising from mixed excitation of coupled BChl Qx and Car X states. However, this feature

has to be studied in more detail for a clearer interpretation. The origin of the negative

signal below the diagonal (at 540–570 nm excitation and 570–600 nm detection

wavelengths) is uncertain, and can be due to ESA from S2 and X states. The S2 ESA

signal has been reported previously (45, 46); however the detailed properties of that

signal are still unknown. The global analysis revealed a 48 ps-1

rate constant (~21 fs, see

Fig. S3) for the 2DEAS-1 component.

The 2DEAS-2 (Fig. S3) has two diagonal peaks at 530 nm and 590 nm, which

correspond to the GSB signals from Car S2 and BChl Qx states, respectively. The positive

S2/Qx cross-peak is due to S2 Qx energy transfer, followed by Qx SE. This peak shows

slight elongation to shorter detection wavelength, which suggests the presence of a S2 X

energy transfer cross-peak hidden beneath the strong negative signal. The positive X/Qx

cross-peak is due to X Qx energy transfer, followed by Qx SE. The strong negative S2/S1

signal is due to S2 S1 energy transfer followed by S1 ESA. Similarly, the negative

diagonal X/S1 signal is due to X S1 energy transfer followed by S1 ESA. Notably, the

X/S1 signal is overlapped with GSB from the X state, and, because of its stronger

transition dipole moment, the S1 ESA signal obscures the bleach signal completely. The

Qx /X cross-peak in 2DEAS-2 is assigned to the same signal as in 2DEAS-1. The decay

rate of this spectrum is found to be 16 ps-1

(~62 fs).

The 2DEAS-3 (Fig. S3) has a simple profile. The three diagonal peaks at 530 nm,

560 nm and 585 nm are assigned to GSB signals from Car S2, X and BChl Qx states,

respectively. The broad negative S2/S1 peak is due S2 S1 energy transfer followed by S1

ESA. The decay rate of this spectrum is 8 ps-1

(~125 fs).

The 2DEAS-4 (Fig. S3) has two diagonal peaks at 530 nm and 585 nm, originating

from Car S2 and BChl Qx states, respectively. The positive X/Qx cross-peak is due to

X Qx energy transfer, followed by Qx SE. Three negative signals in 2DEAS-4 are due to

ESA from the S1 state, populated from S2, X, and Qx states. The overall profile of the

2DEAS-4 is very similar to the raw 2D spectra of isolated rhodopin glucoside (Fig. 3C-

D), if the contribution from the BChl Qx state were eliminated. This is a valuable

confirmation that the 560 nm diagonal feature is due to carotenoid, not

bacteriochlorophyll. The decay rate of this spectrum is 2.6 ps-1

(~385 fs), which is very

close to the hot-S1 state lifetime, well-known from TA studies (11, 29).

7

Fig. S3. 2D evolutionary-associated spectra (2DEAS) of Rps. acidophila and

corresponding population dynamics. The sequential kinetic scheme and rates are shown

to the right of the population dynamics plot. This figure is a replica of Fig.4 in the main

text.

The 2DEAS 1–3 reveal ultrafast decay rates. Previously reported fluorescence up-

conversion and transient absorption (TA) studies of carotenoids/light-harvesting

complexes with excitation to Car S2 state found only one ultrashort lifetime component

on the timescale of 60–70 fs (11, 28, 29). This lifetime is ascribed in the literature to the

S2 SE and GSB signals. We explain the observation of three ultrafast components in the

current work by the better temporal resolution attained using 13 fs broad-band excitation

pulses, as well as by excitation wavelength resolution gained from Fourier transform 2D

spectroscopy. 2DEAS-1 is mostly due to GSB and SE from S2 and X states, revealing S2-

to-X energy transfer. The 2DEAS-2 combines both ESA from the S1 state and SE from

the Qx state, showing that BChl Qx state is populated from both S2 and X states, while

Car S1 state is populated from Car S2, X and BChl Qx states. The ESA signal most

8

probably arises from a higher lying vibrational level of S1 (S1,v). Similarly the ESA in

2DEAS-3 is due to the same or another vibrational level of S1 (S1,v). Whether these S1,v

vibrational levels are different or the same level, which mixes into both 2DEAS, cannot

be determined at present and further studies are necessary to answer that question.

Branching kinetic scheme

From the evolutionary analysis it follows that several parallel relaxation pathways

are active in the LH2 proteins we have studied. The S2 state populates the X state, S1 state

and Qx state. The X state populates both S1 state and Qx state. Finally, the Qx state

populates the S1 state. In addition, all of these states can have alternative relaxation

pathways to states not visible in our experiment (e.g. the Qy state of bacteriochlorophyll).

Evolutionary analysis also revealed that the S2 and X states are not time-resolved in our

experiment and are represented by single compartment. A complex branching kinetic

scheme results from these findings, shown in Fig. S4, together with the corresponding

2DSAS (2D species-associated spectra). The 2DSAS1-3 have similar spectral features,

but of opposite sign, not too different from 2DDAS obtained by lifetime analysis (see Fig

S2). Only the 2DSAS-4 reveals a profile with distinctive features which can be assigned

to physical states of the system. In fact, 2DSAS-4 is very similar to 2DDAS-4 and

2DEAS-4 (Figs. S2 and S3). The likeness of the fourth spectrum among all models is

probably due to the weak mixing of the spectrum of the slowest physical state with

spectra of other physical states. In other words, the component with slowest time constant

dominates in the fourth species independently of the scheme in use, while the other three

components can show significant mixing in species 1-3 depending on the kinetic scheme.

In the current work it was found that the evolutionary kinetic scheme produced the

most meaningful spectra, i.e. only the 2DEAS features can be easily identified while

lifetime analysis and branching scheme analysis failed to produce resolved spectral

features. However, the 2DEAS in this work (Fig. 4 and S3) as well as several

independent TA studies (10, 19, 47) have shown that several branching energy transfer

pathways are present in LH2 proteins. Therefore the evolutionary scheme does not

represent the actual energy transfer scheme. Nevertheless by arrangement of the species

in the order of their relaxation rates the evolutionary scheme reveals quantitative kinetic

parameters of the system and allows resolution of spectral features for physical states.

Moreover, according to the location of these spectral features (positive and negative

peaks) in the 2DEAS, it is possible to identify the key energy transfer pathways. It was

not possible to determine the actual kinetic scheme with branching energy transfer

pathways in the current work owing to imperfections of the data (presence of noise,

limited time-range, etc.) as well as indistinguishable temporal features of some of the

physical states. Owing to the high sensitivity of the 2DSAS profile on the parameters of

the model as well as particular pathways within the branching kinetic scheme, the

demand for the quality of the experimental data is very high.

9

Fig. S4. 2D species-associated spectra (2DSAS) of Rps. acidophila and corresponding

population dynamics obtained using scheme, shown on the bottom right hand side.

Arrows in the kinetic scheme indicate relaxation pathways. Corresponding rates are

shown in ps-1

. The resulted lifetimes are: 37 fs, 63 fs, 125 fs, 385 fs. Ratio of initial

population at t2=0 in species 1 and 2 was 1:0.7.

10

Fig. S5. 2D spectra of Rba. sphaeroides measured in 0–400 fs range with 5 fs step. The t2 times

of each spectrum are shown in the top. These data are not a replica of the data plotted if Fig. 2A,

but represent a separate measurement taken at identical excitation conditions.

11

Fig. S6. 2D spectra of Rba. sphaeroides measured in 0–400 fs range with 5 fs step. Replica of

data plotted in Fig. S5 but in contour representation.

12

Fig. S7. 2D spectra of Rps. acidophila measured in 0–400 fs range with 5 fs step. The t2 times of

each spectrum are shown in the top. These data are not a replica of the data plotted if Fig. 2B, but

represent a separate measurement taken at identical excitation conditions.

13

Fig. S8. 2D spectra of Rps. acidophila measured in 0–400 fs range with 5 fs step. Replica of data

plotted in Fig. S7 but in contour representation.

14

Fig. S9. (A) Time traces of Rps. acidophila (measured at 0–200 fs, 1 fs step). (B) Fourier spectra

of Rps. acidophila corresponding to time traces in (A). Blue solid line show the time trace at S2

diagonal peak ( exc= em=530 nm), red dashed line – the time trace at X diagonal peak

( exc= em=560 nm), green dash-dotted line – the time trace at Qx diagonal peak ( exc= em=585

nm). For peak positions see Fig. 2B. The Fourier spectra are normalized to the amplitude at 1590

cm-1. The time traces represent raw data. Prior to Fourier analysis the exponentially decaying

term was extracted from these time traces.

Fig. S10. (A) Rephasing, (B) non-rephasing and (C) absorptive 2D spectra of Fourier transformed

data of Rps. acidophila (from Fig. 2B of the manuscript) along t2 scale, integrated over 1000-

1700 cm-1 frequency range. The Fourier transformation has been applied after extraction of the

exponential term.

15

Fig. S11. (A) Time traces of Rba. sphaeroides (measured at t2=0–400 fs, 5 fs step). (B) Fourier

spectra of Rba. sphaeroides corresponding to time traces in (A). Blue solid line show the time

trace at S2 diagonal peak, red dotted line – the time trace at X diagonal peak, green dashed line –

the time trace at Qx diagonal peak. For peak positions see Fig. 2A. The Fourier spectra are

normalized to the amplitude at 1590 cm-1. The time traces represent raw data. Prior to Fourier

analysis the exponentially decaying term was extracted from these time traces.

16

Fig. S12. Selected experimental time traces from Rps. acidophila data shown in Fig. 2B (blue,

green and black dotted lines) together with time traces obtained using evolutionary global

analysis (red solid lines). Data points of first 15 fs have been excluded from the analysis.

17

Fig. S13. Feynman diagrams of selected Liouville pathways contributing to the 2D spectra shown

in Fig. 5. Ri – non-rephasing pathways, R’i – rephasing pathways: GSB (i=1), GSB via common

ground state (i=2), SE from locally excited state (i=3), SE after energy transfer to lower excited

state (i=4), ESA from locally excited state (i=5), ESA after energy transfer to lower excited state

(i=6). Ground state denoted as ‘g’, hot ground state denoted as ‘gv’.

18

Movie S1

2D data of Rba. sphaeroides taken at t2 = 0–400 fs delay times with step 5 fs (replica of

data plotted in Fig. S5-6). The spectra were plotted in Matlab using the default color map

with no noise filtering.

Movie S2

2D data of Rps. acidophila taken at t2 = 0–400 fs delay times with step 5 fs (replica of

data plotted in Fig. S7-8). The spectra were plotted in Matlab using the default color map

with no noise filtering.

Movie S3

2D data of spheroidene in acetone taken at t2 = 0–400 fs delay times with step 2 fs

(replica of data plotted in Fig. 4A-B). The spectra were plotted in Matlab using the

default color map with no noise filtering.

Movie S4

2D data of rhodopin glucoside taken at t2 = 0–400 fs delay times with step 5 fs (replica of

data plotted in Fig. 4C-D). The spectra were plotted in Matlab using the default color

map with no noise filtering.

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