Chapter 1

Introduction

1.1 Photosynthesis

The word photosynthesis is derived from Greek ϕωτo photo ‘light’ andsynthesis συνθεσις ‘putting together’ and defines the process in whichcarbon dioxide is converted into organic compounds utilizing the en-ergy that is inherent to sun light. This process occurs in a broadvariety of species, ranging from deep-sea algae over cyanobacteria liv-ing in salty, harsh environments, or dark and cold deep lakes to thehigher plants on the earth’s surface. But no matter where the organ-ism lives, photosynthesis can, in general, be divided into two groups:oxygenic and anoxigenic photosynthesis. In the former process, wateris required, and oxygen is released as a product of the reaction. Thelatter process however, does not involve water splitting, and hence nooxygen is released.

1.1.1 Oxygenic photosynthesis

In green plants, photosynthesis is oxygenic. It can be described by theoverall equation

1

2 Introduction

6CO2 + 6H2O ⇀︸︷︷︸energy

C6H12O6 + 6O2 (1.1)

The apparatus that performs the various steps involved in photo-synthesis is embedded in the thylakoid membrane, which is situatedin the chloroplasts in higher plants and in the intracellular space incyanobacteria (1). Four (I-IV) transmembrane proteins (Figure 1.1)work together, each having a specific task.

PQ

Fd

PQH2

PCH2O

1/2 O2

2e-2e-

2e-

FNR

NADP+

2H+

H+

NADPH

H+ADP ATP

2H+

2H+

2H+

2H+2H+ H+

Pi

Stroma

Lumen

Membrane

PSII + Antenna b6f PSI + Antenna ATP-synthase

Light Light

Figure 1.1: Simplified representation of the photosynthetic processesoccuring in the thylakoid membrane, and the systems involved.

The initial step is the absorption of photons by the chlorophyllsand carotenoids of the antenna complexes associated with the photo-systems. After ultrafast transfer from the antennas to the reaction cen-ter of the photosystems, the photochemical reaction is induced there,that leads to charge separation, and to the release of an electron. Two,by Photosystem II (I) sequentially released electrons, are injected into

Chapter 1 3

the electron transport chain. From left to right: The transport to-wards PS1 occurs via several steps. First a plastoquinone (PQ) isreduced to plastoquinol (PQH2). The loss of two electrons in PSIIis compensated with two electrons obtained from splitting water into4H+, 4e− and O2. The plastoquinol diffuses into the thylakoid mem-brane, where it reduces the cytochrome b6f complex (II). Then, the b6ftransfers its electrons via a plastocyanin to Photosystem I (PSI)(III).Here, these electrons reduce the oxidized primary donor of PSI andvia ferrodoxine and ferrodoxine-NADP+-reductase result in the pro-duction of NADPH. Another process occurring in parallel and directlydependent on the charge separation is the ATP production by ATP-synthase (IV). The 4H+ created when splitting water create a protongradient across the membrane. Most electron transfer steps also in-crease this gradient. The energy contained in this gradient is used todrive the ATP-synthase. In the Calvin cycle those two compounds areused to create glyceraldehyde-3-phosphate (G3P)(C6H5O3 − PO2−

3 ).Through a series of chemical reactions, glucose, sucrose, starch andvarious other compounds needed by the plant are formed. In short,CO2 has been removed from the atmosphere, the plants need for car-bohydrates satisfied, and oxygen is released; and that all just drivenby the power of the sun and some water.

1.1.2 Anoxygenic photosynthesis

Anoxygenic photosynthesis is the phototrophic process where light en-ergy is captured and stored as ATP without the production of oxygen,as the name indicates. The process is somewhat less sophisticated thenoxygenic photosynthesis, involving just a single photosystem. This re-stricts to cyclic electron flow only, hence they are unable to produceO2 from the oxidization of H2O.

4 Introduction

1.2 Pigments in photosysnthesis

1.2.1 Chlorophyll

The name chlorophyll is a combination derived from the Greek wordschloros χλωρoς ‘green’ and phyllon ϕυλλoν ‘leaf’. Chlorophyll isfound in virtually every plant, alga or cyanobacterium on earth. Itis the molecule that allows plants to obtain energy from sunlight. Itsstrongest absorption is in the blue part of the visible spectrum, around450 nm, followed by the second maximum in the red at about 660 nm.The lowest absorption is found in the green (530 nm), which directlyexplains the green color of the pigment. Fig. 1.3.

N21

4

5

6

N22 9

1011

N23

14

15

16

N24

1920

1

78

12

131718

2

3

R2

Mg

132 131

O

OO

O

R1

O

CH3

O

R1 =

Chlorophyll a : R2 =

Chlorophyll b : R2 =

Beta-carotene

Lutein

Neoxanthin

Violaxanthin

Figure 1.2: Structural formula of chlorophyll, nomenclature accordingto IUPAC (left) and the structure of the carotenoids found in PSI andLHCII (right).

Chapter 1 5

The vibrational modes of chlorophyll

For the chlorophyll molecule, there is a set of vibrational modes knownfrom the literature (2), which are observable in the mid-infrared regionbetween 2000 and 1500 cm−1. This is also the region that is mainlydiscussed in this thesis; hence, all other modes in different regions areomitted for the sake of simplicity. Figure 1.2.

For example the infrared modes of bacterial chlorophyll a/b, ofwhich the structure is shown above in Figure 1.2, in solution are:

• 1735 cm−1 for the 132 ester C=O stretch vibration

• 1686 cm−1 for the vibration of the 131 keto C=O

• 1654 cm−1 the mode of the 31 C=O stretch

This modes vary slightly between the different kinds of chlorophylls(a,b,c etc..)

Our main focus is on the 131 keto stretch1, because this modeis very sensitive to hydrogen bonds. The presence or absence of ahydrogen bound can induce a shift of up to 30 cm−1 (3, 4)!

When we combine the knowledge we obtain from the crystal struc-ture about the number of hydrogen bonds between the (groups of)chlorophylls, it enables us to link our time resolved mid-infrared tran-sient absorption difference spectrum to specific chlorophylls involvedin the energy migration and or charge separation in the systems thatcomprise chlorophylls’ as their chromophore(s).

1.2.2 Carotenoids

Carotenoids are the second group of pigments found in photosynthet-ically active organisms. They are formed by an polyene chain of al-

1For historic reasons, the old nomenclature, which denotes this keto mode as9-keto is still very often used and will also be found in this thesis

6 Introduction

terning single and double bonds, a ring at both ends. There is a totalof over 600 carotenoids known, but only Lutein, Neoxantin, Violax-anthin and β-carotene are found in the complexes from higher plants,studied in this thesis. Therefore, we will focus mainly on them.

400 500 600 7000.0

0.2

0.4

0.6

0.8

1.0

400 500 600 7000.0

0.2

0.4

0.6

0.8

1.0

Chl a Chl b

Abs

orpt

ion

[nor

mal

ized

to 1

]

Wavelength [nm]

Lutein Neoxanthin Violaxanthin β-Carotene

Figure 1.3: Absorption spectra of Chl a and b (top) and of the rele-vant carotenoids (bottom) in the visible range in 80% aceton at roomtemperature

The spectroscopic properties are different from those of chlorophyll.They absorb in the region 400 to 520 nm, but do not have any other ab-sorption bands in the visible. Their functions are to provide structural

Chapter 1 7

stability to the proteins, light harvesting, contribute to the assemblyprocess (5), and protect the system from radical oxygen (6). Due tosymmetry rules the transition S0 → S1 is quantum-mechanically for-bidden. Hence the absorption at 400-500 nm arises from the S0 → S2

transition.

1.3 The candidates, getting to know each

other

This section serves to get the reader a bit acquainted with the threephotosynthetic proteins studied in this thesis, plus the fourth protein,which is some sort of an exotic in this content, as it is a lightsensing,rather than a lightharvesting photoactive protein.

1.3.1 LHCII

The trimeric light harvesting complex II (LHCII) is the most abundantprotein in the thylakoid membrane of higher green plants and greenalgae. Approximately 50% of the chlorophylls (Chls) in the thylakoidmembrane are contained in the LHCII complex, which is mainly as-sociated with the photosystem II (PSII). Since Thornber and Ogawaindependently discovered LHCII > 40 years ago (7, 8), the complexhas been the subject of intense study, using a broad variety of exper-imental techniques (9–12). LHCII acts to collect solar radiation andtransfers this energy toward PSII and photosystem I (PSI) where itis converted into a chemical, storable form. LHCII thus acts as anantenna, directly increasing the effective absorption cross section ofPSI and PSII by funneling energy toward them.

It has also been established that LHCII contributes to photopro-tection (6, 13) and energy dissipation under high light conditions (14–17). In addition, LHCII is involved in regulating the distribution of

8 Introduction

absorbed energy between the two photosystems (18). Another func-tion of the complex is more structural, it facilitates the stacking of thegrana in the membrane (19).

Figure 1.4: LHCII, view from the top. Chlorophylls are shown ingreen, luteins orange, neoxantine magenta and the forth, not yet un-ambigiously identified carotenoid in cyan. Chlorophylls shown in redare the ones that connect the complex to adjaicent antenna complexes.

In the native form, LHCII is a trimer of three identical monomers,each with a mass of about 25 kDa, see Figure 1.4. The earliest attemptto resolve the crystal structure of LHCII date back to 1987 whenKuhlbrant succeeded in preparing two-dimensional crystals of LHCII

Chapter 1 9

(20). Seven years later he and Wang (21) reported the first crystalstructure of LHCII with an atomic resolution of 6 A. From this earlywork, it was revealed, that LHCII is a trimeric construct of monomericsubunits. Three α-helices were found to span along the membrane.The resolution was improved to 3.4 A in 1997. Now it was possible tomodel 12 chlorophylls in the electron density map. There was still adiscrepancy to what biochemical analysis showed; namely the presenceof 8 Chl a molecules and 6 Chl b molecules was expected to be found.

For this thesis, the knowledge from the 2004 crystallographic datadescribed in Liu et al. (22) with 2.72 A resolution (and later on with2.5 A resolution in (23)), was available. It is now known that ev-ery monomer contains six chlorophyll b molecules, eight chlorophyll amolecules, and 232 amino-acid residues (see Fig. 1.4). Furthermore,four carotenoid binding sites were found, in which two luteins and oneneoxanthin were unambiguously identified, but no precise assignmenthas been given for the fourth carotenoid, which is thought to be in-volved in the xanthophyll cycle. The LHCII carotenoids play a majorrole in the stabilization of the structure.

In this thesis, the energy transfer between (groups of) chlorophyll aand chlorophyll b molecules, the exact pathways via which this transferoccurs, as well as their timescales, are the main focus when investi-gating the system.

1.3.2 Photosystem I

PSI is one of the largest membrane proteins of which the crystal struc-ture has been resolved (24, 25). For Thermosynechococcus elongatus,which is the sample used in this thesis, the best resolution available is2.5 A. See Figure 1.5 (25). The heart of the whole complex, the PSIcore, is composed of 12 proteins that bind 127 cofactors; 96 chlorophylla molecules, 2 pyhlloquinones, 3 Fe4S4 complexes and 22 caroteniodsplus 4 lipids and a putative Ca2+ ion. So far, it has not been pos-

10 Introduction

sible to isolate the RC of PSI without this remaining core antennacomplexes.

Figure 1.5: The structure of PSI from the side (left) and bottom view(right). The reaction center is in green, the iron-sulfur clusters areyellow. Antenna chlorophylls are presented in black.

The electron transfer chain (Figure 1.6) is arranged along two largeproteins, PsaA and PsaB in a symmetric arrangement similar to theone found in PSII. These two proteins also bind the majority of thePSI core antenna pigments. The electron transfer chain consists of 6chlorophylls, two pyhlloquinons and 3 iron-sulfur clusters. The twoparallel-orientated chlorophylls on top, are denoted as P700.

For quite long they were considered to be the primary electrondonor to the system, and hence named accordingly. But since recently,more and more people are convinced that the A0 chlorophylls situatedunder the P700 are the primary electron donor (26, 27). The chapters 3and 4 of this thesis address this issue amongst other intriguing featuresof the primary charge separation.

Just right under the P700, symmetrically arranged are the acces-

Chapter 1 11

P700

ACC

A0

A1

PQN

FA

FX

FB

Figure 1.6: RC

sory chlorophylls, denoted ACC, and underneath the so-called primaryelectron acceptor A0. The two phylloquinoines (PQN, vitamin K)form the secondary electron acceptors. No matter which branch theelectron has taken so far, the last 3 steps are always occurring alongthe single chain of the iron sulfur clusters FX,FA and FB.

1.3.3 Rhodobacter sphaeroides

Rhodobacter sphaeroides is a purple bacterium that belongs to a groupof bacteria which can obtain energy from photosynthesis. It has beenisolated from deep lakes and stagnat waters.

It contains a reaction center, which is surrounded by peripheralpool(s) of light-harvesting complexes II (LHII) and center-light-harvestingcomplexes I (LHIRC). In wild type (WT) Rhodobacter sphaeroides,the LHI together with the reaction centers (RC) and the polypeptidePufX form curved, eight shaped dimers with the RC in the center of

12 Introduction

each loop (28). The monomeric complexes used in this study are evenmore simple in geometry. The LHI ring, formed by 24-30 chlorophyllpairs encloses one RC. In the presence of PufX however, the ring isnot fully closed, but yields a small gap. Figure 1.7.

x RC

A

RC

B

Figure 1.7: Arrangement of the LHI antenna. Open ring with PufX(left) and closed ring arrangement (right) when lacking the PufXpolypeptide

The RC (Figure 1.8) itself is formed by a series of cofactors, thatform two symmetrical branches, denoted A and B. Only the A-branchwas found to be photochemically active (29, 30). The primary electrondonor, a dimmer formed by two bacterial chlorophylls PA and PB, islocated near the periplasmic side of the membrane, and is common toboth branches. Next in the chain is a monomeric Chl, denoted BA andBB respectively, and is situated just under the special pair, symmetricon both sides. Underneath a bateriopheophytine can be found onboth sides, denoted HA and HB . The last link of the chain in eachbranch is a quinone, denoted QA and QB, which are situated nearthe cytoplansmic side of the membrane. Between the two quinones,roughly equidistant to both, a non-heme iron atom is located. Afterexcitation of P, thus creation of P∗, the electron transits within ∼3ps to BA, forming P+B−

A, and 1 ps later P+H−A . The next step, the

transfer to QA and formation of P+Q−A, takes about 200 ps. Electron

transfer between the two quinons takes about 100 µs. In the meantime,the P+ is reduced by an external cytochrome c2+2 protein in just 0.8 µs.A secondary absorption of energy initiates a second electron to reach

Chapter 1 13

QA via the chain already described, that is then also transferred to QB

in ∼1 ms via proton coupled electron transfer after which the protonis rapidly bound. In detail: PQ−

AQ−B → PQA[QBH]− → PQAQBH2.

The reduced QBH2 then diffuses out of the RC and is replaced by anexogenous quinone (31–33).

QAQB

Fe

HAHB

PAPB

BABB

Figure 1.8: Arrangement of the pigments in the reaction center ofRhodobacter sphaeroides. The BChl, BPhe and ubiquinone cofactorsform two membrane-spanning branches, denoted A and B. The dottedline shows the axis of twofold symmetry, arrows indicate the pathwayof electron transfer. The picture was created using the PDB 314Dstructural information

1.3.4 Photoactive Yellow Protein

Photoactive Yellow Protein (PYP) is a rather small (14kDa, 125 aminoacids) protein , first found in 1985 by Terry E. Meyer (34) in the

14 Introduction

bacterium Halorhodaspira halophila. Its structure has been resolvedwith a resolution of 0.82 A via X-ray crystallographie and reveals atypical α/β protein structure; (35–37) a six stranded antiparallel β-sheet as scaffold, flanked by several helices. Two hydrophobic cores arepresent, one of each side of the β-scaffold. The smaller one containsthe N-terminus cap, whereas the larger one forms the chromophorebinding pocket .

Arg 52

Cys 69

Glu 46

Thr 50Tyr 42 P

P*

I0

I0#

I1

I2

Figure 1.9: The cromophore of PYP in the binding pocket (left) andits’ photocycle (right)

The cromophore is a p-Coumaric acid (pCa). In its ground state,the cromophore is in a trans configuration, and at neutral pH, in adeprotonated state. Upon blue light activation, PYP enters a fully re-versible photo-cycle, consisting basically of three steps, shown in Fig-ure 1.9. The first step is the cis-isomerization of the cromophore lead-ing to the intermediate I1, followed by protonation of the chromphorevia intermolecular proton transfer and structural changes of the pro-

Chapter 1 15

tein leading to I2. The last step in this cycle is the re-isomerizationand deprotonation of the chromophore and refolding of the protein.Because the protein is very stable, rather small and water-soluble ithas become a model system to study primary photochemistry of lightsensing.

16 Introduction

1.4 Experimental techniques and tools

The initial processes in photosynthesis take place in a time range offemtoseconds to nanoseconds. In order to be able to follow those primeevents, a technique with sufficient time resolution is necessary.

The fastest speed known to mankind is the speed of light. By us-ing the light itself as yardstick, it is possible to create techniques thatare capable of performing measurements’ with sufficient time resolu-tion. The tool used in this thesis is transient absorption differencespectroscopy, commonly referred to as pump/probe spectroscopy

1.4.1 Pump/probe spectroscopy

The amount of light absorbed by an object is best described by Beer-Lamberts law.

I = I0 · 10−c·ε·l (1.2)

The intensity I0 of the incident light, which, as a direct result ofits’ interaction with the object, is attenuated, is measured as I. Thedegree of attenuation is directly correlated with the materials inherentproperties, namely the extinction coefficient ε, its concentration c andthe length l of material passed by the light, The product of ε, c andl is commonly referred to as optical density (OD) of the sample inquestion and is wavelength dependent.

OD = −log(I(λ)

I0(λ)) = c · ε · l (1.3)

In transient absorption pump probe spectroscopy, the first stepis always to excite the sample under examination with a pump pulse.When the photon energy of the incident light coincides with the energydifference of the electronic transition from one or more cromophores

Chapter 1 17

ground state to one of its excited states (S1..Sn), the photon can beabsorbed, the ground state thus depleted and the electron promotedto a higher, excited state.

GSB

ESA

SE

S0

S1

Sn

ν1

ν2

νn

ν1

ν2

νn

ν1

ν2

νn

Figure 1.10: Jablonski diagram of the energetic states of a molecule.Small arrows indicate vibrational transitions, whereas big arrown elec-tronic transitions.

Subsequently, a second pulse is applied to the sample. The spectralregion under observation is determined by this second pulses’ wave-length and pulse width. This pulse has a relative low intensity, sothat no additional photoreactions are triggered by the probe pulseitself. Depending on the probe pulse wavelength, slightly different in-

18 Introduction

teractions take place. In case of a probe of 4-6 µm wavelength, inthe mid-infrared region, the vibrational states belonging to an elec-tronic state are the source of interaction. This is represented by thesmall arrows in Figure 1.10, indicating transitions between differentvibrational levels (ν1..νn).

If the probe-wavelength is chosen to be in the visible region, inter-actions between electronic states can be accessed. This case is repre-sented by the big arrows, crossing from one electronic state to another(S1..Sn). Both probe regions are used in this thesis, and therefore thedifferent nomenclature is introduced here to make the reader aware ofthe differences.

By variation of the path length traveled by the pump pulse withrespect to the probe pulse, time dependent spectra can be measured.The speed of light is 3 · 108m

s, meaning for every micrometer difference

in path length between pump and probe beam we create a time delayof 3.3 fs. The actual limitation in our case is not the mechanicalprecision with which that difference is created, but rather the crosscorrelation of the pump and probe beam, that finally results in a timeresolution of 150 fs.

By determining the difference in OD between a sample that hasbeen excited with the pump beam (Ip) and one that hasn’t been ex-cited (Inp), it is possible to monitor the changes in the population ofenergetic and vibronic states as well as the migration of excitons toother states.

∆OD(λ, t) = OD(λ, t)p −OD(λ)np = log(I(λ)npI(λ, t)p

) (1.4)

The result (Figure 1.11) of this ∆OD measurement will result innegative and positive bands. The common definition is: Negativebands represent bleached ground states (GSB), the population, andthus its absorption, is less, as the pump beam has depleted this en-ergy level. The probe also triggers emission from the excited states

Chapter 1 19

vibrational modes, to energy levels that coincide in energy differencewith its wavelength. This is then called: stimulated emission (SE)

Figure 1.11: Shown is a simulated absorption spectrum, composed of 3gaussians representing the GSB, ESA and SE, that together contributeto the spectrum, which we measure in our experiments.

The bands of stimulated emission will generally be shifted towardslower energies with respect to the ground state bleach, representingthe loss of energy by thermal cooling and other non-radiative decayprocesses they were subject to. The third possible interaction is thepromotion of an excited state into an even higher energetic level. Thisabsorption caused by an excited state is called excited state absorp-

20 Introduction

tion (ESA) and appears as positive band in the difference absorptionspectrum.

1.4.2 Technical realization

The setup used for most experiments performed in this thesis is drivenby a Ti:Sapphire oscillator-regenerative amplifier laser systeme (Hur-ricane, Spectra Physics Inc.) which provides a 80-90 fs laser pulse of800 nm wavelength at a repetition rate of 1 KHz. The output energyis ∼0.8 mJ. The beam is split into 2 beams with a ratio of 60:40. Thelarger portion is used to pump a tuneable optical parametric generatorand amplifier with attached difference frequency generation (TOPAS,Light Conversion). The TOPAS output is a spectrally broad mid-infrared beam (200 cm−1) with a central wavelength tuneable in therange of 2.4-11 µm and an output energy of 3-15 µJ. This beam isnormally attenuated (neutral density filter F) to about 1 µJ beforethe sample. The smaller portion of the initial 800 nm beam is directetinto a non-collinear optical parametric amplifier (NOPA), which canproduce pulses between 475 and 870 nm. Replacing the NOPA with aBBO frequency doubling crystal allowes for 400 nm excitation, whiche.g. is used in chapter 7. The here generated, and by the delay line intime relative to the probe beam variable pump beam, is then focusedonto the sample. The probe beam diameter is 120-150 µm, and thepump somewhat larger. The pump is chosen to be a bit larger to ensureall measured sample is pumped. The polarisation of the probe-beamis set with a Berek-polarizer to be 54.7◦ (magic angle) with respect tothe probe beam to provide anisitropy-free measurements (38).

After passing the sample, the probe beam is dispersed by a spec-trograph (Chromex) onto a 32 channel Mercury-Cadmium-Telluride(MCT) detector. Three different gratings with 150, 75 or 50 grovesper mm can be selected and provide a spatial resolution of 3 cm−1

or better, depending on the wavelength region. A homebuild inte-

Chapter 1 21

Figure 1.12: Schematic representation of the setup used to performefemtosecond visible pump mid-infrared probe spectroscopy experi-ments. This setup as been used in chapters 2,3,4,5 and 6 with someslight modifications depending on the experiment.

grate and hold stage feeds the detector data to a PC, equipped witha data-acquisition card (National Instruments), where the signals arerecorded. A chopper, operating at 500 Hz is placed into the pumpbeam, so that the difference in transmission between pumped andunpumped sample is measured and the difference in absorption calcu-lated. To determine if the beams have maximum spacial and temporaloverlap, a thin piece of GaAs is placed in the beam, at the positionof the sample. The visible excitation pulse creates free electron carri-

22 Introduction

ers in the semiconducting material. This results in a large change inthe index of refraction. Hence, big changes in the transmission of themid-infrared probe pulse can be measured. Maximizing the amplitudeof this signals ensures optimal spectral and temporal overlap. Thecross-correlation of this two pulses in GaAs is ∼150 fs. This time de-termines the instrument response function. The whole setup is placedin a plexiglass-box, that is constantly flushed with nitrogen, to keepthe absorption of the mid-infrared beam by humidity at a minimum.

1.4.3 Data analysis

A very important step in the interpretation of our results is a properand reliable analysis of the measured data. This is neither trivial norquickly achieved. Hence, some words on this topic seem in order.

The data we collect is normally 3 dimensional. Wavelength λ andtime t are our independent variables, whereas different transient ab-sorption ∆OD - the intensities we measure, is the dependent vari-able. The less complicated scheme we apply is called ‘global analysis’.Global in the sence that all data are analysed together.

The basis of global analysis is the superposition principle, whichstates, that the measured data Ψ(t, λ) result from a superposition ofthe spectral properties εi(λ) of the components present in the systemof interest weighted by their respective concentration ci(t) .

Ψ(t, λ) =

ncomp∑i=n

ci(t)εi(λ) (1.5)

The concentrations ci(t) of all ncomp components are described bya compartmental model, that consists of first-order differential equa-tions, with as solution sums of exponential decays.

We will apply two types of compartmental models in the frameof this thesis: (I) a sequential model with increasing lifetimes, also

Chapter 1 23

called an unbranched unidirectional model, resulting in Evolution As-sociated Difference Spectra (EADS), and (II) a full compartmentalscheme which may include possible branchings and equilibria, yield-ing Species Associated Difference Spectra (SADS)(39). Figure 1.13illustrates this.

k11

1

2

3

1

2 3

4

k1k12

k2 k21 k22

k3 k3

Figure 1.13: Schematic representation of the global (left) and target(right) analysis. k are the decay constants of the compartments, whichrepresent decay states of the sample

The latter is most often referred to as target analysis, where thetarget is the proposed kinetic scheme, including possible spectral as-sumptions. This technique is only applied when additional informationon the system is availible, e.g. from flourescence experiments, and theresult has carefully to be checked against the raw data to see if theunderlying model is valid.

With ultrafast spectroscopy the instrument response function (IRF)can usually adequately be modeled with a Gaussian with parametersfor location and full width at half maximum (FWHM, typically 100-

24 Introduction

250 fs). The dispersion of this location parameter is described by apolynomial. All exponential decays from the above models have to beconvolved with this IRF (39).

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25

26 Introduction

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30 Introduction

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