 works. Thank you Nicoletta and thank you everyone for having the strength for the last one half hours of lectures at the school. It's nice to see you here and so we stopped at the at the carotenoid functions. We already learned that carotenoids have two excited states and their absorption is occurring from the ground state to the second excited state and they that they are important for the photo protection for quenching the triplet states and getting rid of singlet oxygen in photosynthetic systems. Now the application that I want to show you is related to this role of carotenoids in excitation quenching. Now this is a phenomenon well known from for now probably more than three decades. If you look at the and this is experiment you can actually do at home just if you have a piece of red transmitting glass I don't know a glass of wine may do the trick so if you have a red filter and some blue light to put on a plant and if you basically have a plant and illuminate it with a strong flash of light what happens in the plant of course that the fluorescence from the leaf suddenly increases even if you have switched the light off it the next flash will produce more fluorescent. The reason is that all the reaction centers are now busy processing the light that they received from the previous flash so you close the reaction centers and therefore you know they cannot use the photon energy anymore and so this energy comes out of the leaf partly as fluorescent. Now this is not so interesting this called a photochemical bit of quenching so basically the closed reaction centers will increase the fluorescent but if you keep the light on so at the level where you have you know on a sunny day falling on the grass you will see that the fluorescence of the leaf will gradually decrease and even if you add larger bursts of light the top amplitudes will be a lot less than than the initial one for the dark adapted leaf. Well that's called a quenching of excitation and that's a part of a protection mechanism for the plant to be able to work under both high illumination conditions and low illumination condition so at the bottom of a forest floor it's really on a cloudy day it's relatively dark whereas at the top on a sunny day is very very bright so the plant needs a mechanism to adapt to this and so when you put a lot of light on a plant on the leaf even you will see that it sort of starts dissipating throwing away more and more light without radiation so the fluorescence level will go down in order to compensate for over illumination because if you if you let the reaction center work too hard what happens it produces a lot of oxygen the oxygen catches a chance to oxidize it and then you have to invest the energy from the cell to repair this protein and so basically there was a long time there's a long time discussion still standing what are the mechanisms of this and there are a number of them apparently but one of them is actually a photochemical alteration of carotenoids so the supposed mechanism is that one carotenoid that sits in the leaf in the photosystem it gets epoxidized which means that it's basically the conjugated double chain of it grows from 11 double bonds to 12 double bonds so you increase the length of the molecule and you basically what you are doing you are bringing down its excited states and so we wanted to look at it in a model system this is back in 2006 I think and the reason we wanted to do that is that this is the photosystem 2 photosystem 1 I'm sorry it's a 100 chlorophylls lots of protein lots of carotenoids not a chance to understand something if you just start with this mess so we said okay let's make it make a synthetic light harvesting complex consisting of a chlorophyll like pigment that's of talocyanin and the and a set of carotenoids covalently attached to it so this is a carotenoid molecule that goes here and you can make them synthetically shorter and longer and even longer what happens to the absorption spectrum of the carotenoid that of course it shifts to the red but basically nothing happens to the talocyanin absorption spectrum which sits here at 680 nanometers now what we wanted to look at is whether or not the lifetime when you excite the the chlorophyll like molecule the the talocyanin will it change depending on the length of the attached carotenoid and so if you look at the energy transfer time so you excite the molecule you look at the different absorption spectrum you see these not rotenoid signatures the ground state bleach of the carotenoid the s2 spectrum which is this black line here and then s1 appearing and going down at the same time as a carotenoid is transferring excitation energy to the chlorophyll to the talocyanin you will see the top ion in bleach band grow in so and that takes you know about 20 picoseconds so this is a nice model system for the energy transfer from carotenoids to porphyrins it works if you look in fact if you look at the at the plant photosystems it's almost exactly the same picture the spectra a little bit different because you have chlorophylls and not talocyanins and the carotenoids are different but but generally that the features that you see are exactly the same now if you look at the lifetime so this is now four nanosecond timescale and lifetime lifetime of talocyanin depending on the length of the attached carotenoid you see that if we if it has 10 double bonds it does not change the lifetime of the talocyanin at all if you increase the length by one it already quenches a lot of excitation so on the nanosecond timescale this dark state that has now moved below the excited state of of talocyanin is now acting as a sink for the excitations where they arrive and are quickly dissipated so the lifetime of talocyanin becomes shorter and it's even more so when you increase the length of the carotenoid by two double bonds so that was basically the synthetic argument why this mechanism can actually be true and basically in this artificial light harvesting antenna we proved that if you elongate the carotenoid by even one double bond you already lose you know a really large fraction probably about 90 percent of all the excitations that are there now so that's nice it shows you that your pump rope spectroscopy can monitor both excitation energy transfer dynamics and photo photo protection function of the carotenoids or at least mimicked in the synthetic systems now another example this is really classical work that from photosynthesis it is the energy I'm sorry the electron transfer change in photosynthetic reaction center so if you remember where you excite these two chlorophylls situated nearby here the electron hops here here here and here so now I'm going to tell you about the experiment that that actually told us that this is the way it is happening for a long time it was not known whether the electron actually goes on this accessory bacteria chlorophyll so and if you look at the lifetime it's it's easy to see why so it takes three picoseconds almost four picoseconds to go from P to BA from to the accessory bacteria chlorophyll but after that it only test it takes less than a picoseconds to go on so basically the accumulation so if you think about it as a as a as a pool with a with a very narrow pipe filling it in and the very broad pipe for the water to flow out so basically the population never accumulates here because it's coming with four within four picoseconds and leaving with one picosecond and so it was a question whether electron actually goes here or goes directly on here and the work that answered it was done by Wolfgang Tsint in Germany and so what he did he looked at several wavelengths so this is the special the absorption of the special pair so this is the pair of pigments that initially received the excitation this P then the accessory bacteria chlorophyll absorbs somewhere here and the bacteria pf5 sorry wrong button bacteria pf5 has a clean and nice absorption band indivisible over here and now so if you look at the absorption of the steam of the special pair at the stimulated emission of the special pair you see that it decays quite quite quickly you're excited it arrives very quickly and it's gone within 10 picoseconds it's banished completely now if you if you look at the induced absorption this is actually a mixture this is both special pair and accessory bacteria chlorophyll you can see that the curve is sort of doing a wiggle here and if you look at the bleach of the bacteria bacteria pf5 items this is actually there so you produce it and it's sort of you see the signal growing in here and then leaving again as the electron moves away from it now what Wolfgang Tsint did they try to model it using either two-stage models so directly from special pair to bacteria pf5 and that's your dashed lines and a three-stage model where they said okay first from special pair to accessory chlorophyll then from accessory chlorophyll to bacteria pf5 and then to the keynote and so basically this was the model that produced this the solid curves and it's obvious that the dashed curves even though they work here they definitely don't work here and also they don't work here if you if you try to fit the data and eventually they were able to deduce that the lifetime for arriving on accessory bacteria chlorophyll is about four times longer than leaving it and so and this this lifetime was clear so that was 220 picoseconds but again this was state of the art back in 1999 I believe and so they actually drew the picture of how the electron is moving in the photosynthetic system of bacteria across the membrane so to speak now you may have noticed that this was even with the data you cannot really answer it just from looking at it and so I will spend some time talking about data analysis of ultrafast measurements now it's always fun to watch PhD student when he first does an ultrafast measuring and is confronted by a host of spectral looking like this so these this is a single measurement two polarizations so it consists of about 150 time points and about 300 wavelength points so it's really a big array of data and now you have to do something with it you can of course drew draw it like this you can also draw it in terms of kinetic traces so the time dependence is in specific probe wavelengths and it's not much better I can tell you it's again something that can fit on your screen but analyzing it by eye forget about it's not possible now what if you are an expert in the computer graphics you can produce a nice looking carpet which will give you an overview of the entire data set however it's not very useful as an analytical tool it just looks nice yeah it's a three-dimensional thing and as my supervisor said when I was a PhD student that you know if you want to get to publish a nice paper you should make a lot of 3d plots and contour graphs that's the way to success in science as well so this is one way to success in science but it's not very nice because for example you cannot read what's going on behind this crest here right so it's not you can make a movie by it rotating but then again you are too slow to analyze it so it's hopeless again now and so you need tools for data analysis yeah and you need a model and the models can be different we already talked about it during the question session it can be on one hand they can be they always reflect some sort of imagination about what's going on with reality in our minds but they can be phenomenological and then they are sort of intuitive hand-waving sort of simple problem is no deep physics in them but they give you good description of data on the other hand you can as a couple of colleagues suggested always start with honest ab initio approach write down a Hamiltonian good luck doing that for ten thousand atoms and try to diagonal diagonalize it and and do a time dependence so again it will be an honest good approach but it's not going to bring you results so while it's first principles base and meaningful in terms of physics but it will be an intuitive because of sheer size of it and it will almost certainly not describe the data so that's and you are as a scientist you have to use what tools are available to you so you sort of compromise between the two and sometimes you first do this and then as you progressively move towards the better understanding and maybe refine the data you can actually move to this side of the division yeah so yeah Feynman diagrams propagators big sums tensors everything we loved from the second year at the university now but you can also look at it in a like I'm an experimental science I like to look at it in a simplistic way so imagine you excited the molecule and it's doing its business whatever it is salvation isomerization electron transfer something like that so how about it's just going like this and we approximate this dynamics by a series of steps across this potential and approximate them by first rate kinetic equations now this is a an approach of a chemist it's probably not true in this strict scientific sense but it will yield you a description of the data that is essentially all contains in on contained in four boxes with four numeric parameters meaning that the transfer times from year to year to year to year to year to year so and implementation of this on time results spectroscopy is called global analysis of time result data and you have to you should use it when you yeah you don't know any better for example you are that that student who got this big number of spectra and now you have to make sense out of it and so basically this is the way to parameterize the large amount of data in a concise sort of manner so you reduce your data set of you know 300 spectra into four spectra for example of four lifetime components that's already something you can plot on a two dimensional graph and read that's nice and basically this is also the way of conveying your data to the collaborators who might want to calculate something and dumping you know the whole matrix of numbers with you know 10,000 values in it is not the way to to progress so you have to somehow present the data let's say in a more concise fashion okay so what you do what you still see what I still see being done sometimes so that the student takes a brute force approach so it takes takes a sum of exponentials and analyzes every single wavelength and then puts them in a table so that basically just converts one table into the other table never reduces the amount of data well it wastes some student time so that's good because he's busy and or she's busy but a better way this is not what should be done and global analysis is actually a way of you know putting the data in terms of a simple kinetic scheme that is later can later be used used to actually convey it in a parametric fashion so what you do is you draw a connectivity scheme where you postulate how these boxes or the states in your model if you want to the first rate kinetics are connected so it can be sequential it can also be in parallel for example if you are measuring on a mixture of molecules that are not communicating with each other you can let them decay in parallel you can also have branch kinetics which is the way it works in the reaction dynamics so when you excite a molecule it can sort of take one pathway along excited state potential maybe the other pathway and then after postulating this you solve the kinetic rate equation these are first-order equations even if you forgot how to solve them there's more from alpha and other tools that will do it for you yeah so first or the rate equations and then what you fit the data to is the spectra of each box times the concentrations that you have determined from solving the equations yeah and this turns out there is a software that that can do it for you so one option which is free is the glottaran package from my former university the free university of hamster down there's a program of which you can have a demo from light conversion so that's a demo is free for the program unfortunately if you don't buy one of their spectrometers you have to pay but there are also other three alternatives but what I want to emphasize is that is so simple as drawing this connectivity scheme and then putting in the parameter values for the rates corresponding to each arrow here and it does the fitting for you you can look at all the traces together with a fit see if it fits well or doesn't fit well and what you get is a fit consisting of the let's say the concentrations of time dependencies that you saw of these so how these boxes are populated and the spectrum assigned to each box so let's take a fake data set fluorescence models so let's say our from fluorescence spectrum starts like this and then it sort of becomes more intense and narrows down like this and the time profiles are depicted here so this 10 and 30 picoseconds and you have a basically a two-dimensional data set of fluorescence that is synthetic here and let's try to analyze it using is this global analysis approach so we draw a scheme a goes to be with our one be decays without to and this is the data we are trying to fit we look at the fits about one particular wavelength we can look also at the other ones but anyway they will always be a good fit because that's what we put in it fits perfectly and it produces the spectra just what we put in so a lower amplitude broader spectrum narrower and more intense spectrum just what we put in so we nailed it right so it's if we get the right model we get the right spectra that's all fine and dandy wrong answer if we choose a scheme like this and try to fit it we again get the perfect fit and that will happen every time as long as the number of components we put in is equal in both models so we the model is not the good fit is not the only thing that tells you whether your model is right or not and if you look at the spectrum now of course this is well something that you can immediately see something is wrong here because your spectra of fluorescence are negative so you have a spectrum of fluorescence that is negative negative fluorescence does not exist so but it fits equally well so the lesson to take from it is that actually it's both the spectra and the time dependencies that that determine whether the model that you used is right or not and so basically the if you have three compartments it gets even more complicated it can be sequential can be parallel can be branched this way or can be even branched that way yeah so there's a number of models you can come up and it grows quickly as your dynamics becomes more complicated so if you do a three component analysis again three time constants 10 30 and 50 picoseconds again a normal stoke shift of fluorescence and you analyze it with the right model come on is this and the spectra come out right but if you do it with a different model of course you get nonsense now with fluorescence it's easy to see because fluorescence cannot be negative but if the if the signals are of different science like in transient absorption you can have both decrease in absorption upon excitation and increase then it becomes really complicated and what you what you get there is a sort of a mess and the only way to figure out whether the spectra that you got a right or not is actually looking at the physics of the molecule so if you take the absorption that transient absorption spectrum of the identical system you can fit it with the right model or the wrong one so I fit it here with the wrong one with a parallel model by the time dependence as well like this of course these spectra are nowhere near the ones that we put in okay so this is just to say that the fact that you are fitted your data doesn't mean you have got the meaningful result and there is though a set of models which allow you to sort of use them as a parametrization tool without deeper physical meaning on the model and that's called evolutionary model so that's where you populate first state and then it goes sequentially what it does it simply assumes that your data consists of a number of spectra that morph one into the other with the life with the time constants that correspond to these arrows so this should be first model you try when you don't know anything about your data yet and you don't want to start speculating about the physics you just want to an overview how the data looks like and then if you are start if you start putting physical meaning in these pathways so for example you assume there are two species two different sets of molecules in a sample one of them doing this and one of them doing this that's already a physical meaning ascribed to the model and the analysis of such fitting is called target analysis and that you have you have to judge based on the spectra on the species associated different spectra that you produce and you have to build your intuition about this the only way around it is to look at the spectra and to see okay what else do I know about these molecules that will that will sort of make the spectra real so first of all you may have a different measurement you may have steady state measurements of of transient species that you are observing so then your your intermediate spectra should look like the ones that were measured using different methods of course we saw that when you have salvation your spectrum should shift to the red of course the ground state spectra cannot be negative that you cannot have stimulated emission from the ground state so the only negative contribution in the ground state spectrum ground state bleach spectrum is the bleach of the absorption that you have of your sample and of course basically you have to think about physical processes that induce these physical changes so basically the take home message from this bit is that not all the processes that you see in time results spectroscopy are exponential but a set of exponentials is a nice basis to start your description from and if you get a good fit with ridiculous spectra is much worse than a worse fit with reasonable spectra that just means that you missed let's say the dynamics part of it maybe it's non-exponential maybe you missed an expo a step somewhere but but you should look for the physical reality in your analysis so just to give an example of what what has been done so this is a photoactive yellow protein it's a protein in in bacteria that are photosynthetic they like to feed on the light but they also like to avoid the UV light so if you put the UV light on them they escape yeah unhappy bacterium and it expects escapes from the UV light and so this is the the picture of the number of bacterium this is the illumination but this is where the beam is of the mutagenic mutagenic UV light and a bacteria are escaping away from it now the pigment in this protein is also an isomerizing pigment is called thymetal paracumeric acid doesn't matter what it is but this is a fluorescent spectrum of it and it does isomerizing solution as well so what we saw when we measured fluorescence up conversion on on the stigma is that we initially create a spectrum that is not very much shifted then it in one step in one exponential step it basically shifts in a hundred femtosecond it sort of shifts here a hundred femtosecond is a typical lifetime of water salvation water salvation is very fast and so this is the salvation shift then you have another bit of shift with a little bit of decay so this is this we thought was a start of isomerization and then in the end you have a 30 picosecond decay 2 picosecond I'm sorry decay when the molecule isomerizes and the excited state is lost okay so this is this is about the basic stuff and so we as you might have noticed we've stayed mostly in the in the last millennium maybe the start of this millennium and now I'm going to review the advanced ultrafast spectroscopy so basically the games that you can play with molecule thanks to the arrival of very nice lasers and very nice optical devices if we've seen it in imaging that the cameras that you know digital camera back when I started in 2000 was still something you had to borrow from a guy and he said don't break it it costs you know 1500 euros of guilders in Holland at the same time and nowadays everybody is throwing them away with their old mobile phones and the cameras they are throwing away is actually much better than the ones we used to have now so let's first look what happens if we can add an extra laser pulse into the into the pump probe scheme so we have our pump probe experiment one excitation pulse one monitoring pulse that is measuring the absorption and the problem with the pump probe spectra is that the bands are usually very broad and there's no way of figuring out what they are for so you need an extra way to somehow figure out what do the different bands in the transient absorption spectra represent and this was not our idea but we developed it in the early 2000s it was adding an extra laser pulse so this is like an extra handle that you can tweak in your experiment and an extra interaction with the material during its evolution on the excited state potential which you can use to deduce new things about about the photo observed photo dynamics and basically it helps you to identify these connectivity schemes of this additional parameter in the experiment will allow you to figure out whether you should connect these boxes in way one way or the other it allows you to separate overlapping spectra because if one part of the spectrum is sensitive to this pulse and the other is not you know that they are due to two different things in going on simultaneously in your sample you can separate the dynamics you can separate between the ground and excited state and even produce a new reaction pathways so let's see what what the concept is so suppose you want to monitor some students and you want to know the student reaction pathways for students walking from university and going to the dormitory for example you're going home the question is does the roads lead through the pub because some of the students are always drunk when they arrive home or does the road branches off now the way to do it is one way to do it I'm not saying that this is the way this should be addressed is placing a lion in a pub so if you if you have a lion that eats some of the students who visit the pub then your number of students basically will depend arriving home drunk will depend on the presence or absence of the lion so this is your extra laser pulse so basically and this the result will be different depending on whether the student has decided to go to the pub and had to branch off and these students will not be affected by the lion in this case and they clearly will be affected by the lion in this case now let's switch back to science away from lions and students and so if you excite the molecules and they evolve on their excited state potential basically what will happen if you have this sequential isomerization scheme and you add an extra pulse that is resonance with a stimulated emission of the molecules it will make some molecules go back to the ground state yeah and in the end the end result of the amount of the photo product the isomerized product that you see will depend on the presence or absence of this pulse on the other hand if you if the molecules decide very early in the photo dynamics which way they are going to go so you are dumping the ones that do not isomerize and the ones that do isomerize remain unaffected in that case of course the number of photo product will not depend on the presence of this dump pulse and by playing with a wavelength and the timing of this pulse you can actually map out these reaction schemes in quite a great detail and that's this is what what basically the idea behind this experiment is so you excite the molecules what you can do is first as I said you can demote them back to the ground state and the experiment is then called instead of pump probe it becomes pump dump probes because you dump the molecules down back to the ground state you can produce another excited state so you can re excite them that's called pump repump probe and what you can also do is you can prepare the system in the in the excited state and do an expert another pump probe experiment on the molecules with some of them already in the excited state and this is the way to address how different excitations interact in your sample that's called pre pump pump probe well anyway let's focus on these two because this is what we want to look at and again this is our friend the green fluorescent protein I told you it has it does proton transfer in the excited state and this was known before we started this is the pump probe that again showing the kinetic isotope effect on the growth of their mission so if you use hydrogen it grows in with 20 picosecond lifetime if you use deuterium which is two times as heavy it takes much longer to produce the emitting state if you look at the pump probe spectra this is the way they look you produce a red spectrum here and then progressively as you watch it over time your mission in the green is developing so this band is going down this one is going up this is not changing and then at long times of course it decays back down to the ground state now an interesting thing is of course that it does excited state in the pro does excited state proton transfer but of course to return to its ground state it has to do the same in the ground state so it emits a photon when it's still in the excited state and then you can either wait for it to return to emit the photon and do a ground state proton transfer but we what you can also do you can dump it back to the ground state and that is the way to prepare a ground state with a separated proton with the proton in non-equilibrium place and this is the way to measure of the rate of the ground state proton transfer which is a fundamental biochemical reaction probably more than half of biochemistry is NADs turning into NADPHs and vice versa and so proton transfer is very important and how fast is it that's a question and so this experiment is actually will actually allow to shuttle the proton in the excited state it goes away from the pigment and when you dump it back to the ground state it should go back and this is what we wanted to observe so that's what we did in 2004 basically we waited for the proton to be separated so for the green fluorescence to develop and we applied the green dump pulse at 20 seconds after after the excitation and what we saw is that what the emission was severely depleted so that we lost the emission that's good means we removed the excited state put the system back in the ground state and what happened then if you if we looked at the wavelengths right next to the to the blue from this emission we saw this huge absorption appearing so you at 510 nanometers if the emission disappears but to the blue from it right next to where the deprotonated state should absorb we see this huge increase in absorption and that decays with about 400 picosecond life and not only that if you do this in the H2O you see that this state that you have produced this is now nanosecond time scale so basically I this is a logarithmic scale this is a linear scale this is the decay of the this is the return of the proton on its mother molecule now and that takes about 400 picosecond if you the replay and to show that it's really the proton transfer you replace it with deuterium and it's tremendously slow it's really we within 5 nanosecond it's barely happening so if anyone asks you how fast is the proton transfer in the ground state we now have the answer and at least in this system it's it can be as fast as 400 picosecond of course we did the global analysis of the data using the methods that I just described and we got so the dotted lines and the solid lines here which are almost indistinguishable are the spectra that we got for for deuterium and hydrogen samples so the spectra are really the same it's chemically the same species that we are working on but the lifetime is really really different so instead of 400 picoseconds for the ground proton ground state proton transfer you have 5 nanoseconds in the in the deuterated sample okay another application of this pump dump probe spectroscopy is is playing this ping-pong game with the excited states of carotenoids again and this time it's it's a carotenoid called pyridinin and it's found in the marine algae this is a light harvesting complex of an alga and it contains a lot of carotenoid molecules and only two chlorophylls per monomer it relies on carotenoids to do its light harvesting now this is a strange carotenoid because it has all these carbonyl groups and that as was shown by my friend Donata Zigmantas in Lund that results in a state that in polar solvents in near-infrared emits light so this carotenoids contrary to other carotenoids it has their mission band in the protein and also in the in the near in the solution in the near-infrared so around 900 nanometers 950 nanometers so what we did first we tried to dump this state and the problem by the way with this since it's very sensitive to the solvent we people thought that this is a charged transfer state so it changed a state with a very large change in dipole moment but the problem is that they could not distinguish it from the standard as one state of of paradigm they were decaying as one entity both the emission and as one excited state absorption and there was a big discussion what's the role of the state in the light harvesting and are these states really separate or is it as one fluorescence that we are seeing and what is the deal with it so what we did with basically we tried to dump the emission in the near-infrared and what we saw that after the emission signatures decreased they after a while they got replenished so that sort of signifies that these two states are different and if you take the population away from this one you shift the equilibrium and basically you get it transferred back to the size CT state so that we did that with pump dump probe in 2006 and we wanted to see what it is like in in PCP and that's a very recent result is actually was only accepted several weeks ago so if you look at the pump probe spectra of this light harvesting complex you again see the carotenoid signatures and the chlorophyll signatures this is the energy transfer so we first make a lot of carotenoid and then you see this band developing here which is a chlorophyll bleach band where you see the chlorophylls receiving excitations from the carotenoid at the same time in the near-infrared you see this emission band developing together with this band so you can't really distinguish it this is the S2 signature this is by the way a logarithmic scale so it's very fast within a few hundred femtoseconds you have this emission appearing so what we did in this protein we tried to remove the population from this emissive state again and what we saw was actually that when you arrive with your pulse resonant the stimulated emission here you do lose a number of excitations here so the signal gets decreased but this signal which is a normal pump probe signal which is unaffected this actually the dumped signal is sort of has a bit of a delay doesn't want to really decay quite as fast as this one and if you use a higher power for depletion then you can actually see the the emission signal so here you basically deplete it to zero and your emission is actually coming back so this is the same mechanism as in the pigment this is the S1 state so this state supplying the excitations back to this one and they are interconnected by the equilibrium and so okay well skip the details but the idea is that again you have the equilibrium between these two states and it's on the the equilibration time is sub picosecond or maybe one picosecond and so these these two states when you remove the excitations from here you sort of get the influx again from S1 and in PCP of course it's a bit more difficult because everything is still at the same time transferring the energy to the chlorophylls so it's a bit harder to disentangle but we could do that and we saw what we saw actually that the main energy transfer channel from the carotenoid to chlorophyll is not as one state but the ICT state and it is even if you put this rate constant to zero or the time to infinity you still can describe the data relatively well now so and you can do the global analysis and you can compare a spectra I'll skip the details and because we want to talk about different techniques and the other twist on the pump rope spectroscopy is that the approach which is meant to unravel structural dynamics now the electronics spectroscopy is that we looked at are all very nice they give you very nice kinetic information so the time resolution and a signal to noise is very good and you can so have the behavior the time dependence is very nicely the problem is you don't have a clue what your spectra stand for so you need either modeling or different experiments to give you this clue alternatively you could use a structure sensitive type of spectroscopy which of course is vibrational spectroscopy so you can do either mid-infrared probe or Raman spectroscopy I will talk about Raman and it was introduced yesterday so if you look at what essentially is Raman spectroscopy is is the scattering process in elastic scattering by the molecule where a photon comes in with a frequency new and it basically drives the molecule up and down and it's the molecule ends up in the excited vibrational state and you lose exactly one vibrational quantum in your Raman scattering process so you basically shift the frequency of your scattered line now it's a problem if implemented in an ordinary fashion is that vibrational lines are very narrow vibrational lines are narrow so typically of the order of 10 reverse centimeters and that is the spectral width of the laser pulse that you are allowed to use if you want to measure a Raman process and 10 reverse centimeters is corresponds only to a couple of picoseconds so you cannot address very fast processes using that now the way around this is femtosecond stimulated Raman spectroscopy developed by Jesus I forgot the name it will come back in a second rich Matthews in Berkeley in California and what the way it is done it's like a pump probe experiment but then your probe is a pair of two pulses so one of them is long and narrow spectrum and the other one is very short and broad spectrum and so what what this pair of pulses does is basically driving your molecule which you excited with your pump pulse up and down and producing a vibrational coherence and now since you have the signal only where these two pulses overlap so it's a fifth order process essentially so you get your spectral resolution from a narrow band pulse in the spectrum and your time resolution from your narrow duration pulse in the time so you need two pulses superimposed on each other and you can measure Raman spectra with femtosecond time resolution by using this as your probe light so what you need to do in your three pulse experiment you need to basically stretch this pulse in duration and narrow down the spectrum that's what you have to do and then you can get structural dynamics of your molecules with femtosecond time resolution now this is very complicated but the idea of the setup is that you do have a long pulse and then short probe pulse and the pump pulse so that's let's forget about the technical details of the setup and this is the seminal paper of Matthew's group by Phil Kokura in science in 2005 where they looked at the isomerization of rodopsin so again this is the retinal protein that sits in the retina and the isomerization of this thing is sub one picosecond so you cannot follow it using normal Raman spectroscopy and they first published the vibrational features so if you look at this band here these are vibrational fingerprints of isomerization and a shift of a band here as well and of course these spectra are wonderfully detailed and they provide really nice information information what's happening to the structure of the molecule when it's doing its business on the excited state potential so another applications that we did was we wanted to look at the certain class of photoactive molecules that become colored when you put the UV light on them and then revert back to transparent when you put them in the dark so when we entered the field come on batteries down okay so the photo again for photochromic molecules they have this by stable ground state where you have one ground state minimum and the other one and this one will absorb in the visible and this one will absorb in the UV in our case okay so when we enter the field these are these are endolobenzoxazine these are the molecules that were advertised by the group in Florida as very fast photo switching molecules so basically they they have the forward switching is somewhere in the femtosecond regime the backward switching was 25 nanoseconds which is very fast for a conformational dynamics typically you have lifetimes you know of milliseconds microseconds this is 25 nanoseconds so that's quite nice now the funny thing was that these molecules were synthesized by a group in Lithuania very long time ago that basically made them describe them in some journal himia get there it's a glitches kicks in any in Russian back in God knows when actually 1989 and like 20 years later these papers starting started appearing that these these are a new and promising and very nice molecule photo switching and so on and basically what they said that okay if you can open this molecule and make it so what happens when it absorbs light it breaks the CO bond here and it produces this chromophora group this thing absorbs in the in the blue so it's basically makes the solvent look yellow it's a para-nitrophenolate and what they said okay if you add the base to it the tetra butyl ammonium hydroxide you get this spectrum which you can measure and if you put light on this you get the spectrum that is depicted in the dots here which you could also measure what they said with Italian flamboyancy was that this looks the same I mean this is probably the same so we said okay so they are opening optically and also chemically and so the closing process that they measured was of the order of 20 nanoseconds and so these were new photo switches what we the guys in Kona they said okay is are they these molecules really so nice and could we do more research on them if so they said okay we could do spectroscopy it's nice to have something photoactive doing their business we actually spent probably seven or eight years not understanding what's going on and mostly because of because we believed that this picture of photoisomerization was true now the first clue that may not be true came from oxygen dependence experiments and if you look at the lifetime of these molecules in nanoseconds you take the solution you measure the decay here and then you measure it after bubbling the solution for 15 minutes with argon and you see the blue curve and you would say well it's probably just the systematic error you have to take the sample out bring it somewhere measure it and so you bubble it with air again and you measure the red curve which is exactly the same as the black one different molecules the effect is also there it turns out that it probably is the effect of oxygen on this supposedly ring open state that is supposed to be appearing on photo excitation and oxygen as I already told you interacts very strongly with triplet states so maybe it's a triplet state we said in a paper that we published a couple of years ago but we didn't know so again when you do with these experiments more carefully saying that so this is the red is the spectrum of chemically open form and the blue dots are the ones of optically open form now you have to be very brave to say that this is the same I mean if the data quality is like this then maybe you can ignore this couple of points but but here it's not possible anymore if you change the molecule a bit you even get a band here which is completely absent in the chemically open form so we said probably it's not opening this thing when you put light on it to check that we did the femtosecond Raman experiments on it and what we saw is that most of the action was going on around 1600 nanometers and it matched quite well with the optical femtosecond resolved absorption so you can see this going up and down in the kinetics both in the Raman scattering kinetic traces and also in the visible now but if you it turns out if you compare these Raman spectra that you see at different delay times with the Raman spectra of chemically open form there's nothing in common so really everything is completely quiet here whereas here you have this forest of bands appearing and when you chemically open it if it's the same process as the one that optically induced that is optically induced you should be able to see the same bands appearing here and you don't so we have concluded that these things are alas not photochromic and yeah this 15 or 20 papers that the group in Florida published we think we're wrong but well somebody made a career out of it so that's okay I guess and that brings me to the third twist that is coherent spectroscopies in the last half an hour I want to discuss those now we already saw that what the nice additional information can be gained from these molecules and semiconductors we'll mention them in a brief while by you by cleverly manipulating the light pulses that you put on your samples so basically the best implementation of it are coherence spectroscopies and they start with a modest and very bread and butter technique that is used in semiconductor physics it's called light light induced transient grating so what you do is you do a pump probe experiment only your power is our two beams crossed at an angle in your sample what happens then is they produce an interference pattern in the sample so sort of a grating that is inscribed on the sample because where the intensity is high the sample will absorb more where the intensity is low the sample will not absorb so basically I'm making a grating on your sample and you modulate the density of the charge carriers in the semiconductor in this case by the same law as as you get from intersecting two beams at an angle so yesterday we saw a super resolution coming out of it today we have basically an information about carrier drift and diffusion in semiconductor so the carrier recombination will do a upon carrier recombination the grating will sort of gradually disappear in amplitude so it will reduce amplitude of the grating now and the carrier diffusion will also reduce the amplitude of the grating but in a different sort of way so basically this is a very nice way to characterize your semiconductor materials without having to put electrical contacts on which is an additional step and when you're growing materials it's not always easy to do that because if you do it at one angle you have a grating with large spacing if you do it at another angle you have a grating with with fine spacing a fine spacing grating will disappear faster due to the diffusion because the carriers don't have to diffuse quite so much but the lifetime of recombination will not be changed so by doing this experiment with several different angles and taking the angle dependence of the signals of the transient gratings different grating spacings you can distinguish between the diffusion and the recombination lifetimes and so this is what a group of my colleagues is doing in Vilnius they work on the growing semiconductor material and characterizing it using this technique this is a fast and very nice contact less technique come on all right so and basically if you do the temperature dependence and okay I will not go into the detail on how you model the data but basically it's a continuity equation which you want to fit the data to and you can determine the non-radiative lifetime the band-to-band recombination rate and the Auger recombination rate and so basically you characterize the behavior of the electrons and holes in your in your semiconductor in a very nice fashion so that's a transient grating it's a bread-and-butter technique and of course you know physicists would not be physicists if they didn't try to improve on it so the first attempt to improve it was saying okay what happens if we separate the two pump pulses in time so if you do that you not only get the interference in space but you also get the beatings in the frequencies so if you take two pulses to light pulses and you spread them away in time the spectrum the Fourier transform of these pulses will become a modulated a beating will it's very similar to to you know to several frequencies detected by your radio when the selectivity is not very high and if you separate them further away the spectrum will be modulated with the fringes in the spectrum will be more dense so you have more fringes and so basically the direct implementation of this is called a photon echo experiment and what you do is you have two pulses and which are shifted in time with respect to each other and you have a third pulse after a longer time that arrives and gets scattered on the great thing both in space and in time so the spatial grating gives you the phase matching direction so your signal is coming out in the direction that is basically some of the two wave vectors minus one of the other ones and so you detecting your signal here and your signal is proportional to the both the number of fringes in the grating and the let's say the time that you have waited because molecules and solution they they are not sitting still they are getting pushed by the environment all the time and they forget their original frequencies there are let's say excited state energies if you want to and that leads to the washing out of the frequency grating and the disappearance of this phase match signal so basically what you do you have three delay lines now three delay lines imagine that and you basically put three beams on the sample and you detect in the phase match directions with a couple of diodes and so the idea of the experiment is if you have a very fine grating so not a very large time shift between a very large time shift between the two pulses the grating has many many fringes then if you are if the frequencies of your molecules in the sample are changing it will be destroyed very quickly yeah because it's a very fine grating and it in the frequency domain it will disappear very very quickly so that basically you will forget about about the original frequency if you put the pulses closer together the modulation will be wider spaced so it will disappear more slowly due to the same process of spectral diffusion and that allows you to tell how fast the frequency of the molecule the transition frequency is being modulated as a as a function of time because it's seeing its environment now of course this coarse grating will not scatter as well as a fine grating yeah you know that for a good optical grating you need many many lines per millimeter five lines per millimeter do not produce a nice a nice interference so need many lines per millimeter to have a good signal but on the other hand if you have too many they get washed washed out too quickly so that basically is a is a compromise that the maximum signal is when these two pulses are shifted away in time only a little bit and this is this echo signal the non-linear polarization that comes out of your sample and this is basically what you are monitoring in the photo echo experiments so this is actually the picture that I took with one of the first digital cameras back in 2000 when I started my career you can see from the quality that you know this is something that costs one a half thousand euros back then and you have three beams that are producing the interference and here you have the three other beams that are the diffracted beams from this frequency grating so these are the photon echo signals of the molecules and what you are effectively measuring is this shift when you have the maximum signal so that this the separation between between the two pump pulses when you have the maximum signal as a function of the waiting time for the third pulse to arrive and this effectively maps out the memory of the system so how well it remembers the face of the electric field that excited the molecules in the first place and now while this is a nice technique you do get some problems with it and I drew pictures of some confused people here from the other side of the ocean so basically what you end up with is just one curve so this memory loss of the system and the experiment is time consuming you have to do it you have to analyze the data in just one curve my professor was telling me you need to produce contours to be successful in science that's one reason why this got abandoned more or less at the start at the at the let's say 2000 early 2000s 2005 or so in favor of more sophisticated technique and the data interpretation requires a microscopic model of system bath interaction which is very very difficult in terms of calculation so it's it's it's a big model so and the data is difficult difficult to interpret but people are inventive and the lasers improving and the detection systems improving have led to the idea okay well if we what we do is we don't measure the integrated intensity of this echo signal what we do it we do that we want to resolve the electric field the time dependence of the electric field radiated by the molecule as a as a function of of the separation between these two pulses that produce the frequency grating and awaiting time of the other pulse and so this gave birth to the 2d electronic spectroscopies that are all the rage nowadays and really a booming field I would say and I will spend some time talking about it now so the first paper was describing the technique that actually worked was back in 2004 by Tobias Bricksner and from Graham Fleming's group and in Berkeley they worked for a long time to get this going with they did photo-neco experiments for a decade before that and in the end they got it right so the way you do it is you need these three pulses and another pulse to have to make interference with but because what you want is you want to interfere to interfere your signal with a laser pulse to be able to deduce the spectrum of the signal that is emitted by your sample and you do the Fourier transform with respect to this delay tau and you do the measurement as a function of this waiting time for the third pulse to arrive which is called capital T now the setup of it is is quite complicated you use the fractive optics so that a grating on which you put two beams to produce four beams in total for identical beams replicas of four beams the time delays are realized can be done in the separate several ways the original ways by shifting two wedges of quartz inside the beam so basically you take the amount of glass inside the beam and if you push in one wedge a little bit further in you have more glass and glass delays the lights more than air does so you can produce really sub wavelengths delays by by having this this sort of optical gear as they call it so you push the glass wedge by a micron and you delay your light by lambda over 20 or lambda over 50 or something like that and so you intersect the four beams in the sample and you detect the field that's coming out of the sample and you do a measurement by mixing it spectrally with a local oscillator field to have an interference with a spectrometer and do it as a function of the delay between these the first two pulses and that delay has to be controlled with interferometric precision which is what they did so you can calibrate the delay as a function of wedge position so you push the wedge for I don't know for 200 microns you see that you only pass several wavelengths and that gives you a very good idea so lambda over 50 type of idea what delay you actually have between the two pulses now the advantages of this spectroscopy are numerous so first of all you do a Fourier transform with respect with a waiting time with the two pulses that you have delayed with the wedges so that's one frequency and then you do you have a spectrum recorded by a spectrometer of the emitted lights again as a second frequency so you have two frequency axis and this is why it's called 2d electronic spectroscopy and so you can think about it as a pump probe experiment that is being done using all the excitation wavelengths at once normally you just have in pump probe you have a laser pulse at the center that is specific wavelength and now you just have a very broad band pulse that is that's that is covering all the system so it's like 2d and mr or if you want an analogy you take you want to determine the resonant frequencies of a bell what you do is you take a hammer and you bang on the bell and you record the how it's ringing and you do do the Fourier transform of that of that ringing and you get the spectral light lines on the bell and so basically it's like many pump probe experiments in one go and in fact it has the advantage that the time resolution is not limited by the pump pulse so because your the pulse that is emitted by the sample is not directly Heisenberg connected to the excitation pulse there is no limitation as it is in pump probe that long pump spectrum or very precise pump wavelengths means bad time resolution with respect to the excitation way so it doesn't have this drawback so this is a nice spectroscopy and come on yeah the problem with it that is that it produces a lot of data and again a microscopic relaxation model with a lot of equations is needed to interpret while pump probe you can sort of interpret in ping-pong sort of way as I showed you so they take molecules put them here they evolve you put them here and that's all clear now here it's all coherently coupled so you have to do the calculation involving the electric field itself calculate the nonlinear nonlinear polarization and then look at the terms and find one diagrams that actually correspond to your signals at specific wavelength and it does gives jobs to a lot of theoreticians which is nice but the problem with it for an experimentalist is that you look at your data and you have no idea what's going on so but yeah and the application that I want to show again from Fleming's group in Berkeley is the one that produced a minor scandal in the field so this was again a light harvesting complex called FMO and basically back in 2007 I think was it yeah in 2007 a student came to me and I was teaching a class and he came to me during the break and he said did you hear about this coherent energy transfer of coherent photosynthesis something he had read in a news release and on a technology website and the paper was titled evidence for wave-like energy transfer through quantum coherence and photosynthetic systems now this is you know a very strong statement because what it says it says that it actually people could observe the role of electronic coherence which normally lives the spectral diffusion from all we know from earlier experiments is on the time scale of about 50 femtoseconds maybe even 10 femtoseconds part of it so electronic phase memory is lost in the in the in the molecular systems on a time scale of 10 femtosecond there's no way that the energy transfer process which takes you know pico seconds and tens of pico seconds can be governed by this short memory phenomenon at least from a you know really common sense point of view and they claimed of course the opposite this is how they got into nature and so basically this is a bag of chlorophylls again this is a tightly coupled so you have a nice separated absorption bands in the electronic absorption spectrum and they did these two-dimensional electronic spectroscopy on this and I told you in order to be successful in science you have to produce as many contour graphs as possible so this is what they showed so these are the frequency axis and these are the signals to think of it as an excitation wavelength and probe wavelength so what what is nice about the spectroscopy is that you can see correlations so you excite here and that one starts talking to you you excite here and the other one says so you can actually see the electronic couplings between the pigments if they are connected in this way and what they saw was actually this off diagonal peak so really two talking two talking electronic transitions that are talking to each other they saw it oscillate yeah now oscillations this is actually the signals that they looked at so in order to produce more colors they have colored them by you know a non-linear color maps which is nice because there is a lot of detail in the spectra what I didn't like about this publication the first time I saw it was this yeah so when I was I think in my first year as a student I had to measure in the practicum I had to measure the resonance acoustic resonance in the steel rod by you know making a resonance curve and I did three points in frequency and I drew a resonance curve through them and I went to my professor or associate professor showed it to him so there's a nice resonance and he said you know I could draw a circle through these three three points that you have met go and measure 15 time point now turns out that if you are from Berkeley you can interpolate your oscillations like having a point here and there and maybe there and you know your oscillations was time instead of decaying they get stronger but that's all okay if you have a nice model to go with it so they said okay we see the oscillations I'm pretty sure they actually saw the oscillation but the quality of the data that they are presenting here is really appalling and they should have never meant made it to nature before being re-measure now and this so what they said is that the quantum coherences so really entanglement between electronic states are playing a large role in energy transfer and photosynthesis and probably were optimized by nature to be you know such and such so nature was solving Schrodinger equations in evolutionary fashion to optimize the energy transfer now that of course is garbage and I think most of the people in the field they know it because if you ask them about this they they are quiet politely because they want their grant money flowing in but they are never really except from the first offer from this paper who made a career out of it of course they're never really addressing it in such a blatant fashion anymore but if this gave gave actually arrives to a new field called quantum biology I hope it subsides soon now speaking of oscillations you remember the oscillations I showed you with a wave packet packet moving around all you need for an oscillation to be in your pump probe spectrum is one vibrational degree of freedom coupled to the electronic transition that turns out that I did this experiment which I never published in 2000 again on this light harvesting complex I saw the oscillations there if you do it with enough time resolution at 77 Kelvin which is what they did with FMO you do see the oscillations appearing nicely there and if you look at it this is your wave packet moving and it's all clear and probably more than one one vibrational mode here there's relaxation again they they stay around for several picoseconds nothing surprising in there I the reason I didn't publish it because it was published already in 1996 now but that of course doesn't stop people from seeing the same oscillations which are probably of vibrational origin at the best they're probably of mixed vibrational and electronic origin in these two-dimensional spectra and and publishing publishing them in nature in 2007 and giving rise to a new field but and don't get me wrong I don't want to say that the technique has to is to blame for this I just think that it was misapplied in this case and over sold severely but I think the two-dimensional spectroscopy will sort of phase out maybe or maybe not the pump probe experiments for very complicated systems where where you really need to know more details where the coherence is mixed electronic vibrational playing a role I think there's a big let's say application field for it waiting in in nanostructures like nanotubes and things like that so the technique is not a place a very nice technique I like it but I think it was misapplied in this time and for reason now instead of conclusions and I think I saved some time I would like to say that the time results spectroscopies are time savers and they are instrumental in understanding what's going on dynamically with the molecules solid states and nanostructures and they come in different guises from modest single photon timing which you can attach to your microscope and map out the surfaces of cells or wafers and semiconductors to all the way to 2D and electronic spectroscopies which require extremely stable lasers the experiment that you saw presented is probably run for 10 hours maybe at low temperatures lots of work required and but it gives you new insights new information especially by playing with the material and playing with your laser light that you shine on the material you can get new sort of way fronts in science and I think we all deserve a break after these intense two weeks and I would like to thank you for patiently listening to me to yap on for four hours thank you