 I thought it would be good to start with a general overview of scattering because my understanding is that the students, they don't have that much experience. So even if some other teachers have already, you know, talked about the basics, I think it's good to remind them. And then I will talk mostly, I will give some examples of kyber hybrid gels. So I don't know about that. Okay, so I think if Anne is going to listen, if you mean this, we rather get started now. So it's my great pleasure to introduce Marta Martinez-Sanz, that I met the first time I think in Sydney. Nice, but Elliot Gilbert. So Marta comes from Spain, but he's almost half Australian, I think, because you spend a lot of time in Australia doing a lot of nice work on carbohydrates or using neutron scattering and other techniques as well. So without further ado, please go ahead Marta. Thank you, Tommy. So it is my pleasure to be here in this course participating. I want to congratulate all the students who have been chosen because I think this is a great opportunity to learn about scattering techniques. Not so many scientists can say they are experts on these techniques. So you are very lucky to be here and please take the opportunity to ask as many as many questions as you need. If you want to come right in the chat or if you want you can wait until we finish and then we will have discussions. So I will tell you a little bit about how we can apply scattering techniques to the investigation of the structure of carbohydrates. And as I was explaining, I would like to start with a brief introduction of the basics of scattering techniques. I think most of you haven't got so much experience, so I think it's good to, you know, talk a little bit about what are the basics of these techniques. Then I will go on talking about what types of structures we can find in most of the carbohydrates. And then the application of scattering techniques in the particular case of carbohydrates. And finally I have several practical examples, but I don't know if we will have time to talk about all of them. But I think it's best if we leave some time for questions so we will see as we go. So first of all, as you know, scattering techniques are based on the interaction between a source of radiation which can be light x-rays or neutrons and the particles in our sample. So what we do in scattering techniques is that we analyze the scattered radiation and so that it can give us information on particle size, shape, orientation, interactions between particles, etc. So here you can see a very basic scheme of what scattering techniques are. And as you may have heard along these past days, the scattering angle is related to the scattering vector which we typically use in scattering experiments. And at the same time, this parameter is related to real space dimensions. So what this means is that depending on the type of structure or their size range that we want to study in our sample, we can also find different scattering techniques. So for instance, if we have our detector position closer to the sample, we will be able to register higher angles and that means we are working with wide-angle scattering techniques. And these techniques are going to reveal structures ranging from 0.1 to 1 nanometer. That means, for instance, in the case of carbohydrates, we can study crystalline structures. Then if we move the detector farther away from the sample, we will be able to register lower angles. So we are talking about small angle scattering techniques. And in that case, we can study size ranges from 1 to several hundreds of nanometers. So again, in the case of carbohydrates, that means that we will be able to study the structure of polymeric chains, so the size, conformation, interactions. And finally, we have also ultra-small angle scattering techniques. They are based on a different arrangement in terms of instruments. But we can also study size ranges from several hundreds of nanometers up to 10 microns. And that's a real advantage in the case of carbohydrates because sometimes we have larger structures. For instance, in the case of gels, if we want to study the mesh size of some polysaccharides, we will need to use ultra-small angle scattering techniques. And then, as you also know, we have two main sources of radiations, which are x-rays and neutrons. And this is very important. I'm talking all the time about carbohydrates, but this can also be applied to other types of materials. But in the case of carbohydrates, this is especially important because x-rays are going to be able to highlight differences in crystalline and amorphous domains because they are sensitive to variations in electron density. So for instance, you will see later on that in the case of cellulose, x-rays are going to generate contrast between crystalline cellulose and amorphous cellulose. While on the case of neutrons, since they are scattered by the atomic nuclei in the atoms, they depend on the nuclear structure. So that means we can play around by substituting hydrogen atoms in our sample with Euterium, and in that way we can generate contrast. So that's a very powerful tool we will also see later on, contrast variation. And in the case of carbohydrates, since we have many labile hydroxyl groups, this is an advantage. So we need to know that the source of radiation that we are selecting for our experiments is very important and we have to select it on the basis of the sample composition and the structural features that we want to investigate. In this context, the scattering density is a very important parameter, we will talk a lot about that because that's something you need to take into account before you plan your experiments and also after you analyze your data to know what is the contrast that's being generated between the components in your sample and the solvents that you are using. So here you can see graphically an example of how you see neutrons or x-rays will highlight different components in our sample. In the case of neutrons we can see the hydrogen from the plastic components in a camera while in the case of x-rays we will see more clearly or we will have more contrast in the metallic components. So why are the scattering techniques so important for us that we want to investigate the structure of materials or biological samples? We know that microscopy techniques are typically used to investigate the structure of many types of materials. I know in biology because I think many of you have experienced or background in biology. The scattering techniques are widely used but they have several issues. First of all, we are limited to a very small region of the sample so we never know if that's representative of the whole sample. Secondly, in most of the times we need to make a sample preparation meaning that we probably will have to dry the sample and this is a big problem, especially when working with carbohydrates as we will see later on. And on the other hand we have scattering techniques which evaluate the change in the direction of a source of radiation as it interacts with the material that we are analyzing. So we will get a volume average of the sample that we are analyzing that means we can have a more representative results from the structure in our sample. The complex part is how to analyze the data but if we are able to analyze our data properly we can extract a lot of structural information which is going to be representative. So very briefly, the advantages of the scattering techniques is that they are non-destructive as I said they give us an idea of the bulk properties. We need minimal sample preparation and then in the case of carbohydrates we also have the possibility or the advantage, well not in the case of carbohydrates but it means that in the case of carbohydrates it's a great advantage that we can use the contrast variation technique or selective deterioration of the samples. And then these advantages mainly is that many people do not know how to interpret the scattering data so that we need to understand very well what we are doing, plan our experiments very well and then be able to extract as much information as possible. And then I would also like to highlight that it's always advisable to combine scattering methods with other techniques so that we can know for instance when we are doing our data fitting, we need to know if the results that we are obtaining are representative of our samples so that it is always advisable to complement with other methods, it could be X-ray diffraction, microscopy, spectroscopy, whatever you need to gather some information on the structure of your samples. So let's start with carbohydrates, I don't know if any of you is particularly working with carbohydrates in their projects but I will start with the basics. So carbohydrates are biomolecules which consists of carbon, hydrogen and oxygen and as you probably know we have the basic unit which are the sugars, monosaccharides and then we have also these saccharides which are the combination of two monosaccharides. And then these monosaccharides could also combine to form very large chains which are the polysaccharides so these are the complex chains that we know as polysaccharides, some examples that I'm sure you have heard of is cellulose and then you also have the structure of amylose which is one of the two components of starch. And why are carbohydrates so important? Well we can find them as major components in fruits and vegetables where they account for more than 90% of the dry content and also in the food industry they are very, very important because they are used as additives due to their several functionalities, they can be used as sickeners, yelling agents, also to encapsulate bioactive components or even additives for low calorie products. And those carbohydrates can have different origins, we can find them in plants and some examples would be cellulose, hemicellulosis, starches, pectins. We can also find carbohydrates in seaweeds and these carbohydrates are nowadays very important for the industry because mainly for food industry they are being used, there is a lot of interest in them because they have jelly properties which are very good for some food products. So in that case you probably have heard of agar but we have also alginates, carcinans, fuccoidans, etc. And finally carbohydrates can also be produced by several microorganisms and some examples would be shantans, I'm sure probably you have heard of shantan gum because it's also used in the food industry. Pululans, gelans, carcinans, several types of gums and here I would also like to mention bacterial cellulose because it's possible for some microorganisms also to synthesize cellulose. So most of these carbohydrates, we find them in nature as hydrated systems, they are always or almost always associated with water. So as we were talking previously, drying the samples for any type of analysis is going to be problematic because we are not going to get information on the native structure of the carbohydrate. So that's something that you always need to keep in mind. And then the other thing is that while some of the carbohydrates might present crystalline structure, so they are impermeable or insoluble in water, and that might be the case of cellulose. Some other carbohydrates have an amorphous structure, and therefore they are soluble in water, and that may be the case for instance of phycocoloids. So agars, alginates, they are soluble in water, we can disperse them and then they form gels when they are subjected to a specific temperature conditions. So that's one particularity of some carbohydrates that they can form hydrogel structures and that's one of the examples that we will see at the end, because they are very interesting for several industries. And here I'm showing you the some examples of the hierarchical structure of two major polysaccharides, the first one is starch, so that you have an idea of how complex the structure of one carbohydrate can be. And as you see here in this scheme, we can go from the atomic level on how the starch polysaccharide chains are arranged, forming crystalline structures. And for that we can use wide angle scattering techniques, we can also use x-ray diffraction. And then these crystalline structures are arranged in a very specific way, which is known as lamella. So they are ordered, forming these structures, which are typically around nine nanometers, and they are organized in such a way that they are combined also with amorphous domains. So this size range can be typically covered using small angle scattering techniques, such as SACs. And then we would move to the next structural level in which these lamella are combined with the amorphous domains, forming the growth rings in starch. And this structural level, if we want to study, we would have to go to ultra small angle scattering techniques, but we can also of course combine them with microscopy and spectroscopy methods. And then we also have major polysaccharides such as cellulose, which you can find in many vegetable products, in grains, in fruits. Depending on the source cellulose will have a different structure and it will also be combined with other components so that the presence of these components will at the same time affect the structure of cellulose. But here we have the basic structure. And again, we can cover different size ranges and we need to keep in mind when we are doing scattering experiments that we are covering a specific size range, but we have to take into account that the other structural levels are also having an effect. So in the case of cellulose, we also have the crystalline arrangement of the cellulose molecules to form the crystallites. This is typically studied by wax or x-ray diffraction. And then these crystallites are arranged, forming what is known as cellulose microfibers. And these microfibers have a very specific structure. Depending on the source, they can have a different cross-section, which is more similar to a parallel pipet or it can be modeled, as you will see, as cylinders. And the dimensions are different depending on the source. And then these microfibers are interacting with each other, with water, and with other components to form the cellulose bundles. And this is going to be completely different depending on the source that we are analyzing. And finally, in the case of plants, we have the primary cell walls and secondary cell walls, which are the structures in which cellulose is combined with other polysaccharides, proteins, polyphenols, etc. So another important aspect is to take into account that the carbohydrates can form different types of structures. Here I'm showing only some examples, so that you have an idea. As I was telling you before, some carbohydrates can form hydrogels. And these hydrogels are formed when we need to have first a solution in which the carbohydrates chains are randomly distributed, so they are typically salt in water. And then if we hit these solutions, it happens sometimes that these carbohydrate chains, they can arrange forming double helices structures, and this is the first step in the gelation process, so that we have this order structure of double helices. And these double helices, if we keep, if we then call the sample, they can interact with each other to form this type of bundles in which they are interacting with each other, typically by hydrogen bonding. And they form a very, well, a more or less strong structure, depending on the polysaccharide, so that it has physical integrity, so something like gelatin, for instance, I think. So that's one type of structure that we can find in carbohydrates, but we have also some other things that we can find, for instance. If we are working with polysaccharide, some proteins, so the mixtures of both, they can form interprenetrating networks, and this could be of physical nature or more chemical interactions, but the idea is that we have some kind of winning of the chains of our polysaccharide and the other molecule, it could be polysaccharides with different surface charts, or it could be that we are working with one polysaccharide, for instance, anionic polysaccharide and then a protein with positive changes, so that they can interact with each other. And finally, I would also like to mention that we can also find polysaccharides in emulsions. So, although typically to stabilize oil droplets, we find proteins, we have been working lately on the development of emulsions, which can be stabilized by either the combination of proteins and polysaccharides, or just polysaccharides, which are able to gel and then form domains at the surface of the oil droplets. So, this was just to give you some examples of the structures that you can find, but in all these cases, you will see that scattering methods are very, very powerful to give us an understanding of the structure that we have. So, as I was telling you before, in all the types of materials where we find carbohydrates, it could be food, could be biological systems, solutions, emulsions for food industry. We also have water, and water is going to be intimately interacting with the carbohydrates, and that's something we need to keep in mind always for preparing of our experiments. Because drying affects strongly the native structure of carbohydrates, and this is something we know for sure, for instance in the case of cellulose, we know that once it has been dried, we cannot go back to the initial state because the hydrogen bonding that is generated when drying cannot be disrupted if we want to rehydrate the material. So that's something very important. In that sense, scattering techniques are very powerful because we can study the structure of carbohydrates in their native state, we don't need to dry the samples, as you will see. So, what are the phases when we are planning or we know that we are going to perform scattering experiments. First of all, I would advise that you carry out some preliminary characterization, but by using other methods. We need to have some idea of the structure of our samples. And in the case of carbohydrates, it would be very good if we have some characterization by microscopy. I know I said that microscopy is not the best method to study the structure, but it can be helpful to complement our scattering experiments, you will see later on with the example of cellulose. It is also very important to know what is the chemical structure of our sample and for that we can typically use FTIR or NMR in the case of carbohydrates. And it would also be good to have some idea of the particle size because that is also going to determine what type of technique we want to use for our experiments. And so, once we have some information of our sample, let's say that we start our scattering experiments. So, important things that we need to take into account is that sample preparation is going to be very important for a successful experiment. In the case of carbohydrates, it is critical to avoid sample drying. I know I have said it several times, but I don't want you to forget about that because it's very important. And so, we need to use suitable sealed sample holders. Here you have an image showing some of the sample holders that could be typically used for a sun's experiment. So you see in these cells, the first one starting from the left, that would be good when we have more liquid like samples. And then the second one we used a lot this type of cell for gels. We will see later on that in some cases it's good to soak the gels in different mixtures of water and D2O. So in this case it is possible to fill the cell with a hydrogel with a small piece of the hydrogel and then add the solvent. So that is soaked in this solvent. We also need to determine the range of concentrations, which is going to be good for our experiments. We want to avoid having a lot of noise in our signal, but we don't want to saturate the signal either. So if you haven't done experiments before with your material, I would advise that first of all you do a set of experiments just checking on the concentrations. And then we need to set up the experiment, all the parameters in our experiment according to the expected range. So if we are doing sacks or sands, we can modify the queue range within the possibilities of the instrument, but we can modify it so that we make sure that we are going to cover the size range that we are interested in. And finally, and especially critical for sands, so when we are using neutrons, it is very important to select suitable solvents, which are going to give us enough contrast. And how do we do that? So in this case, it is very important to know the scattering intensity of our material, because that is going to make us choose a suitable solvent to generate contrast between the sample and the solvent. And the scattering intensity, I'm sure you have had some other lectures giving you some of the basics, but just very briefly, it is related to several parameters. So the number of particles, the volume of the particles, the scattering length density, and then the form factor and the structure factor, which are related to the particle shape and size and the correlations. But as you can see, the scattering density is one of the parameters that is going to affect the scattering intensity, so we need to take it into account. And so in scattering experiments, we need to have enough contrast between the sample and the solvent to see the components in our sample. And the contrast is going to be generated taking into account the scattering length density of the sample and the scattering length density of the solvent that we are using. In that image, you can see an example in which we have the same refraction index of the solvent and the beds, and another example in which they are not the same so that we can generate contrast. It's something like that. This is a visual example of what we can do when using different combinations of solvents, especially in the case of neutrons. So the scattering density, this is the formula. And as you will see, it's not very complicated. You also have online calculators, I will show you later on, to estimate the scattering density of the main component in your sample. So things that we need to know, the scattering length, you can find in tables from all the atoms forming your material. We need to know the physical density of the material, and that may be a little bit more complex in some times. You can find, typically, you need to find for literature to find a value for this. And then the molecular weight. That's very important also. We need to know. So with those values, we can calculate the scattering length density of our material. And here is an example where you have the calculations for the scattering length density of water and detour. Both for X-rays and for neutrons, because you know the scattering length is not going to be the same, depending on the source of radiation. So I'm not going into detail with this. You can check on the presentation later on, but just important to highlight that while in the case of X-rays, you see that the scattering length density of water and detour is the same. So that means we cannot play around with contrast. In the case of neutrons, the scattering length density of water and detour is completely different. So that gives us the advantage of selecting what type of solvent we can use depending on the scattering length density of our sample. The objective may be to generate the maximum contrast possible, or we can also look for something that is giving us almost zero contrast, because we want to highlight the structure of other components in the sample, for instance. So here's an example of an online calculator that I use a lot to calculate the scattering length density. Here's the example for cell loss. And you can see we only need to put the molecular formula of the material, the density, and then we can calculate the neutron and the X-ray scattering length density. So this is very good. And here's an example of how we can manipulate the scattering length density contrast in our samples. This is an example for cell loss, but it can be applied to any other carbohydrates. So as you can see, using the online calculator and just knowing the molecular formula and the physical density, we know that the neutron scattering length density for cell loss is 1.87, whereas for X-rays it's higher, it's around 14. And then on the right side you have the scattering length densities for water and for detour. As you see, if we are going to do an X-ray experiment, it doesn't really matter if we use water or detour. So typically we perform the experiments in water because that's the solvent we typically find in nature, so that's fine. And then if we are going to do a neutron experiment, you can see, if we look at the scattering length densities of the cell loss, water and detour, you can see that we will have higher contrast if we use detour as our solvent. But as I said, we may not want to generate maximum contrast because imagine that we have several components in our sample and we want to make cell loss invisible. In that case, we can calculate what would be the mixture of water and detour that would make cell loss invisible. And that is known as a contrast match point, meaning that we will have zero contrast. Cell loss would be invisible. In this case, it would be 35% detour, but we have to calculate this for each carbohydrate. And then another advantage of neutrons is that we can play around by substituting some of the hydrogen atoms with the children, and that is known as selective deuteration. We can replace all the hydrogen atoms in our carbohydrate and that's typically done by chemical approaches, but we can also substitute some of the hydrogen atoms, so have a certain degree of substitution. In the case of cell loss, here I'm showing you how the scattering length density would change if we would have a fully deuterated cell loss. As you see the molecular formula obviously is different because all the hydrogen atoms have been replaced with the material. And then the scattering length density in the case of x-rays would be almost the same. So that's why this approach is not useful for x-rays, but in the case of neutrons, as you see we have increased a lot the scattering length density of the cell loss. In this case, we would have more or higher contrast between the deuterated cell loss and water rather than detour. So this may be helpful also when we want to highlight the structure of other components and we want to play around with the contrast between the cell loss and the solvent. So how can we estimate the contrast match point for our carbohydrate or our sample in general? We know that the contrast match point is the point at which the scattering length density of the sample equals that of the solvent. So in samples with labile hydroxyl groups such as the case of carbohydrates, we have to keep in mind that the scattering length density values will change depending on the ratio of detour that we are using in our solvent. Why? Because imagine that we take a piece of cellulose hydrogel and we soak it in a mixture of 30% detour and water. As soon as the hydroxyl groups from cellulose find the detour, some of these hydrogen atoms are going to be exchanged with the material. The sample is changing and therefore we can only have an estimation of the contrast match point because as you will see later on, the theoretical curve that we should have when representing our data is going to be affected by this exchange. So that's something very important. So to calculate the contrast match points from a sample experimentally what we can do is we can take our sample and measure it by sums using different mixtures of water and detour. And then once we have our data, we can go and measure the intensity at a certain Q point. We need to find a Q point which is not being affected by any structural features. So typically we can go, well, not typically in the case of cellulose, I know we can move in the low Q range, but you will have to see for your sample for your particular sample. And then we can plot the square root of the intensity versus the percentage of detour in the solvent so that we will have a straight line or we should have a straight line like this. And the point at which the square root of the intensity equals zero is the contrast match point. Another possibility as we will see later on with cellulose is to plot what and more advisable I would say when we know that we have a substitution of some of the hydroxyl groups is to represent the intensity term versus the amount of detour in our sample. And we should have a parabolic function in that case with a minimum point and that minimum point would be the contrast match point, but we will see. So then after we have done our experiment, the next step would be data processing. We will talk a little bit about that as well and mobilization so this is the difficult part, I would say, because we have our scattering data that this is our one example of the scattering pattern from from cellulose. And now what do we do with that. So here comes the problem many times we don't know what to do with this. So the objective is to find a mathematical function which is able to describe our experimental data. And we can perform a very basic analysis of the data just to, you know, determine the slope in the in the low Q range. And to compare between our samples that's going to give us an idea if we have more branch structures or not, but we can go more in depth in the analysis and in that case, we need to find a fitting function. This fitting function. I don't know what software you will be using but in many cases what you have is like a library of functions, and you need to find one which is suitable for your sample, according to what you already know of your sample. For instance, in the case of cellulose we know that we have micro fibrils at these micro fibrils we could modelize them as a cylindrical objects. So then maybe we can start doing some trials with a function for cylinders that would be a very good start point so we need to have some idea of what we are looking for. We need to fix or constrain as many parameters in this fitting function as possible, based on the knowledge of our sample. In the same example, I would say if we have an idea of what are the dimensions of the cellulose micro fibrils, maybe we can constrain the cylinder radius because we have an idea of the range that it should be. So, as we were talking before the scattering intensity is related to several factors, one of them is the form factor, which is related to the shape of the particles in our sample. And therefore we have several fitting functions which are based on these particle shapes. For instance, we have Gaussian coils, that would be the case of amorphous polysaccharide solutions. We have spheres, rods and disks. The rods would be a very good start point in the case of fibrils, or if we have, for instance, jelly polysaccharides which form double helices. So, but for each type of sample we will have to go and try with different possibilities and see which one works better. And then the other parameter important for the scattering intensity is the structure factor which is related to interactions between our particles. And that's going to affect how the scattering patterns look so maybe we can have a peak which is related to interactions taking place in our sample. And it is especially important for particles which are electrostatically charged. So, for instance, if we are working with protein polysaccharide coacervates, that term may be important. So, we also need to consider this when looking for our fraction. And finally, as we were seeing previously for starts and cellulose, we also have to keep in mind that most carbohydrates have a hierarchical structure. That means that even though we are studying a certain size range, it may be possible that we are losing some information in the higher size range or in the smaller size range. And this may also affect our scattering patterns. So, for instance, in the case of cellulose, it may be that we have very large fibrils with very large dimensions on the longitudinal axis. So, we are missing this information on the ONASAMS or SACS experiment, but the scattering intensity in the low Q range is going to be affected by these structural features which we are not seeing in our experiment. So, that's also something you always have to keep in mind. And with all of this, maybe we can go into some examples. So, you see how all of this is applied in the reality when we are working with different types of carbohydrates. In the presentation, I prepared three different examples, but I think maybe we will have time for one or probably two, I don't know. But the important thing is that you really understand what we are doing, why we are doing these experiments and that you ask many questions at the end. So, the first example is cellulose. The second example is some experiments performed on starch and microalgae blends. And the third example is related to sulfated polysaccharide hydrogels such as agar, but we will start with cellulose. Why cellulose is so important is the main structural component in plant cell walls, where it is combined with other components such as semi celluloses, pectins, lignin, proteins. Well, depending on what kind of tissue we will have other components, but cellulose is the main structural component. And it would provide different functionalities depending on if we are talking about primary cell walls, which are the growing tissues, or secondary cell walls, which we can find for instance in wood. So, going back again to how cellulose is organized hierarchically, we have the cellulose crystallites, which can at the same time present different crystalline allomorphs. So that's something that will also depend on the source that we are working with. We know for instance that beta cell, the one beta allomorph is more abundant in some vegetable tissues while the one alpha allomorph is more abundant in seaweeds or bacterial cellulose. And then these crystallites are arranged into microfibers with different cross sections and dimensions depending on the source. And finally, we have the ribbons. So if we have several size ranges that we want to study, we will have to combine different characterization techniques. And what is the approach to study the structure of cellulose? Well, since we find cellulose implants combined with other components, if we want to isolate the structure of cellulose, we will have to remove the other components. So what could be do in sometimes is to perform several extraction steps so as to isolate the cellulose from the plant cell walls. But we can also use an interesting approach, which is the use of a model system, in this case bacterial cellulose. And why is it interesting? Because in that case, the bacteria synthesize only cellulose. We feed the bacteria with sugars and they are able to synthesize cellulose in the form of a hydrogel. But we can also incorporate other components into the culture media so that the bacteria will be able to produce cellulose in the presence of that component. And we will be mimicking somehow the synthesis process that is taking place in the plants and also be able to isolate the effects of that particular component that emits cellulose on the structure of cellulose. So I'm telling you all of this because I will start with some results from bacterial cellulose hydrogels, which are the simplest model that we can use because it's only cellulose and water. So I'm going back to the scattering lens densities because that's something very important when we are planning our experiments. You see that we will find contrast between crystalline cellulose and amorphous cellulose when using x-rays, while in the case of using neutrons, we can generate contrast between the cellulose. And the different degrees of exchange using dethrone to soak our pellicles or by producing fully-butyrated cellulose. So in this case you will see that the approach that we followed in these experiments was to soak the hydrogels in different mixtures of dethrone and water so that we will have different degrees of exchange. But we also tested the possibility of producing partially deuterated cellulose by feeding the bacteria with deuterated glucose. So here is the experiment setup. You see that it's very simple. The idea is very simple. We take the cellulose hydrogel, a small piece of the hydrogel, and we soak it in different mixtures of water and dethrone. We were using the cells that I was showing you previously, and we performed some experiments. So you see here the sun's patterns for our samples in the different D2O-H2O mixtures, and so now what do we do with these results? Well, we wanted to extract as much information as possible, so we started by trying to find a theoretical model to fit our data. So at this point we need to think about what do we know about our system. In the case of cellulose, we know from literature that the ribbons are assumed to have a flat ribbon morphology with a cross-section of 7 nanometers by 20 to 16 nanometers. So these are the possibilities that we have. We may use a parallel pipet, a large disc model, and in fact, at the time that we performed the experiments, there was one paper using a large disc model to fit the data from cellulose, but this model needed to use a polydispersity of 95% to get reasonable fits. So you have to be very critical with what you are doing, and if you see that you need such a high polydispersity index, that probably means that the model is not suitable for your samples, and that's something that I want you also to keep in mind. Many models could work for your sample if you adjust the parameters, so you see if you put 95% polydispersity, maybe a model can work to fit your data, but then you have to be critical and think if that makes sense, it makes physical sense, and also based on the knowledge that you have for your sample, the parameters that you are getting from the fit, you have to assess if they also make sense. So in this case, we tested a different model based on the cylindrical morphology. As you can see in this plot, none of the models that we proposed was suitable to fit our experimental data, and so we thought that maybe we were doing something wrong, and we were forgetting about something in our sample, and we thought that since we know that the structure of cellulose ribbons is actually formed by smaller components which are cellulose microfibers, and that we have a lot of water in our system, it may be possible that the cellulose microfibers are not solid objects, but that we have a solid component, and then a water layer hydrating the microfibers. So we tried to find a model to account for this hydration layer, which at the same time could be changing when we are soaking our samples in different solvents. We could have regions with different scattering density values, and therefore we proposed to use a model, a core shell cylinder model, in which the scattering density of the inner region is not the same as the scattering density in the outer region, because in the outer region we have more labile hydroxyl groups which can be easily exchanged when we are soaking our samples. So this model had a lot of parameters, as you can see there, but since we already had some knowledge on the sample we were able to fix or constrain some of them. The ones which are involved, they were fixed, and some of them were also constrained between values that we thought made physical sense, so that in the end we didn't have a model with 19 parameters that we don't know the range, we didn't even know the values that they should have, that would be, you probably won't get good results if you try to feed your data without constraining any of the parameters. So, as you can see this model worked much better to feed the data from cellulose. You can also use cracky plots, sometimes, you know, the structural features are not very evident in the scattering data. In that case, you can always plot the cracky plots where you would see, for instance, if you have shoulder features they will appear more evident, so that you can check on the suitability of your fitting. And then the next point would be using the contrast variation experiments to do a simultaneous fitting of all the data. If you test a model, it works well for one sample, then the next step would be to use this model to feed all the data from contrast variation experiments. And if it works, that's a very good sign, that means you found a very good model for your sample. So in this case, as you can see, the model worked very well to feed simultaneously the data from cellulose in five different solvent conditions. And from the parameters that we obtained, we were able to extract structure information on how the core of the cellulose ribbons is arranged, the amount of solvent that we have interacting with cellulose, the dimensions, and the same for the shell region. And as I said, another interesting approach would be to generate a higher contrast by replacing some of the hydrogen atoms in the cellulose with deuterium. In this case, we have the advantage that since we were using the bacteria to synthesize cellulose, we could feed the bacteria with deuterated glucose so that they were incorporating these deuterium atoms into the cellulose that they were synthesizing. And if you are planning on doing something like this, you also have to check on how the structure of the polysaccharide is being affected by the deuteration, because it may be the case that it may be changing. In this case, as you see the structure of the hydrogenated and the deuterated cellulose was very similar. And I'm showing you here the patterns, the sans patterns from in blue, you see the hydrogenated cellulose, and in red you see the deuterated cellulose. So as you see the shoulder feature that we were detecting in our sans patterns in the hydrogenated cellulose was much more evident in the case of the deuterated cellulose. And that allowed us to test our fitting model with higher certainty, because obviously the feature is more evident so that we know if the model is working well. Also important, and with this I think I will finish, is that as you can see the type of information that we get from neutrons and x-rays is completely different, but in the case of neutrons, we were getting information on the ribon structure, in the case of x-rays, since we were generating contrast between the crystalline and amorphous domains, we got structural information on the cellulose microfibers. And finally we can also perform, or it would be advisable if you have semi-crystalline carbohydrates to do some x-ray diffraction, because that is also going to help you a lot, especially if you are doing sax experiments. I think I will finish here. You can check on the other examples later on, but since we have 10 minutes for questions I think it would be good if you ask anything you want. Okay, thank you very much Marta, and I mean it shows clearly shows that the usefulness of neutrons for studying carbohydrates, so is there anybody who want to have questions to Marta? I can start with some questions. Yeah. First similar question, what is the difference between hydro gel and gel? No, there is no difference. I mean hydro gel is the term that is typically used for carbohydrates because you have a large amount of water in the structure, but you can use, I mean you can use both terms. It doesn't really matter. In the case of hydro gels, the thing is that the liquid face will always be water. Okay. Yeah. And you also mentioned that we can by looking at the structure function, we can see the interaction between the samples. So if in the case we have two different materials and we want to see the interaction, do we need to deuterate one or we can just see from the intensity directly if these two materials are interacting with each other. I mean if you have, for instance, a mixture of two components, well that will depend on the scattering density of each component. If both of them have very similar scattering densities, you won't be able probably to distinguish between the structure of both of them. Let's think of cellulose and hemicellulosis. Since they have a very similar scattering density value, if you do a sex or sex experiment, you will have an idea of the structure of the whole thing, you know, not the structure of cellulose and then the hemicellulose. For that, you probably would have to deuterate one of the two components so that the scattering density is modified and then you have contrast between the two components. Otherwise, you will get an idea of the whole thing. The last question, you use the cylinder to see the structure of these cellulose, but in the solution, there are many cylinders of these ones and then they interact with each other differently. And then do we need some like more specific parameters to fit this model because I see there is a page but I don't really fully understand all the parameters that we need to fit the data. Well, in the case of this model that we particularly used for cellulose, it is quite complex, but we didn't have any parameter accounting for interactions between the fibrils because we know that in the native cellulose hydrogels, the interactions between the fibrils are not so strong, meaning that the interaction between cellulose and water is more important than the interaction between fibrils. If you try out the material, that's another story because in that case you eliminate water and probably you will have interactions, close interactions between the fibrils. So in that case, you may need to adapt the model, but in this particular case of the hydrogels, we only have into account structural parameters from the dimensions of the cylinder. As you can see the radius, the thickness of the shell, the length of the core and the shell, the volume of cellulose, meaning the volume fraction, so meaning how much cellulose and how much water or detour we have. And then we also took into account the degree of exchange, meaning how many of the labile hydroxyl groups were being exchanged when we were soaking the hydrogels in the different solvents. And then power low exponents to account for the for the low Q region. So, as you see, there was no interaction parameter in here, but because we know that it was not the most important thing in this case. So, if I understand right in the picture, the circle here is the model that you use and these little blocks like pink ones they are, they are the cross section view of the cellulose. Yes. So on the top part of the slide you mean. Yeah. Yeah, so what you have on the inner region would be the cellulose microfibers. And you see, it's a little bit complex okay because the cellulose structure is hierarchical, as I said so that we have different levels of structure. So the smallest structural feature would be the cellulose microfibers, and that's what you have inside the black circle. And they contain crystalline domains, which are the dotted parts and then para crystalline regions, which are in blue, in light blue, and they are interacting with each other. But this is not what we see, or this is not the structural feature that is being probed in SANS. What is being probed in SANS is the larger structural feature, which would be the ribbons, meaning the combination of all these microfibers into one larger cylinder if you like. This is a model, right, so it's not a cylinder as such, but we can consider it to be like that. And in this larger cylinder what you have is an inner region where there is not so much hydrogen exchange. Why, because the microfibers are interacting with each other so that we don't have so many labile hydroxyl groups. In the outer region, we have the labile hydroxyl groups from the outer part of the microfibers and they are easily exchanged. So that's why we have regions with different discontinued densities. And that's why the simple cylinder model didn't work in this case, because you have to take into account that the exchange is not going to be the same in the inner and the outer region. So the beneficial of using this one instead of X-ray scattering is because we can use different contrast and then we can fix them like to convince us that this is the structure of this hydrogen. Exactly, yes.