 Asa, thank you for arranging all of this. Trevor, thank you also. And I'm excited to actually talk to you this morning. I'm not a morning person, so I ended up putting my alarm clock two hours in advance because it takes me a good hour to get out of bed. So I didn't wanna miss any of this. So as Tommy has mentioned, we've done a lot of work on the membranes over the years and my research changes depending on what flavor we have depending on the students that I have. And years and years ago, when I was at Chalk River in Canada before coming to Oak Ridge, I had a very good student called Jeremy Spencer who's now works for the nuclear industry as a reactor physicist. So your careers could change. So he started out doing light scattering and membranes. Eventually he came to me and we started doing neutron scattering at Chalk River. And he really introduced me to small-angle scattering for the most part because his thesis was on small-angle scattering. And then together we started looking at nanoscopic domains but we just didn't have a lot of money at that time to get the right material. And in fact, we didn't even have the right instruments which was a sands machine. And I know that many of you have access to that. And in Europe, you have some of very good machines at the ILL in Berlin and now eventually you're gonna have the ESS who's gonna have great capabilities. And so that work remained dormant for quite a while because I moved on with another students of mine developing the scattering density profile model which is now used extensively by simulators and other experimentalists. But when I came to Oak Ridge, they, we had to find something that was unique to neutron. So, and really that only neutrons can do extremely well and not X-ray scattering because when you're dealing with scattering techniques, X-ray scattering is always at the foremost of people's minds. And the reason for that is because they have been exposed to X-rays either through lab sources or by going to synchrotron sources that are plentiful around the world. And then you're dealing effectively with a flux problem because neutrons are really weak as far as flux, number of particles or photons per unit area. Neutrons really are very weak on the many orders of magnitude down with respect to X-ray. So the one thing that neutrons have and this is the take home message today is that you are able to change contrast very, well, I wouldn't say very easily, but much, much easier than any of the other techniques that I know of. And of course, all techniques for the most part require some contrast, all kinds of imaging techniques and certainly a lot of all the scattering techniques require contrast. And changing contrast with these other techniques is very difficult with neutrons. Fertuitously, because of protons and the isotopes of hydrogen, you're able to do that. And so by the end of this talk, I would like you to sort of think, okay, this is what we need to do. And I want you to interrupt whenever you want, just ask questions, feel free to ask questions. And hopefully I'll know the answers. Sometimes I don't know all the answers, but hopefully I'll know the answers. And I want you to be able to then think that, where does the field go? What if you're interested in this kind of work, then how could you contribute to this? So when I came here then, I was given two postdocs. This is not 2010. And I realized that the work that I had done with Jeremy Penser was the work that would be ideally done here now, at Oak Ridge with the resources that we had, both financial and also from an experimental point of view, that we can actually now exploit neutrons to look at nanoscopic domains. Something that is done, cannot be done or is done poorly sometimes with all these other techniques, but neutrons are ideally suited for that. The student that I had was one from Jerry Feigensen's lab. His name is Fred Heberle, and he's currently now a professor at the University of Tennessee in Chemistry. And he and I started this lateral heterogeneity program that then expanded to the work that I will show you with Jonathan Nichols and Living Cells. So any questions, everybody? So I just wanted to give you a little bit of a history that things don't always go in a straight path. You basically start something, you get people, and this is where interactions are extremely important collaborations. You get young people coming into your lab. They have ideas of their own. You basically set some boundaries, but they're allowed to go beyond those boundaries, not too far, because then we don't know what we're doing anymore. And before you know it, research has evolved, new ideas have come, and hopefully the field has moved forward. So as I mentioned, I've looked at membranes for effectively since I've been a graduate student. So I started as a graduate student in 1984. Probably some of you were not even born back then. And not that I knew anything about membranes, but I certainly had to learn quickly, and I've stayed in membranes ever since. And some people find that strange that somebody would spend their entire career looking at one particular superstructure within biology, but I think it takes that much effort to really make a headway. And when you look at your research, when you start and when you look at your research, when you're ending your career, hopefully they look very differently, but in the same sort of vein, but they are extremely different from what I did in 1984 as a graduate student to what we did in the last 10 years here at the Oak Ridge. So membranes, what are they good for? Well, of course, many of you may know that membranes form barriers and they do that. And of course, they're selective barriers. So they can don't let everything in and they don't let everything out. So they choose what they can do with regards to allowing things. So there are selective barrier. And of course, if you destroy them, then they'll let everything in and let everything out. And then they no longer function and the cell dies. So you've got that situation. Then the surface of the membrane, the plasma membrane, so the exterior of these cells has recognition. So carbohydrates are there to look at, recognize either foreign objects, pathogens or cells of their own type. And then of course, the plasma membrane contains all kinds of other things where you could enable cell adhesion, cell surface marker identity, selective channel lines, as we said, enzymes, et cetera. Can you actually see my cursor moving on the screen, isn't it? Do people see it? Yes. Okay, good. Okay, so this way I'm not, it's just not for entertainment for myself. So that is the plasma membrane. Of course, some cells like bacteria only have a plasma membrane, or a lot of them. And of course, depending on what bacteria is, they may have more than one membrane on the outside. They may have a double layer. But eukaryotics, you know, eukaryotic cells like us, mammalian cells, then have many membranes within because there are all kinds of organelles. And that makes it much more harder to study. And the other thing is that eukaryotic cells are very difficult to grow in D2O conditions. They reach a level, and after that, they just don't, you know, they either die out or they stop growing. As you will see later on, we used prokaryotic cells, specifically bacillus. Which is a gram positive bacteria with a single membrane. And so that made life a lot easier for a lot of the studies that we want to do. So the other thing is, remember I said that I've been in this business for a long time. Well, you say, well, why would you, you know, so, you know, John, you've spent over 30 years, don't you get tired of it? Well, it's, you don't get tired because there's always something to discover. So here I'm just giving you a little history of the membrane as we know it. And we still don't know many, many things. But you could see that I'm giving you a timeline of a hundred years where it's taken us a hundred years to go from the point in 1925 where Gorder and Grendel effectively came up that the membrane is a bilayer to the current state where we started thinking that the membrane is a lateral heterogeneous superstructure. And all the iterations in between where we have the sandwich model made by Daniela and Daphson started in 35 modified in 57. And then the fluid mosaic model which many of you would have heard through your biochemistry classes or biophysics classes with Singer and Nicholson in 1972. So you see that even a simple, just getting the arrangement of the various biomolecules the lipids, the proteins, the carbohydrates, et cetera in a fashion that actually makes some sense took us a hundred years. So things take time some, we don't learn as fast as we think we do. So a hundred years to reach the point where Linwood and Simon while wrote this review but it was really almost in 1997 or in the nineties that we started really thinking about lateral heterogeneity in a very serious way as a functional thing. People sort of had inklings about it in the seventies but we really didn't start thinking about it till much later on as far as function. So what are these things? Well, the plasma membrane as we said is a heterogeneous structure. Both as I will show you in this direction which I will call the transverse direction and this is I will call the lateral direction. So you see here there are basically lipids that are different from the lipids here. So this is what they call a domain or a raft because if it has functionality they will call it a lipid raft or a lipid domain, whatever you want. I mean, there are many terms that are used but nevertheless we're talking about something that has chemistry that is different from this around. And of course they are thought to enable function of these proteins. So when proteins come into this domain made of different lipids of specific composition then protein could be enabled to perform its function and when this raft disappears then the protein no longer functions or its function is reduced. Now of course it's a chicken and egg argument. Does the lipids attract the protein or does the protein attract the lipid? I think the jury's still out and there may not be one universal answer. They may be very different from system to system. So here I'm showing a model system using fluorescence. So this is now micron scale. So this is on the order of a thousand times bigger than what we'll be talking in this talk, but effectively we're gonna be looking at something similar to this, but on the nano scale. And I'll tell you why that is important. But of course membranes are not static. And as many of you would know and anybody who doesn't know, we'll tell you these are very dynamic structures. With different months scales. So these molecules are moving at different time scales on the different months scales. So you have effectively very quick rotations. So this is on this side. Then you have diffusion, you have protrusions that you get over here. You have stretching modes, bending modes, et cetera. So all of these things are happening at the same time. So there's a hierarchy of time scales and length scales that the membrane is undergoing all the time. Now in some places, of course, they may favor one over the other, but you're talking about a cell with many surfaces. These things are probably going on all the time. And of course, one of the things that, and I'm sure you will learn through this course that Trevor and Tommy are putting, you will learn that the neutron capability can actually look at practically all of these length scales and all of these time scales, reasonably well. I mean, there are time scales that, on the very long scale millisecond or much less than microseconds, let's put it this way. NMR can do this on the order of nanoseconds and hundreds of nanoseconds, almost to the micron. Second, you've got neutron spin echo and maybe somebody would talk about neutron spin echo techniques. Then on very fast motions, you got quasi-elastic and backscattering techniques. And of course, when you're looking at scattering, the structure, these are the dynamic components that I've just discussed. But if you're looking at the structure that you have small angle neutron scattering, you have diffraction techniques that you could do reflectometry techniques, et cetera. Now, of course, the cell is also, you can actually get flip flops, but those, meaning that lipids go from the inside to the outside and vice versa, but those are very, very, very slow motions. Now, people, there's a huge debate in the model membrane system about this, but in biology, these are extremely slow. And you'll see, and the reason is, because membranes, there are processes in biology that enable these to do this. They don't just happen by themselves. There are enzymes, there are biomolecules that allow this to happen. And in fact, the membrane is not symmetric. And this is something, a slide that I have at some point. I'll show you. So it took us a long time to get to the fluid mosaic model. It took us about, was it about 15 years or so to get to the fluid mosaic model from the notion of the bilayer. And then it's taken us a long time now to figure out the lateral heterogeneity part of the membrane. And of course, there are many studies that have been done to bring us to this point. So there have been studies where we have looked at extracting detergent resistant membranes and inferring that they have to be domains and antibody tests and fluorescent studies, et cetera. So it's taken us a long time to get even to this little subsection of the membrane. So besides sort of putting all the biomolecules in the right place spatially, figuring out what the in-plane scattering, what the in-plane structure is, has been another big grand challenge. And we're not done with it. There are many open questions. Although, like everything else, people get tired of studying something and then they move on. And then the field drops off for a while and then there'll be another reason and it'll pick up again. So these things come in waves, it's like fashion. I'm sure before, we may not go back to polyester of the late 70s and 80s, but you see, for example, bell bottoms coming back into fashion and things like that. And that's just because human beings like change and they get bored after a while. And science is no different. That happens like that in science too. But if you stick with something, then you're always, if it does come into fashion again, you're ahead of the pack. So don't always go with fashion. I don't always recommend that you go with the latest in fashion, but be cognizant of what the recent trends are because they may be applicable in what you do. So the other thing is that if you have worked with model membrane systems, your membranes are always symmetric, almost always. I mean, there are exceptions I have now, for example, Fred Heberle, Drew Markard, London, Irwin London, and others people that I don't know that are working on asymmetric membranes in model systems. But it's, they're not easy to make. So we're trying to figure out easier ways to make now or mimics of mimics that are easier to make. But you find out that in the 70s, now this is from 2009, but it was in the 70s that I can't remember, I think it was a Danish group that effectively, I should put that reference in here, came up with the lipid composition of the different member of the bilayer leaflets. So what is the lipid composition on the outer leaflet, the versus the inner. And you see that you have lipids like single myelin, phosphatidylcholine dominating the outside part of the plasma membrane. Well, phosphatidylserine, phosphatidylethanolamine, and PI, inositol and PA are dominant on the inside. Well, I would say this is an equal, I should say. So it's interesting, for example, if you start seeing serine on the outside of the cell, then it almost tells you that there's something going wrong because serine is usually fine on the inside. And the other debate that is unresolved in one of the reasons that people are interested in studying asymmetric membranes in model systems is that it's not clear to us to this day whether these lipid domains only happen in the outer leaflet and the inner leaflet is more randomly distributed or do lipid domains trend occur in both bilayers and are they correlated in both leaflets? I should say this is a single bilayer. These are the leaflets. So is a domain, does it happen in both? So this is an open question. You're thinking we would know that, but we don't. And there's a lot of debate about that. And one of the reasons that I think asymmetric membranes for are have a lot of appeal in model systems. And in developing those model systems is because we're hoping to answer that question or at least get something. And there are some data that shows that you can actually form membranes on both domains on both bilayer leaflets. But we don't know that for sure that this is happening in biology because biology has a lot of things that we don't have in model membrane systems such as for example, the cytoskeleton that we don't have. Now you could try to mimic that by making vesicles with polymers on the inside or acting on the inside. You could do that and people have tried that. But things get complicated very quickly. So even with a simple system in model systems things get very complicated and difficult to make reproducible day in, day out. So that is my introduction about membranes, sort of how we've got there, how I got here. If there are any questions, feel free to interject at this point and just shout it out. Because I'm not gonna keep track of hands going up or anything like that. I usually just shout questions out, but then this is me. So let's now start thinking about some neutrons. And what makes neutrons? And I'm sure this is Thursday so you've had lectures Monday, Tuesday and Wednesday. So by now you're very well versed on the neutron key things. And I'm not gonna talk to you about very much about that. Except to show what you've already learned is that X-rays sort of scatter in a very monotonic way. In other words, they're very predictable. You just look at the number of electrons and you know the scattering. Neutrons, not so much. You actually have to, it's much more difficult to know what the scattering ability of a neutron is as far as an element is concerned. So the elements are randomly distributed. Now you could say, well, that's kind of confusing. It can be because you have to actually go and look at tables to find out what is the coherent scattering? What is the incoherent scattering? What is the absorption? Well, with new X-rays you could get that just simply by knowing the atomic number. You get a very good idea. You don't have to be looking at things. But the nice thing about it is that it has a lot of flavors then. Then you're into a lot of flavors, right? And this is just showing you things that have negative scattering cross-sections and positive in green. And the thing that of course is of interest to us, at least to most of us here, is hydrogen because you have effectively proteome and deuterium that have different flavors. And that really opens up things as far as contracts. So the other important thing to note, and this is a technicality, but it's important to know is that with X-rays, you're always scattering from a fairly large object compared to the wavelength of the X-ray. So you have what they call a form factor and I don't know if people discuss that, but you have a form factor, which means that you get a lot of scattering and then it drops off very quickly. And that's a technicality, but it's important because you basically have to do corrections for that or form factors. And also you actually lose a lot of intensity of going to the higher angles. Luckily, there's a lot of X-rays. With neutrons and because they scatter from atomic nuclei and atomic nuclei are effectively point sources, your form factor is a straight line, doesn't change as a function of angle. And that's interesting and important. But again, that's another topic and we're not gonna, but I'm hoping that somebody did talk about form factors and I'm sure they did and that they pointed out that in one case you have what they call a form factor coming from the electron cloud versus a point source where you don't have a form factor and it's effectively a straight line across. So here I'm showing, I put a nice little box and I'm showing you proteome and deuterium. And you see this difference here. This is very important to know. And that minus sign is what will be very important for this talk. That little minus sign is what enables then this contrast variation that we're gonna talk about in that. I think makes neutrons as far as biology, but even in other sciences, even in hard condensed matter science, very, very unique because you're able to change that contrast. So this is something that you've seen and this is just something to show you what is a cross-section and what is a scattering one, but that's not that important right now. In fact, it's not that important as long as you know the basic concepts of it. So what am I talking about contrast? And so contrast is something that we deal with every day in life. You can see me because I have this contrast between me and my background. If my shirt was the same color as my walls, you wouldn't be able to see my, from the neck down. And that is lack of contrast. We're basically have no contrast at that point. And here I'm showing you something with visible light. In other words, the refractive index of light in air and in water and those beads. And what happens is what does happen? Well, you know that there are beads, but when you put them in water because they have a matching contrast, so their contrast batch, they disappear. So you wouldn't know by looking in there that there are actually beads in this water. It's only when you put your hand in there and you feel around, oh, there's something in there that I can't see. And only when you take them out can you see them, right? Because the contrast in air and the water are different and they're not contrast match with the air. And that's all I'm talking about. And this is what I'm gonna be talking about now for the rest of the talk. And that's gonna take us whatever amount of time the next hour and 15 minutes. As we talk about and you're gonna ask questions about contrast and that are bothering you by the, when I'm finished giving you this lecture. So in biology, you have effectively water, DNA, RNA, protein, cholesterol, lipids. There's other stuff, but you know, these are sort of like the basic components. And that you can see that if we took a biological system, a cell, because it has all of the stuff in it, and we put it in 100% H2O, just regular water, then you will see scattering from all of these components because none of those components, none of those biomolecules have been contrast matched. At 8% or so, 10% D2O, you see that this line that belongs to lipids is now crosses the D2O, the water line I should say. And that is a point of contrast. Meaning that at this point, at this percent D2O, everything will scatter. Neutrons will see everything except for the lipids. And that is applied then as we're going along. Proteins are around 40 some odd percent, 41%. Then you have DNA, then you have RNA, and then, well, there's nothing. And this is where wherever that water line crosses that biomolecule series line, then that group of biomolecules becomes invisible to neutrons. So is that clear to everybody? But what does it say? It means that there is no time, there is no H2O D2O concentration where we could mute, in other words, turn the volume down on all of these biomolecules at once. So there will always be a group of molecules that always has their volume up. They're always scattering. So here is just a simple, well, it's not a simple equation, but this is what you're always measuring. You're always measuring this, or at least in most cases, you're measuring the intensity of your scattered beam one way or another. And most of these things are dictated by the instrument, and that is up to the ESS people and the Berlin people and the ILL people, they've dictated this to you. You don't have, when you, as a user, go to one of these facilities, you are stuck with a lot of these things because they're instrument related. They built those instruments in a particular way. You may be able to, I don't know what you could change, but not very much, you could change the wavelength. But this is where you come into play, this part here. This is where you come into this differential cross-section is extremely important. And I'm hoping somebody described this to you, but this differential cross-section blows up into this over here, which then has all kinds, it has all the stuff that you need to do the experiment. So this differential cross-section is equal to this, whereby now you have all kinds of interesting things, but what importantly for this talk, you have neutron contrast. So neutron contrast is folded into the intensity through this differential cross-section. So what happens, as we said, if you have contrast, you actually get a signal, right? You don't know what that signal is coming from necessarily, but you will get scattering if there's contrast. If there is no contrast, you will get, in an ideal world, of course, you will get zero scattering. That doesn't always happen because there are always fluctuations. It's extremely hard to get something perfectly matched, but you can come very close. And we'll find out how you can do it. How are we gonna do that? So this term here is extremely important. And this is the one that you, as a scientist, as a researcher, has control over. This is where the facility people have control over, but they don't have any control over this because they don't know what necessarily what you're gonna do, but you got control over this, and this is where all the interesting things are really happening. Now, the sample-dependent things are, it's interesting. This is just, I think I've mentioned to Tommy, many more than one occasion, is that facilities are more than happy to spend millions of dollars to control this term here, but they're not prepared to put anyone, or very little money, into controlling this part of the function, which is interesting. So that means that we have to figure out ways to deuterate materials, because deuteration, we will find out, is then part of this, contrast part of this term. And that is something that we wanna be able to control. So there is, I think you've got now the gist of where we're heading. We basically have systems that scatter very differently in a real cell, because that's a real cell, we'll have all of these, a living cell, we'll have all of these components, and it wants to restart my computer in an hour, and I won't say snooze. And then, we know that we could change H2O, D2O, and turn off some of the signals. So this is something I'm sure somebody has explained it, because you guys did all of this data analysis, didn't you, yesterday or the day before? You did all kinds of data analysis, and this is a simple experiment. I have a magnetic field, but don't worry about a magnetic field does not apply to these things. But you effectively have a neutron beam that could have, you know, delta lambda over lambda, meaning that it's not purely one wavelength, but it has a little bit of wavelength on either side of that main wavelength. It meets your sample, it scatters, you get this nice diffraction pattern, they all look the same at first glance, but then when you massage the data, then you can actually analyze it. And one of the useful expressions that you should all know is simply that Q is equal to two pi over D, because this is something that you, if you go to neutron facilities, that is something that they will always talk about, because it's wavelength independent. Q is wavelength independent. It's already folded in, and so you could get a lens scale by knowing Q, or you could get Q by knowing the lens scale. So it's something you should know when you go and do your first experiments, or if you're already doing it, then you know the importance of having something like that, because you're always looking at intensity versus Q for the most part. I mean, people plot things in different ways, but that is a very standard way. So the other thing that I want you to be as young scientists is don't just go and look at do things and look at the data. You know, nature has given you eyes that are very sensitive, actually, much more sensitive than any neutron, and they're connected to this. And if you use this and this together, then you could do a lot of things. You can actually figure out lots of things that then will convince you that what you've done with your experiment also makes sense. So what do I mean by that? Meaning that when I look at something, you know, I should be able to, if I know enough physics, I should be able to figure out, okay, this thing is made up of this kind of stuff. I should be able to know that. So in other words, look at milk when you have milk. Why is milk the way it is? Why does it look white in most cases? Sometimes my milk looks yellow, but why does it look white in most cases? Well, it doesn't matter what color it is. It means that there's a lot of big stuff going on in there and that's why it looks white because it has different sized objects that scatter. Right? So it means that I have stuff that is on the order of microns. And that's why it's got that color that it does because effectively you're scattering visible light into your eye and it looks like that. So imagine you do an experiment and you say, no, no, this milk has nanoparticles and they're all the same size. Well, we know that's not right because your eyes and your brain, if you know anything about scattering, we'll tell you that's not the case. So it doesn't matter what neutrons tell you at that point. But it would be very beneficial when this, this and that data all makes sense. All comes together and makes sense. So without doing any fancy measurements, somebody, a beam line scientist gives you a nine versus Q data set. You should be able to look at it and say, oh, you know, if I took the slope of something here and I looked at a peak over here and I look at the slope over here, you know, I could get a good idea as to what this is. And if I looked at it with my eyes, does that then make sense with the story that I got over here? And then if I actually did some polarized microscopy, does that also tie in together into one unifying story? So I put this slide because to show you that sense, even though they look weird and you have to do a little bit of analysis, they actually give you results that are consistent with what you should see. So here is a milky substance made out of multi-lamelar vesicles. And why is it milky? Well, it's milky because these objects are micron size. No different than your milk. So it has these big objects that scatter light. You do scattering, it gives you at a Q to the minus four, which if you know a little bit of theory, then you know that these are 3D objects. And then you have effectively these multis crosses that you see over here in microscopy, which are another fingerprint for these kind of structures and so on and so forth. And when you get to structures that are like this, where it's clear and very dilute like alcohol, it means what? I know it's difficult to have this. This is the one thing about Zoom, you have this. I can't see your faces that you're thinking, what is he talking about? But effectively you have now very small particles that are not interacting. And they are, as far as your eye is concerned, there's really nothing there. But as far as neutrons are concerned, there's something there. Okay. So that's my little summary as to trying to get all of the data coming from your eyes, either looking at the thing under a microscope and then neutrons, and that all of those data points, all of that picture has to be cohesive. It has to make sense. Otherwise, there's something wrong somewhere. And that's part of science. And we find out that sometimes we just can't reconcile some of these things. So here it is. I'll describe this as the experiment. And given at the end of this, I have three papers that I would, you can take a look at and read. All of the information that I'm gonna translate to you is in those three papers. And we'll discuss, if you read them, I had a time, I'm not sure if Aisa got them to you, but I sent them late at night. You probably haven't read them. That's okay. You got many days ahead of you to read these things and you could always send me questions. But effectively, this is one of the experiments that we did with Fred Heberle when he came here and it ties a few concepts together. So one of the first things that we did is we wanted to look at nanoscopic domains. And the reason is, we wanted to make sure that we're able to see them in a very clear way, which we had done with Jerry Pencer, but not so clear because we didn't have the money to get deuterated compounds. And we didn't have the money here, either, and I will explain. And this is gonna be part of your questions that I'm gonna ask you later on. But Jerry Feigensen's group at Cornell had done a lot of phase diagrams. And so these are these phase diagrams. Actually, I had to go and look at how to read these things again because I had forgotten. And you could get all of this data and create a phase diagram using various techniques. One of them is, for example, differential scanning calorimeter. You could also use fluorescence techniques, et cetera. But they're very tedious because you need hundreds of points to create one of them. In other words, you have hundreds of individual experiments to make one of these phase diagrams. And Tom is very familiar with these things. And I'm sure some of you have already done some. So we wanted to take a look at a canonical, meaning a prototypical system that we know has phase separation. And in fact, the important thing about a phase diagram is that you know the composition of a particular point. You know exactly the composition of lipids in it, the ratio of lipids in that particular phase. And that is actually quite important. So in this particular system, Fred looked at this phase diagram made out of DSPC, which is a very biologically irrelevant lipid, but this is in lieu of Sviglumilin, POPC, which is a very biologically relevant lipid and DOPC and cholesterol. So these two lipids are really are not biologically relevant for the most part. These are, and that part of this is because we didn't have the money to make, for example, Sviglumilin and deuterate it. But this is the canonical diagram that we took from Fygansson's group. And so Fred, if you decided that we needed to induce contrast, right? So imagine if this is now proteated. Imagine if this is proteated, meaning all green like that. Can you actually differentiate easily one from the other? The answer is really no because they're pretty similar in chemistry, right? So you're gonna have now a system that yes, chemically you could have DSPC here and the rest of them here, but because they all look the same, there's no contrast, you can't tell, you can't tell. Even though the phase diagram is telling you that there should be phase separation. And so the phase diagram here is showing that there's an LD liquid disordered phase and a liquid ordered phase. So there's a coexistence. In other words, we're basically seeing this, what we're seeing here in this movie. And as you could see, the contrast here is fluorescence. Now, of course, if it was totally contrast matched with neutrons, this thing would be this vesicle here would be totally black, blend into the background. Now, of course, because it's fluorescent, it never really does blend into the background. You always see a bluish, so here it is, right? No domains, but you could still see the blue because you're always looking at fluorescent. But if you didn't have any fluorescence, then you would see nothing for the most part, right? And that's what we wanna do. So what Fred did is we didn't have the money to deuterate the head groups. So, but we were able to get this. And what he found out is that you could calculate now, right? You could calculate the scattering length density out of for every single component, knowing the atoms that are making this liquid and their volume. And then you get what they will call a scattering length density. And by doing an Excel sheet, whereby you're a little bit of arithmetic, you could find out then if I took this vesicle, I can actually calculate the scattering length density of that vesicle. And then what is the water H2O D2O ratio that I could make it disappear when the vesicle is randomly distributed? So are we all on board on that? If there are questions, now's the time to answer because I wanna move. I have question about this video. So which of the lipids is tagged with dye? Is it the DSPC? The one that is targeted with, it's interesting that the liquid ordered phase here is the dark phase. And the liquid disordered in our experiments, in theory, it should be the DSPC, right? That should be the target. I don't know exactly in this video, but the DSPC is supposed to form the liquid ordered phase which forms the domains. In our case, because of the composition and the size of the vesicle, we find that it's actually inverse. We actually find out that the liquid disordered phase is the one that actually forms the domains which is actually inverse to what is commonly thought and thought to be formed in biological systems. So you can talk to say that it's sort of an inverse to a conventional phase in a way. It's an inverse to them, but for the purpose of this talk, it doesn't really matter. The fact is that the domain here is the one that has the fluorescent tag. Otherwise, you wouldn't see it, right? Nicole, are you okay with that? Yes, just wondering. So what is the fluorescent tag maybe? Oh, that I don't know. I mean, it's irrelevant really. What is the fluorescent tag? I mean, it has nothing, no bearing. It's just a fluorescent tag. Like, you know something? Who cares? Okay, so the tag is actually locked to the lipid. It's not a preferential in the liquid state. Correct. Now that's a very good point. What fluorescent people do and why is it important to know the phase diagram sometimes than when you're doing the neutron scattering is that they will put a tag and they know it's attached to a particular lipid then that has a preference for a particular phase, right? And in fact, when they're using tags that are not attached to the lipid or let me say this, they don't have to attach it to the lipid. They could put a fluorescent tag in a molecule that will preferentially partition into that phase. So you could do it two ways. If you're doing fluorescence. So, and Tommy is better to answer that question than I am, but effectively then you have that particular molecule that prefers to be in that phase. So it's a proxy. It's a proxy for that phase in that set of molecules. And of course, you don't wanna put too much of that because then things are affected, right? And we have found that in fluorescent studies, for example, that you can actually form domains when there really shouldn't be domains or something like that because the fluorescence has created some kind of reactions that then result in that. But people now have a very good understanding of how to avoid it using fluorescent studies before it wasn't very well understood. Now, in our case, we don't have that. Well, we don't have that problem because in theory, we don't have a probe. We don't have a fluorescent probe. But don't forget that proteome and deuterium are not exactly the same. The hydrogen bonding between proteome and the hydrogen bonding between deuterium is different. The bonds are different. The bond strength is different. The bond length is different. But for the most part, they're as close to putting something that is isotopically labeled without a probe as you can get. But there are differences and actually we're looking now at how deuterium affects other things in another program that we have. But so yes, so this fluorescence here, you could either have the tag on a lipid or you could have it in a probe that then preferentially partitions into that domain. So you could have it both ways. But it's important, what I'm trying to show is it's important for when you're trying to do a study to show proof of principle and also to understand something else is that it is useful to know the phase diagram because you know exactly how many lipids of something is in this phase versus this phase. And that you could get from the phase diagram. So Fred did all these calculations in which I said that any one of you can do. And if my computer kicks me out because I can't put it on snooze, I'm gonna have to come back again. But we're not there yet. He then mixes them and what you wanna do is you want to be able to contrast match it and see if he actually did his calculations correctly. Now you could either, like I said, calculate or you could actually do a titration where you take those vesicles in the disordered phase, right? Because you don't want them to be in phase separate. You want them to be randomly distributed and you could then look at the intensity and I will show you later on and follow how the intensity reaches a minimum and then goes up again because it'll reach a minimum at some point of H12 because that's where it is the best contrast match. So he did this and what you wanna do is get zero scattering when you're in this randomly mixed situation because now, even though you have deuterated chains, they're randomly distributed as we had shown over here, right? We're into this phase here except that there's no fluorescence. It's contrast match. This whole thing is looking black and then as you are lowering the temperature then phase separation will take place. And then what happens? Like you start to get contrast, yes. You get contrast, which means you get scattered. Ignore. Right. So now what you do is you start going from, so this is just a set of data where you get, it's this one here and this is the contrast match condition. And you can see the moment that he's come down from 50 or 60 degrees wherever he was to 20 degrees where phase separation is taking place, then you start seeing measurable changes in intensity. And even if you do nothing, you know that there's phase separation taking place. And as what then the experiment was to do is what happens as you are now exchanging one of the unsaturated lipids, POPC with the other unsaturated, polyunsaturated lipid, actually the monounsaturated with two chains, DOPC. What you find out is through Monte Carlo simulations, it's another thing that I'm sure you were told and when you did your sans analysis is that you have to have a model, right? There is no direct transform that you could do from inverse space into real space. You see that the, you see that the scattering intensity is getting larger and larger. And that is due to the fact that you're getting more and more the domains are getting larger and larger and larger. The interesting thing about this thing is that you're able now to model the system. You could get the domain sizes, but also through some separate experiments, you're also getting what people are really interested in, the mismatch in thickness between the domain and the bulk. And as the domains get larger and larger and larger, the thickness difference is getting bigger and bigger and bigger. And which means that the line tension is getting larger. And this is of interest to many people because we don't understand effectively how domains are formed. So we're trying to figure these questions out. And this is one of the first papers that effectively through this contrast that we're using we're able to tie in domain size and mismatch and line tension, et cetera. So you see here you got effectively a yes or no answer without having to do any analysis. And then if you do some analysis, then you get some information that could be extremely useful in of interest to sort of physicists in particular because they're interested in phenomenological type theories as to why do we get domains and how do we get domains and what they take the size. And these are all open questions that we're still trying to figure out. So this paper gets either a lot because of this. It's not because we saw nanoscopic domains per se but because of all the information but it's because neutrons enabled us to do it because of this very unique characteristics that neutrons have between proteam and detergent. So do you understand now how we're gonna do an experiment like that? You could actually calculate in a model system everything and I'll show you later on and we could discuss that. And then, but you could also do it experimentally by taking the same system and putting different amounts of D2O until I will show you later on, you get a minimum. And at that minimum is where you wanna do that experiment when there are randomly mixed, all right? Okay. So you know the question. Yes, so here in this figure, do you need to control the time in, do you have some type of time resolution or how do you say like, because they will grow and move with time, right? No, no, no, no, they'll make a certain size and they will stay there for quite a long time. Yeah, no, they're not, well, we don't know if they are at their thermodynamic minimum per se because those are very hard. It's another very difficult question to answer. But we know that over extended period of time, these domains are fairly stable. And there's lots of studies, like I said, fluorescent studies in different sizes and it's an interesting system which we now are using for other purposes. So as a baseline, because now we have a good baseline for fluorescence and also from neutron scattering in different sizes too. So it's interesting, the thickness of a membrane in a cell is, this is basically our knowledge of it. We don't have any techniques really to measure our angstrom resolution or nanometer resolution features of biology in a living system. We have lots of techniques when they're dead, when we kill them and freeze dry them or in this case, use them on a grid for electron microscopy, et cetera. But when it comes to a living system, we don't have any techniques that I know of, but I mean, people could tell me. There are people that do fluorescence, but that's not exactly on the nanometer. It's on the micron scale. And there are people, of course, that do things on the micronsome, but like I said, the cells have been dismembered and taken apart. So one of the things being in this business for a long time is biologists always like to know about in vivo stuff. So this stuff that Fred did is of interest to biophysicists and physicists, physical chemists, but biologists couldn't care less. They really don't care and that's okay because they got their own stuff. So one of the questions was, can we actually start thinking about doing this particular experiment, but in a living cell? So because this is the best we had, really an electron micrograph of an E. coli that was stained and it's dead and that's okay. And that gives you a good idea. There's nothing wrong with it, but this was the state of affairs until recently. So the question is, what can we look at? Well, it's not easy, as I mentioned, to look at eukaryotic cells or mammalian cells because they're complex, they're very complicated and they're also very sensitive because they have a lot of membranes, a lot of things are happening. And maybe when we're gone, I think the complex organisms are much more sensitive to natural disasters and epidemics as we are seeing caused by things that are very simple. So, and biologists are interested in simple organisms. In fact, there's a huge amount of research that goes on looking at bacteria and pathogens and all kinds of other things. So we've had this idea as to can we take a look doing some of these biophysical studies at the angstrom's nanometer scale in a biological system? So what you need to do is you need to actually interact with biologists because physicists don't know anything about, or we know very little about biology. And we certainly don't know how to manipulate them. This is a field on its own. So we went and find some biologists and told them, this is what we wanna do. And they look at you very strangely, why would you wanna do that? Like who cares? And you say, well, I think it would be interesting to do and how would we go about doing it? Well, then they look at you strangely again and they say, okay, well, I'll speak to you next week. So off they go and they go amongst themselves and they chat, chat, chat. And they come back and they tell you, yeah, yeah, I think we could do something for you. And you say, oh, wow, that'll be great. And so what is that? Well, they came to the fact, oh yes, we'll work on some bacteria because they're simple. They're simple, you can manipulate them and their biologists have been manipulating them for decades. So in the end, we spoke to Jim Elkins here and Bob Standard and we came up to that we should be using a gram positive bacteria because gram positive has only a single membrane while gram negative have more than one, two membranes, which means that it's got one too many membranes. So, and then they said, well, which is a good gram positive bacteria that we should work with? Well, they said, there is, first of all, you gotta work on something that is not gonna kill you or you don't need to wear bunny suits and go in there because then we would have to evacuate the entire site. So we didn't wanna do that to put things that are pathogens on the beam. So they came up with this thing called B. septilus. So B. septilus is, Bacillus septilus is found, supposedly if I go now in the backyard right now, I will find some bacillus and there it is. And I could put my hands in the soil and Bacillus will be there. And it's amenable to genetic manipulation and in fact, biologists had done this for many, many decades that had manipulated these bacteria, the septilus in ways that you wouldn't, they sort of thought didn't think anything of it. And not only that, but you're able to buy stocks of these mutants for nothing, like $10 for a mutant and then you can actually use it to then grow your own, et cetera. So we settled on Bacillus because as we said, we have the single membrane and biologists actually know how to mess with it and change it and control it. And that's what you wanna do. You wanna be able to control it and show that you are in charge of that bacillus, not Bacillus in charge of you. So this is what we wanted to do. Although sometimes I think Bacillus was in charge of us, so it's unpredictable. So remember what I said. Remember that nice figure we had before whereby we had all of these various groups and at some point we're able to cancel that. Now, what did I say before? How do, we could calculate, of course, the scattering intensity of something. And in fact, you know something, we could calculate the scattering intensity of a Bacillus. Why is that? Well, because we sort of know what it is made out of, like they sort of know to a reasonable degree of accuracy, the lipid composition, the proteome composition and all of what it's got. So that's interesting to the fact that you could go into an organism and put its scattering intensity and calculate everything and how it should behave. But what's another easy way of doing it that we said? Just trying to experiment later in a contrast theory. Correct. Just do an experiment, you know. As an experimentalist, you should always do an experiment. Don't worry about it because people sometimes sit there and they yak and they yak and they yak and they yak. When you could just go and do the experiment and get it over with, you know, instead of talking all the time, just go and do the experiment. So good thing to know is if there's much doubt and there's a lot of talking, just go and do the experiment if it's possible. If it's only gonna take you a day or two to find out the answer. And so if you take then this Bacillus and you stick it in water, what are you gonna see? Well, remember in water, we're gonna see everything, right? Everything from that bacterium is gonna scatter because nothing is contrast matched. Now, of course, if we go to the next concentration, then the lipids are gonna not scatter. And as we're changing the concentration of H2O2D2 by increasing the amount of D2O, then there's gonna be the proteins don't scatter. And then as we go later on, then the DNA won't scatter. But of course, all the proteins and the lipids are scattering now. So, you know, you only shut them down for a bit and then you come back, they're back again. So you could only shut them down for that one point. And if you look at the scattering, you do the experiment, I think it was Jen that mentioned we do the experiment. Then you see that there's a minimum, right? So if we're integrating this, just measuring the intensity and integrating, nothing fancy, anybody could do it. You guys could go and do it tomorrow. Tommy's gonna give you beam time and off you go. And what you'll get is a minimum. Why do you, where do you get this minimum? Where is this minimum? Well, the minimum happens to be where there's a lot of protein, right? Because these things really by mass, they mostly have protein. So when you make it to around 40 some odd percent, you've now shut down the protein. Mr. Protein volume is zero now, it's muted. So the proteins can, I can't hear them anymore, but I could hear everything else. So you could say, well, what good is that, John? That's very nice, that's a very nice experiment, but what good is it? Well, it's of no use, you know? Really it's of no use because people know about this. It's not like we, this is the first time, but we got biologists on our side this time. We're not going in there with biophysicists and knowing all of this stuff, we're going in there with biologists and they're gonna help us out. Because now we're telling them, could you please do this? And can you please do that? And can you please do the other thing? And of course they say, yeah, but why would you want to do that? And then you explain to them and then they say, okay, if you say so, we'll do it. I don't understand, they shake their heads. You know, why would you want to do that? So let me just digress for a second. I had a conversation yesterday with the guy that I worked on this project and a new guy that I, and they're both in the same group, they're both biologists. The guy that I worked with this project, when I was talking, he understood everything. The other guy was shaking his head, like the same thing with this guy, the first guy was doing four years before. He was saying, well, why do you want to do that? So what is the moral of that story? You have to collaborate with somebody and then they understand your language and then eventually they understand where you're going and you understand what they can do and what they can't do. So the younger guy that I had worked with four years before, he was, yeah, okay, I understand what he's saying. The other guy was saying, what the hell are you saying? So it'll take me another four years to get the new guy on board. So we did that and then we decided that this is not gonna work, right? Because we have now, if we do these things, we always get some scattering and of course the scattering decreases and then increases. But what they could do is this, and this is very important. What the biologists could do is they can manipulate this bacteria. In other words, it has one membrane, which means the lipids all reside in one place. They don't reside in a million places. They happen to know how to control that bacteria. In other words, they happen to know how to turn off certain path things and turn on certain things, meaning that they could turn on biosynthesis pathways or they could turn those off. And they could also then enables you then to feed it the contrast, the biomolecules that you want into it and it will now take and synthesize and put them where you want them to go. So in this case, what we wanted to do is we grew bacteria in 90% D2O, right? So what is gonna happen? You can see here, all of that stuff here has changed into this stuff here, right? Because this bacteria now has taken all of those proteins that it had and turned them into deuterium. Now it's not the happiest bacteria in the world, but it's still doing its thing. It's replicating and growing. So is it the same bacteria? No, but it's still a very functional bacteria that is deuterated now. And it may have its own function in biology if it was all due to who knows, I have no idea, but it's functional. But what does that do to us? Well, it still doesn't allow us, we now have no contrast, right? Because now we could actually make it to a point where we could effectively mute the entire bacteria. Is that good or bad? That's right, it could be good, it could be bad. It could actually be very good if you then introduce then protein into it, right? Because what is gonna happen now, we're gonna break that contrast match condition because we're gonna now put something in it that is not contrast matched. And we're gonna put that in the membrane, which is what we wanna look at. And how are we gonna do that? Well, we have shut down, you shut down its pathway of making fatty acids using an antibiotic called cerulinin. Now you could do this with CRISPR also. So this CRISPR-19, you could do it using now CRISPR, but we didn't have CRISPR at that time. And you could just shut it down with an antibiotic. This is all known, we didn't invent any of this, this was all understood previous in decades before. And then if you do that, then you will create this contrast over here. Right, because everything else still will be deuterated except for the membrane because it will now, you've shut down its pathways, it needs to survive. You now feed it to fatty acids, which was not the seven fatty acids that it usually has, it has usually seven fatty acids. We feed it to a low melting and a high melting, which will go into liquid order and liquid disorder, we think. And if you do the natural bacillus, you'll see all the different fatty acids. If you deuterate it, you will see them spread out because you will see some of these fatty acids moving. And then if you shut down and now you've got the bacterium the way you want it, you will only see the fatty acids that you've known. It is important to know that the bacteria is still making whatever lipid it wants. But the lipids, the lipid head groups could be whatever they want, but the lipid chains are those that you fed it. So all the fatty, all the lipids will have changed the way the bacteria wants to put them together. It will have head groups that they want, where it wants to put them together, but it will only have those two fatty acids. Do you understand? So you've manipulated, but it could still make, it makes what it needs to survive and to be viable. We have not fed it the lipids. We have fed it the fatty acid. It makes the lipids itself. And then when you do that, you have now introduced contrast, which means what we have now effectively have everything opaque except for the membrane. We help. And when you do those experiments, then you find a beautiful curve that then resembles something from a model system because you have now all of these bacteria that are opaque except for their membranes. And their membranes are two-dimensional sheets. And you could fit that with a Q to the minus two. And then it falls into that short drop off, which is the membrane thickness. And that is a Q to the minus four. You know, very standard stuff that you would expect from a vesicle that you could do that same experiment and get that from a synthetic membrane. So this is sort of like the first measurement of a living system where you're looking at the angstrom or nanometer level. The interesting thing is that it's not much different from the thickness of the dead bacteria measured by electron microscopy. And that's okay. And that's okay. So to start finishing off before, like I said, if I do get knocked out, I will return because I have no control over telling it to snooze for some reason. That's because Oak Ridge is telling me, you know, you shouldn't be talking longer than this much amount of time before we turn off your computer. But we're not there yet. So what is the next step? What is the next step there? The next step is to replicate then that 2013 experiment done by Hepperly, but now in this system, which is a much more complicated system. And what are we gonna do? We're gonna guess at the phase diagram, right? We're gonna take a guess and because we only have two lipids and then we're gonna actually try to be clever in devising an experiment that will give us a yes or no answer because trying to analyze the system would be very, very difficult. So what did we do? Well, what you wanna do is set up a situation where you have a control mixture and this is simply to show that we have, and let me say something very, very, that's very important. These bacteria will take the two fatty acids, this anti-iso, and then say in the same amounts all the time. Meaning that they're very precise, if you grow them in a particular way, they're very precise in incorporating, let's say two of those of the A15 and one of the other ones or vice versa consistently, which means that you know exactly what the scattering is because it's consistently taking the same amount. You understand that? And that is very important. That is extremely important because otherwise you would have no control over doing any experiments. So it's very precise in what it takes. It will do that all the time. There are exceptions, but I think that's when things go off the rails. So you do an experiment and you collect a scattering. And let me tell you, now you're looking at the lateral heterogeneity. Before we were looking in this direction and we were looking at all of the bacteria. Now we're looking at structures that are within the bilayer, which means that our signal is gonna be a lot weaker and this is why we need ESS and STS because we need some flux for these kind of experiments. So then what you do is you collect and we have then here again the fatty acid, that's not important. And then what do you do? What would you do to show if there are domains or no domains? So let's say we start with this. I say, okay, you do an experiment, 30% proteated, 70% deuterated, anti-iso, 30%, 70% and 16, it takes it in that amount, you get the scattering. What do you do next? What do you do next is assuming that these things will go, because if they face separate, they will be in different places, right? If they don't, they'll be in the same place, doesn't matter. So what you do, see I'm always looking here even though, because I see your pictures here, even though my camera is nice. So what you will do is you will change the ratio of the mixtures. And it says that it wants to sign me out such that the scattering length density of this mixture is identical to this mixture. Do you understand that? Meaning that when we add up the pluses and minuses of scattering, this mixture is exactly the same as this. Meaning that the scattering length density has not changed. It's exactly the same. But what would you expect if they're randomly distributed? Imagine that we are now at a contrast match condition, at a condition that, and we have just simply changed where things are placed. Remember that diagram before that we had done? Remember this? Remember this? So imagine now if we take this and the only thing we've changed is where they go, potentially. So what you will see is that if this is one scattering, this is exactly the same scattering length density. In other words, when I add up the scattering atoms, they all are the same. I've just simply changed where most of the deuterium is now. I've taken it away from here and put it to this guy, N16. I've taken it away from the N15 and put it there. But the scattering is the same. And that's important. And what you see is there will be a difference if there is domain formation, you will see a change in the scattering. If there is no domain formation, no lateral, they should be no change in the scattering. You should get exactly the same scattering. So even though we haven't analyzed any data here because it would be very complicated, what you see is through experiment is that you see that there are domains that are formed in bacteria, which is something that people have debated. Do bacteria form domains? And they do. And you do it with a very precise experiment. And there is no questions asked because if they were randomly distributed, nothing would have changed. You would have gotten exactly the same scattering. But because you get a change in the scattering means that the lipids having 16-0 and lipids having anti-iso 15-0 are in different places for the most part. So that's how you create then contrast even though the total scattering of the system is exactly the same, nothing has changed except the species that carry the deuterium has changed. So this is the end of that talk, but I've given you questions that we could go over and I've given you also reading that you could look and these are the three papers that are effectively, this paper here is by, is a take of the first paper in that it looks at the dynamics. But what is important, what I wanted to stress is take a look at this experiment here. I'm gonna go back. And like I said, if I do get kicked out, just bear with me, I'll be right back. Is that okay Tommy? Because it's not like I have any control over. They wanna shut me down at some point. Yeah, okay, maybe we can take a break after this and then we'll go through that. If I mean, it's probably a break. You wanna take a break right now and then we'll come back and we'll continue. I had some questions at the end which gives you to think about and I will use my hands but basically what you're getting is when you're considering about looking at these domains and the scattering, you have to consider that there's gonna be scattering from the in plane and the out of plane. And what you would like to do is minimize both of these components when they're ideally mixed and highlight of course, have the capability that they will highlight when they're face separated. So one of the things that I wanted to bring to your attention is that the data collected by Heberle because of our financial constraints at the time when we're starting, if you look at here, you see it says 35% detool. How could you improve on this experiment by Heberle? How could you improve his experiment? My default answer to that is that that's an awful lot of ordinary water in your solvent. Like if you go to more D2O as having your match point, you're gonna have a lot less incoherent scattering. Very good. And that's exactly the answer. That is exact. So what you wanna do when you're doing these type of experiments or any neutron scattering experiment as a rule of thumb, because most of your sample is gonna be water, right? Because in a lot of these systems, you don't want them to be too concentrated because then you'll get all kinds of other scattering effects, right? You'll get structure factor besides form factors, right? And now I'm sure you covered that during the course that you wanna keep the system reasonably dilute. With enough, of course, signal to get something measurable, but you wanna be able to just get the form factor of the scattering function. Remember that we had that somewhere? That was somewhere here, right? You only want this, you don't want this component. You wanna have this component minimized as much as possible because then that adds to it. It's another complication that you don't want to fit the data. And so you're absolutely correct. And because we didn't have the funds at that time to put more deuterated lipids, to then bring up the contrast match point. Remember, we would be able to do that. We did what we could. And then of course it was great, but the paper that I've shown you later on, this paper here, I'm sorry, I'll just keep it this. This paper by Nichols has that. We got the money at that point. And then we were able to, and his experiments are done at much higher H2O, D2O car. I think I have it somewhere. So this stuff here is from that particular paper where they ended up using, I believe, yep, they ended up using, we ended up using about 93 and a half percent D2O because we had all these various components, right? So you're absolutely correct. So that was exactly the answer I was looking for. But it's a good thing to keep in mind whenever you're making experiments with neutrons, make sure that the bulk of your sample is deuterated because the incoherent scattering, if you go to the NIST cross-section table there, the biggest single incoherent scatterer of all elements is protein and that'll kill you every day. So that was one of the things that I wanted to make sure that you understood that, that when you do these experiments, then you'll be able to do that. So the other thing is, it's not always possible, but if you do know the phase diagram of something, then do take advantage of it. And the reason is, then you could maximize then where you're gonna put your deuteriums and where not to, because you'll wanna maximize when they're face separated, you wanna maximize that contrast. So having the phase diagrams are very useful in dictating what your samples are gonna be like. What else did I have then for you guys? So, and I think we've covered this where you could either calculate things reasonably accurately, but then you have to know the volumes of various things in order to calculate, right? Because in order to calculate, let me see, do I have it somewhere? No, I don't, but you need to know the scattering length and then the volume of something in order to calculate the scattering density of something in you. So, but you could just do an experiment. If you sort of know where you're gonna be at and you've got enough sample, well, then mapping it out, there's nothing like an experiment. All the calculations in the world are nothing like an experiment, but calculations will get you close and that minimizes then how much effort you need to put in in order to get the right contrast for your sample for that given experiment that you wanna do. So I think that was all of the things that you sort of need to know to do these type of experiments. And of course, I think working with living systems, if you can, is useful because biologists really do care about what they're looking at. It's hard to engage a biologist when you're giving him a phase diagram of lipids. But when you show him a bacterial cell and you've done these, they're interested in that. They may not understand that, but they're interested. And then their interest may disappear after five minutes, but nevertheless, they're interested at least for five minutes. Let's just say it's incredibly impressive with your contrast-matched bacteria, like that's super cool, informative as well. It can be. And in fact, now what I want, the conversation I was having with the biologist yesterday is to deuterate a protein complex, a protein complex in the living bacteria and then do something that is relevant and see how the protein complex changes again in a system. That will take us a little bit of work, but that's where we're heading, you know? And then if we start thinking about manipulating then bacterial systems, let's say, because they're easier to manipulate than your karyotic or mammalian systems, you could start then developing the tools and that is necessary to attack these problems. And then lots of people could use them at different facilities to do them. So we'll see. And of course, membranes are used for many, many things, right? And so understanding membranes is extremely important. And people are only beginning to really clue in. Membrane work in the last 10 years has grown tremendously. Prior to that, there was very little because everything was proteins and proteins and proteins. But now people have realized that proteins live somewhere and they live in membranes. And membranes are extremely important and lipid composition is extremely important. So it's a good field to be in if you're interested in biophysical work. So other questions, concerns, anything. I actually saw a question by Tony, Tony sent me a message, which I have to read afterwards about some domain work that he had done. Bacteria, but we also have, I think, capsules, virus particles, et cetera. And these tend to be, I guess most membranes are fairly high in proteins as well, but my notion is the virus capsizes and even if the virus with a membrane encapsulated around it, they'll be a bit higher still in proteins than your ordinary cell. So are those viable directions for your future direction with looking at proteins in membranes or is it best to stick to the bacteria for now? The key is this, as we've discussed, you wanna be able to keep your detour a reasonable level. So with a lot of these systems, you need to be able to, then let's say you did an elastics gallery and you did incoherent in elastic. So that you're looking at backscattering or quasi, then you need to minimize the amount of protons elsewhere except for where you wanna look at them. And that's a trick, which means that you need to deuterate everything else as much as possible except for that group of molecules that you want to look at. So this is the discussion I was having yesterday with them but if we're able to do it, then it'll be very, very interesting because, especially if we're able to take some biochemical application, where biochemists know that this is what is happening but they don't know at the molecular level but they know that these things are happening and they understand the chemistry. It would be interesting to look at it then from a biophysics point of view and have some insight. So we have to find those problems. I mean, for a lot of things, we have lots of nails and a hammer. The question is sometimes you don't always have to have a hammer. So this is now a hammer looking for a nail in essence but that's not the way you wanna live your scientific career. You wanna do science. And if neutrons are good, then they're fine. And if they're not good, don't use them. That's what I suggest. So even though I've been at a neutron facilities my entire life, I happen to enable them because they give me a paycheck but there's a lot of things that I do besides neutron scattering. But sometimes it's good to expand the field by finding interesting problems that make people think, oh, neutrons would be very interesting to do that. For example, how many people knew about cryoEM until the last decade, for example, very few. And it was only used by specialists and there was a bunch of people that stuck with it. But now because all of a sudden cryoEM effectively is saying that we may be able to solve problems that you don't need these large crystals needed by synchrotrons and certainly by neutrons. Neutrons require a football-sized crystal which biologists can't deliver. And that's why neutrons and protein crystallography are sort of a mismatch in many ways. But x-rays have been that. But now they're finding that x-rays can have reached the limit. And now in theory, cryoEM could do it. So people like me at a facility that I do science and use all kinds of techniques once in a while have to develop the capabilities to enable neutrons so that a person that is not an expert will say, I want to actually use neutrons because of this. It's telling me that he wants to restart again in one hour and I'm going to tell it four hours. So use, do science, don't do a technique. And if the technique is the right technique, then use it. And neutrons have a lot of advantages. So one of the things that I think that Tommy could help out, and I think you guys have already started and people have advanced is to have deuteration capabilities. But I mean, extensive deuteration capabilities. Because as you saw in this talk, without deuteration, you would not be able to do any of the science of some sort. And of course, if we are going to look at expanding neutrons. So remember this slide I had here. Going back to this slide, remember this, all important, you can see my cursor in the slide. Can you see my cursor? Yeah, sorry, if you put it in a presentation. Better, better. That's better. Okay, so this term here, which is the sample term, which gives us all of this, costs only a fraction of what it costs to build an instrument. Yet we don't put enough money here, but we're more than happy to spend money here. And I think this is very important for this, increasing this budget will allow us neutrons to become much, much more effective. Because right now we deliver instruments, but we do not enable them to do the science. And in our business, we need deuteration. In magnetism or something, they need crystals. Everybody needs, has needs. So I think the facilities have to start thinking how to enable neutrons because they're not commonly used. And in order to attract people, you have to make it, you have to bring down certain barriers and also enable neutrons because they cannot reach their full potential unless you have the right samples. So most of the experiments that are done are done in less than ideal conditions because people cannot afford, as you saw, we cannot afford to deuterate things. Other questions? Please shoot. Shoot, don't worry about it. I mean, Orinel is paying me to do this. So don't worry. I can think of worse places to be. So, John, one of the things that you are always, when you do the deuteration, I think people can do it with bio-deuteration and the challenge is often to separate the damn things. So you end up with a mixture of everything and then in particular, lipid, handling of lipid is rather challenging because they solubilize in some solvent and some solvents, they don't solubilize. And if you treat things here and there, the whole thing precipitate out to undefined gooey, not the very pure, but you can't actually do anything with it. Well, that's why I've written lately two big proposals which have not succeeded yet on making synthetic lipids, deuterated capabilities. And in my opinion, unless we are able to do that, neutrons can never reach their full potential. They cannot. I mean, it doesn't matter how much flux you got. It doesn't matter how fancy people make it up. If you don't have the right samples that make the maximum use of that probe, then the probe cannot shine. It cannot really show what it can do. And to this point, in North America, we failed miserably. In Europe, you failed less miserably. And in Australia, at least they actually have a group that does something, even though it's not extensive, at least they're doing something. And I think you guys also have, Hanna is doing things. Yeah, Hanna is doing. So for those of us who is in Sweden, you can actually contact Hanna and if you want to lipid, that can help you and it actually turns out that one of the persons who do the synthesis, Anna Leung, actually is trained in how she comes from the coming table in Australia. So there is, and also in ISIS, they do. So there is a community. There is, and I think we need to expand because it needs to be much more expensive and much more to people. Because like I said, you can not fully exploit. Like, a perfect example that was this, where we had early experiment with 34% because we just didn't have the capability and buying anything from Avanti, it costs you, you might as well just give away your first two children to them at that point. So we need to do that as a community because we invest millions and billions of dollars in the facilities, but then we invest zero or very little in making sure that we maximize it. And we could say that as far as software is concerned, as far as many things. You know, we have to make it accessible to the novice because the novice is not doesn't understand and think about neutrons. It's not like when I started in the Stone Age, where neutron facilities didn't have user programs, the people at the facility were the world that ran everything and they understood neutrons inside. And now, well, okay, well, that's the way you're going to run things and that's fine, but if you're gonna have a user community, then it has to be very accessible to the person that has no idea and doesn't really wanna run very much about neutrons, they just wanna use it. So we need to do that. So I would like to encourage people to just demand things. I mean, otherwise facilities doesn't improve. And I think that's what we said already in the beginning that contact the neutron science, the beam line scientists. If you want to do an experiment, just contact them, just talk to them and so on. And they're extremely happy to help. And they, this is the way we push the science forward. So they can give cost cause it's up to the user to require things. And the users are actually the ones that pay. We are all tax payers, so we pay for the facilities. So we should make use of them and make use of them to do good science. Absolutely, I think these large facilities, I think, I don't know how many of you are gonna be professors at universities in the end and all of that, or are already, making use of large facilities is a great bang for the buck. Like they say, a great bang for the euro. Yeah, yeah, exactly. The euro, because you get a lot of help that is get provided by the facilities. So you don't have to have everything in your own lab. You can make big uses of these multi-billion dollar facilities. And it doesn't cost you anything. And not taking advantage, you're not gaining anything anyway, but I think of that. But have the right questions, force the facility people to think, what they could do one step above. This way it forces facilities to think, not do the same old, same old, turning the crank, like they say, boring stuff over and over and over or again, the same stuff, make them think. And that can only happen from the user community because the user community hopefully is thinking about doing their science. And this way the facility people then start expanding their horizons because they're starting to say, oh, that's an interesting question. Maybe we should look into it, see what we can do. So I think that's important to think that. And so like I said, do the science. Don't become technique-centric. Even though I'm at a facility, I do a lot of other things, but I also try to expand the neutron horizons because I do a lot of science. And I have my foot in both and I have, I have a lot of toes in different places. So, but that happens with time. So, is there any final question for John? You all exhausted after this is the fourth day. One more day to go. It's just my first talk. I mean, I'm fresh, I'm ready to go. I'm ready. But now, let me look at it. Not only that, but it's one more day after this. Ha ha ha. But I think you're, if you have any question, don't hesitate to contact John. I mean, it's a long, long experience with the facility and eager to help you. So that is. And you can always call me, you know, you can always phone and I'll get a phone call from Sweden. It's not the Nobel Prize, but the next best thing, a good person to talk. Okay. This is my question. You can call Satik here. Helen, Helen, yeah. Yes. Hi. It's not a question. Hi, I just wanted to say thank you for a super interesting and cool talk. It was really nice. Well, thank you very much. It's my pleasure. Like I said, I'm not a morning person, so I had to get up six o'clock my time to make sure that, and so my partner was telling me, you know, are you up? So she gave me the elbow to get it. But then, you know, this is really what I enjoy is interacting with people. And this is the one thing that I've really missed. You know, I used to give lots and lots of talks and in the last, well, like all of us in the last year and a half almost now, we've been doing all of these Zoom meetings, which they're fine, but you don't get to really interact with the people to see your faces and how you, you know, cause you get a lot by seeing people. And so I really liked this. And so I'm glad that Tommy and Trevor gave me this opportunity to speak to you, even though it was the resume, so I appreciate that.