Episode 20 Transcript

We discover how this wavelength of light can help us look back in time and learn how ancient civilisations lived, help preserve works of art, and even help track down the root cause of cancer.

 

Frank Martin: “Maybe stem cells and altered forms of stem cells underlie the pathogenesis of diseases such as cancer; and being able to identify the in situ location of altered stem cells will be pivotal in actually targeting therapy to wipe out those altered stem cells.

 

Meera: Frank Martin will be revealing how such cells could be identified later in the program, when we also here the latest news and events from the light source. Hello, I’m Meera Senthilingham and this is the September edition of the Diamond Light Source Podcast. One feature which helps Diamond stand out from other scientific institutions is the wide range of science taking place there. Research at the beamlines looks at all fields of science, from jet engines and new materials, to viruses, cancers, and even cultural heritage. In the past, Diamond’s beams have been used to investigate the Mary Rose and the Dead Sea Scrolls, and now Diamond’s infrared or IR spectroscopy beamline is offering hope to those trying to preserve ancient works of art, as Gianfelice Cinque, principle beamline scientist on the IR spectroscopy beamline explained on a recent visit to the Ashmolean Museum in Oxford.

 

Gianfelice: “We’re using the Infrared light produced from Diamond, from the synchrotron, to do a couple of things; microscopy, spectroscopy, and we are combining the two techniques. So the infrared is quite a sensitive probe for organic material, so we can really probe the protein content, the lipid content, the nucleic acid, without doing any staining. At Diamond, we have high flux, high brightness produced by the synchrotron, which allows us to do micro-spectroscopy. So we have a very high, spatial resolution which is unachievable by conventional methods. I would like to stress that the infrared light is also very mild, so it’s non-destructive. On the other hand it’s very sensitive to tiny residues on organic materials, or molecular composition.”

 

Meera: “What have been the variety of applications to date using infrared?”

 

Gianfelice: “Most of our work is in biomedical applications. So we’re doing analysis of single cells, tissues, cancer research, chemotherapy. But of course there is quite a strong interest nowadays in archaeology, cultural heritage, and conservation of paintings, fragments of paintings, pottery, plus samples from ancient towns and other things”.

 

Meera: “And how does infrared spectroscopy actually work to provide this great level of insight, and also in a non-destructive way?”

 

Gianfelice: “Well, the infrared is capable of probing the molecular structure. The light is mild enough, but it’s the right wavelength to excite the molecules, and from the molecule absorption we can say what composition you have got in the material. Now the micro-beam available at my beamline allows us to have micron-resolution, so you can distinguish the different composition in a few microns scale. So you can do both the full field microscopy which is called ‘images’, so having a large snapshot of an area and disentangle where is which molecular composition or you can do confocal microscopy or spectroscopy and say what is different in a tiny sample, in different areas of the paintings for example of a plaster section.”

 

Meera: “So, we are in the Ashmolean museum in Oxford at the moment, where there’s a variety of cultural heritage items that this infrared could be used on, and so we have a painting next to us here by Van Dyke, which is gigantic. It’s about 3 metres high, and about 2 metres wide. So I’m assuming, obviously, with this you wouldn’t put the whole thing into Diamond, so how would you set about analysing this?”

 

Gianfelice: “Typically people brush small fragments from an area of interest, and we take these fragments and we micro them. So we cut a very thin slice, and we can go through the analysis of the different layers of the painting, which the painting is made of, and try to understand what is there. What we can do at Diamond is work in the medium frame, which is a different spectral region, in which you’ve got the really molecular formation, you’ve got the one to one correspondence with the absorption bands and the composition, the molecular composition. So in this way we could say where the inorganic layer is, where the dye is, what is the binding layer, or what has been added later, or if there’s a protective layer, or the environmental effect, like degradation over time, owing to the ultraviolet, or just the air surrounding it.”

 

Meera: “So it can tell you about the history of the painting, and also how the painting was formed in the first place.”

 

Gianfelice: “Yes, you can get information about the way in which it’s made and also information about its history, the way it’s been handled.”

 

Meera: Gianfelice Cinque, principle beamline scientist on Diamond’s infrared spectroscopy beamline. Now, conserving works of art is a major challenge to those working in the field. But, as well as conservation, these heritage teams are also keen to learn more about how these works of art were created in the first place, something the non-destructive and probing analysis of infrared can offer insight into. Mark Norman, Head of Conservation at the Ashmolean Museum, discusses what his team are keen to find out.

 

Mark: “Especially, what things are made of. So we’re interested in pigment analysis, pigment identification, constituents of metal alloys is another area. With something like Renaissance bronzes, we might be interested in the paccination supply to the bronze, and a lot of work needs to be done on that. So that’s a project we’ve got up our sleeves sometime in the future”.

 

Meera: “And of the things that you’ve heard Diamond could offer insight into today, what were some of the things that you think might be quite useful with the samples you have here at the moment, so the painting behind us for example?”

 

Mark: “It’s a painting by Van Dyke of Christ being taken down from the cross, and we’ve done optical microscopy, and we’ve taken pigment samples, we’ve done polarised light microscopy. So we can get the basic mix of the pigments that were being used, but each pigment has got a lot of other constituents in it, so we’d like to be able to break that down into those constituents, to get a better idea of the true nature of the paint that Van Dyke is using at that point.”

 

Meera: “And the spectroscopy and the infrared could hopefully do this.”

 

Mark: “That’s right, yes. This is a group of tomb figures from about 1500 BC, and they show butchering, bread making, beer making. And these are the people who fed the deceased in the afterlife.”

 

Meera: “And how old are these?”

 

Mark: “They’re about 3500 years old. They’re made of wood, and it’s covered in a thin layer of plaster, and then the plaster is painted, and they’re a bit like Playmobil people actually. They’re articulated, so their arms move.”

 

Meera: “So what are the kind of things you would want to know about these?”

 

Mark: “I think with these, we’d quite like to know what the medium is that binds the paint, we’d like the pigments confirmed, there’s red ochre, there’s probably carbon black, there’s chalk white, but it would be good to have that confirmed. We’d probably be more interested in the medium that actually binds the paint together.”

 

Meera: Mark Norman, from the Ashmolean Museum. A field of heritage currently benefiting from the analysis that infrared beams can offer is archaeology, more specifically, the building materials used by ancient civilisations, and their manipulation of local resources to improve their surroundings. Emma Anderson from the University of Reading has been using Diamond’s beamline to find out more about the craftsmanship and design skills used by people during the Neolithic era.

 

Emma: “I’m looking at architectural materials from the Neolithic era, which literally means ‘the new stone age’. The Neolithic occurred about 11,300 to 8000 years ago in the Near East, which is the area of Turkey around Iraq, the Mesopotamia area, where they think agriculture really started. That’s what really characterises the Neolithic, the beginnings of agriculture, the domestication of animals and crops by humans, and the beginnings of the first permanent settlements.”

 

Meera: “And what aspects of this period are you researching and focussing on?”

 

Emma: “I’m looking at their architecture and their building materials, and also the pigments that they used in things like wall paintings, and in mortuary practices because they paint skeletons and animals bones in red and black paint.”

 

Meera: “What’s really known about this so far, say, with regards to the pigments used and so on?”

 

Emma: “They tend to build their structures out of natural materials, so the soils and sediments in the areas surrounding the site. So, for example, Çatalhöyük is one of the sites I’m studying, which is located in what is now central Turkey. It was a small town where people lived around 8000 to 9400 years ago. There was probably around 8000 people living there at the height of its occupation. Their houses were built out of mud brick which they’ve then plastered with very pure white plaster on the internal surfaces and on floors. On those surfaces, you find a lot of art work, made out of red and black pigments, in lots of different designs; so they’ve got abstract, they’ve got hunting scenes, different geometric designs and things like that. And they also plastered animal skulls and put them on the wall as well.”

 

Meera: “And so within all of this you’re looking at the actual pigments they use of the walls and the floors?”

 

Emma: “Yes. I’m looking particularly at the red and black pigments in one of the wall paintings at this site, which was found in one house above a platform which contains nine human burials, so we know it’s an important area of the house.”

 

Meera: “And what really are you sampling, what are you trying to find out about the pigments?”

 

Emma: “I’m looking at the manipulation of sediments by these people in order to make and decorate the houses and to form the wall paintings, what they’ve made the paintings out of.”

 

Meera: “So really you’re looking at how they made the most of their natural environment and used it to form these decorations and decorate their immediate environment.”

 

Emma: “Yes. I have sub-samples from different architectural materials, and also I have thin sections through sequences of architectural materials that are on glass slides. They’re very thin. They’re only 30 micrometres thick, so it means that you can look at them under a microscope and look at the microscopic structure of these architectural materials, and also the pigments in the paintings. So in terms of the artwork in the wall painting I’m looking at in this house, I have a cross-section through all the applications of wall plaster, including the actual painting, which has a layer of red paint and then a layer of black paint . The archaeologists believe this was probably through reapplication of paint in a different colour in the same design.”

 

Meera: “And are you really looking to see the structure of these samples, and the structure of these pigments to basically figure out what they are?”

 

Emma: “Yes. I want to know what they’re made of, what these people are mixing together in order to make the pigments to apply to their walls.”

 

Meera: “How do you set about analysing that then? What methods of spectroscopy or analysis are you using to get that insight?”

 

Emma: “I’ve been using a range of techniques to look at these samples, but my primary technique is infrared microscopy, to look at the minerals that they’ve been using, because it’s really good at identifying those, which is really important when you’re looking at sediments and soils, because that’s mostly what they’re composed of. It’s really minerals that are making up the different soils and sediments that they’re using to make the components of their houses and also the pigments.”

 

Meera: “What have you managed to see so far? What were they using during this time to decorate the world around them?”

 

Emma: “Looking at the layers of paint, they’re very very thin. They’re about 20-25 micrometres in thickness. They’re also made of a mixture of components. In order to isolate each individual layer, to isolate the individual components in them in order to find out what they’re made of, I had to use the synchrotron source at Diamond, rather than a conventional thermal source, which I’ve been using to do experiments at the University of Reading. Because of the improved spatial resolution, it meant that I could pick out all these individual minerals. It helped me to find that the red paint actually contains a mix of calcite and clay which is probably from the source which is found underneath the site, so the local sediment. This was mixed with small grains of obsidian, which is a natural type of volcanic glass that is found further away in the volcanic areas; these may have helped to improve the optical properties of the paint. So, for example, they could have made it more reflective. In modern road paint they add small glass beads in order to make it more reflective when the light from your car headlight hits it, so you can see it brighter. In these houses, which were really quite dark inside, the only natural source of light came through an entrance hole in the roof; everything else would have been through fire. Because it was quite dark in these buildings, anything that could make that paint more reflective would make it stand out more in the building.”

 

Meera: “So they really seem to be understanding these materials around them and manipulating them to make their surroundings brighter?”

 

Emma: “Yes. I think they know about things like lime burning and firing pottery. They’re experimenting with these things at this time.”

 

Meera: Emma Anderson, from the University of Reading.

 

 

Meera: You’re listening to the Diamond Light Source podcast, and this month we’re seeing red, infrared, to discover how this wavelength of light can offer insight into research as diverse as ancient civilisations and preserving works of art, to studying cells and the onset of cancer. Still to come, we discover how stem cells can be analysed and given fingerprints for earlier diagnosis of cancer. But first, it’s time for our news update with Sarah Bucknall, starting with the identification of a new material.

 

Sarah: “A group of researchers from the University of Oxford and Diamond have discovered a new material. It’s a calcium hexaboride compound, but it’s crystallising in a previously unknown crystal structure. The ordinary calcium hexaboride is a semi-conductor which has amazing hardness and a high melting temperature, and that’s governed by its high chemical stability. That material has been investigated for a long time, due to its relevance to a lot of industrial production processes such as the manufacture of boron alloy ultra high-strength steels. The new research, which was published in Physical Review Letters, suggests that by choosing the right metal and synthesis conditions, it might be possible to achieve new electronic, magnetic or even superconducting properties.”

 

Meera: “So how did the researchers actually investigate this to see that this was possible?”

 

Sarah: “Well, the Oxford team created the novel substance by imposing high pressures and temperatures on to a normal calcium hexaboride crystal, and they then used Diamond’s extreme conditions beamline, I15, to take the measurements of their crystal. So although the new calcium hexaboride phase was formed under really high pressure, the researchers had to succeed in quenching it down to ambient pressure, so that it would have the potential to be used in applications. So the next step is to develop the material further for potential applications.”

 

Meera: “Away from this, there have been developments in other areas of material science at Diamond, in particular a material for carbon capture. This would capture the carbon dioxide in our atmosphere and alleviate this problem somewhat in the future.”

 

Sarah: “Yes. A group from the University of Leeds faculty of Engineering have been using our high-resolution powder diffraction beamline, I11, to investigate the use of calcium oxide based materials for carbon capture. So these types of materials are low cost and high abundance, and they have a large absorption capacity and fast reaction rates during the chemical process, so they’re a really good candidate for absorbing carbon dioxide. However, there is a need to study them further, because through the process of capturing CO2, the calcium oxide material actually suffers from wear. SO research is underway into making these materials more durable.”

 

Meera: “And how do you set about doing that? How do you try to make a material more durable?”

 

Sarah: “The Leeds team is interested in the current method used to help the material maintain its capture capacity, and that’s a process of hydration. They’re interested in this because it results in the material suffering a reduction in its mechanical strength. SO the idea is to solve this problem so that the material can maintain both its capture capacity and its mechanical strength, so that it will become a longer lasting option for carbon capture.”

 

Meera: “What do their results show? How have they made it possible?”

 

Sarah: “Their results, which were published in the Journal of Energy and Environmental Science suggest a mechanism for the interaction between calcium oxide and water during the hydration process. So the next step is to work on improving these materials, so that they can potentially become a low cost answer for carbon capture on a very large scale.”

 

Meera: “And there’s just one more research story. This moves us more into the life sciences, and the field of photodynamic therapy, which has been around for a while, and it’s quite a good therapy used for cancer treatment.”

 

Sarah: “That’s right. Photodynamic therapy is a highly selective technique for destroying cancerous cells, whilst ensuring that most healthy cells go unharmed. A collaboration of scientists from Diamond and the University of Reading have revealed the binding mechanism of the so-called light switch effect complex, and that’s a type of chemical compound that fluoresces on binding to DNA. SO that’s the kind of compound that would be used in photodynamic therapy.”

 

Meera: “And so how do these compounds actually work?”

 

Sarah: “The compounds are designed to kill cells when activated by means of a light source. So, when the compounds are irradiated they then bind to the DNA and form an ultra-reactive oxygen within the cell which then destroys them.”

 

Meera: “But the therapy has been around for a while, so what’s really been developed here, what’s new?”

 

Sarah: “Well, until now, previous research was only able to determine which chemical compounds would bind with DNA when irradiated, and the details of the interaction were highly debated. So this research, which the team published in Nature Chemistry, explains exactly how the photoreactive metal complex binds to DNA, and that actually reveals that the binding is sequence specific. So the structural data collected on two of Diamond’s macro-molecular crystallography beamlines shows that the compound will only bind with DNA with key A-based pairs, but not AT.”

 

Meera: “And how does this really come together as a targeted therapy?”

 

Sarah: “This means that the group can now look towards targeting an individual DNA sequence, and that can potentially allow future development of highly-specific binding agents; this, in turn, might pave the way for improved cancer diagnostics.”

 

Meera: “So a very current field indeed. Thank you, Sarah. Moving away from the research now, you have an investigators award coming up.”

 

Sarah: “Yes, right now we’re calling for nominations for the 2012 Young Investigator award. This is an award that’s given to an early career scientist who has shown exceptional contributions to a research project using synchrotron light.”

 

Meera: “How do people get nominated?”

 

Sarah: “It’s supervisors who can nominate a young researcher for the 2012 award. They can just use the form that’s available on our website. The deadline for nominations this year is the 30^th September, and the winner will be announced in November.”

 

Meera: “Excellent, look forward to seeing that winner. You’ve also had quite a bit of success this year with your open days, which have been running for as long as this podcast has. But you did something special this year.”

 

Sarah: “That’s right. It’s our 10^th anniversary year. So in June we held a special edition of our open days. They’re a really great success. We had almost 2000 visitors come to Diamond, and they were able to meet our many scientists, engineers, technicians, support staff, as well as our users, who all volunteered their time to explain and demonstrate how the synchrotron works and what it’s used for.”

 

Meera: “Those were special events. That was five days in a row almost. But you’re heading back to your single open days soon. When’s the next one?

 

Sarah: “The next Inside Diamond day is on Saturday 17^th November and the details and registration will be available on our website shortly: that’s diamond.ac.uk.”

 

Meera: Sarah Bucknall, from Diamond’s communications team. Now, it’s time to start seeing red again, as we continue probing into the scientific developments offered by the use of infrared beams of light. We’ve already heard about the range of uses this non-destructive approach can have in investigating our cultural heritage, but its uses span out into the life sciences as well, as Gianfelice Cinque, who we heard from at the start of this podcast, explains.”

 

Gianfelice: “The point is that you can have a sensitivity, a signal to noise, like we say, so a spectral quality. And a spatial resolution which was unachievable, say, ten years ago. So in this case, you can start looking at a smaller scale than before, you’re really looking on the microscopic scale: cellular structure, sub-cellular structure, which before was quite impossible to sample in biomedical applications. Now, in cultural heritage you can look at the inclusions, you can look at the morphology of the sample and the chemical composition, at a level which probably before has been overlooked, because it was just an average measure of some relatively large area, so 100 microns.”

 

Meera: “Now, you’ve touched on the fact that infared is used widely in biomedical applications, samples and testing, could you just give an overview of the examples of biomedical applications.”

 

Gianfelice: “A typical example is cancer research, so the study of single cells. There are plenty of cell lines we can use nowadays for the purposes of mimicking the behaviour and evolution of a cancer. SO the question is how does the cancer evolve. You can test therapy drugs. So you can test the same chemotherapy drug, a different dose on the same cell line, which is identical in all its replicates. And you can assess in which dose you get a response, and to what extent you get a response. So from the infrared fingerprint you can understand the biochemistry which is going on at the single cell level. Now you can also do this in a nucleic area, so you can look at the nuclei of the cell and see if there’s something happening there, or is there something happening at the membrane, or in the cytoplasm, and you can do this with a microbeam, with an infrared microbeam from the synchrotron.”

 

Meera: Gianfelice Cinque, principle beamline scientist on Diamond’s infrared spectroscopy beamline. One promising area of research in this field is the identification of cellular fingerprints. Professor Francis Martin from the University of Lancaster is using Diamond’s infrared beamline to identify fingerprints for and learn more about the cells underlying the entire formation of the human body: stem cells.”

 

Francis: “The major conundrum that we have in stem cell biology is that, despite four decades of intensive research there are no bio-markers that are robust for what we call the stem cell. We know that there are several tissues in the human body that are regenerative. We know that certain cell types have the ability to differentiate into functional or differentiated cell types. But the actual progenitor cell, ie. the stem cell, remains a mystery. And there is a great interest, for a number of different reasons, in identifying a marker, or a phenotype, or a fingerprint of a stem cell, and this could be applied for a number of different purposes.”

 

Meera: “When you say ‘identifying a biomarker within these cells’, how would this be used if one is found?”

 

Francis: “Well, a biomarker would be used to track the lineage of the stem cell. We could identify where the stem cells reside. This would allow us to conduct more fundamental research into their biology. This could then be applied to, for instance, regenerative medicine. What makes a stem cell a progenitor cell? Can it trans-differentiate into other cell types? Could this be used as a treatment modality for degenerative diseases? Or, for instance, might a stem cell be a precursor to disease? If an alteration in a particular stem cell occurs, could it give rise to, for instance, cancer? So if we can identify a stem cell within a heterogeneous mass of cancerous tissue, can we target that cell in order to cure the cancer?”

 

Meera: “And essentially stop it spreading and forming the cancer?”

 

Francis: “Absolutely.”

 

Meera: “So how are you setting about looking for these biomarkers and therefore trying to find these cells?”

 

Francis: “Our hypothesis has been that stem cells have a fingerprint, distinct from that of the other cells within a particular tissue, the trans and amplifying cells, the differentiated cell types. So what we have set out to do is exploit IR spectroscopy to fingerprint different cell types, and we specifically used Diamond for this because Diamond gives us an aperture which is not dissimilar to the size of a cell. And this allows us to interrogate particular tissue types, and compartments within the architecture of the different tissue types; so identify different cells, individual cells. The basis of this approach is that a spectrum derived from this IR spectroscopy approach is a fingerprint, a spectral fingerprint of those interrogated cells. We can then exploit the fact that the stem cell will have its own spectral fingerprint which is distinct from the trans and amplifying and differentiated cell types.”

 

Meera: “Essentially, are you aiming to see the different patterns and the different biological entities that make up a stem cell, and therefore get this fingerprint for it, and therefore be able to identify them amongst a mass of cells.”

 

Francis: “First of all, to identify whether we can extract a fingerprint spectrum or phenotype of what we might call a stem cell. The second thing is to identify whether we can understand the mechanism that characterises the stem cell. Is there an alteration in DNA confirmation that’s associated with this stem cell? Is there a reduction or elevation in RNA levels associated with this particular stem cell? Or is there, for instance, lipid levels associated with this stem cell or not associated with the stem cell?”

 

Meera: “And how far have you gotten with this work? What have you managed to see by way of a fingerprint so far?”

 

Francis: “We’ve been able, first of all, to characterise in situ where the stem cells are, using IR spectroscopy, in human tissue, which is quite a unique experiment. The vast majority of the work has been conducted in mouse models. We’ve applied this to quite a number of different tissue types. We’ve done it in human skin, we’ve done it in human breast tissue, in prostate, in human gastro-intestinal tract, in the cervix and in the metrium. What we’re coming up with is a uniform and consistent biomarker that segregates the stem cells. We can then use this uniform and consistent biomarker to hunt out where the stem cells reside, in these less investigated tissue types.”

 

Meera: “How is it hoped that this insight could be used in the future?”

 

Francis: “Can we, for instance, exploit our identification of stem cells, either to combat disease or to remediate disease? We are living in a world where the population is getting older, as a consequence, we’re suffering more and more from chronic age-related disease. A major means of combating chronic age related diseases would be to develop stem cell biology and regenerative medicine in order to address those problems. That includes neuro-degenerative diseases, cardio-vascular diseases, and diabetes. The other side of that coin is that stem cells and altered forms of stem cells underlie the pathogenesis of diseases such as cancer; and being able to identify the in situ location of altered stem cells will be pivotal in actually targeting therapy to wipe out those altered stem cells.”

 

Meera: So watch this space, as you may soon see some biomarkers or cellular fingerprints in the future. That was Francis Martin from the University of Lancaster. Now, that’s almost it for this month, but before we go, we continue our celebration of Diamond’s ten year anniversary, with scientist Paula Salgado from the University of Newcastle, who’s a regular user of Diamond, and has fond memories of her first visit to the synchrotron.

 

Paula: “As a protein crystallographer, I’ve been using Diamond’s micro-molecular crystallography beamlines for the last four years. My first trip to Diamond was in September 2008, on a sunny Saturday. As I drove on the motorway from London, I was excited to finally see this amazing facility working. Since then, I have visited Diamond often; some trips more successful than others, but each visit a learning opportunity. The discussions with the extremely supportive beamline staff help us collect better data, and, hopefully, contribute to further develop the beamlines and all the associated facilities. It is a joy and a privilege to be part of such an exciting and thriving community. The development of new beamlines will bring new challenges for both Diamond staff and us, the researchers coming to collect new data. What new projects and new ways to probe proteins can we explore together? The future will no doubt bring exciting answers.”

 

Meera: Paula Salgado from the University of Newcastle. Now, that is it for this edition of the podcast, but do join us again at the end of October, as we bring you more research and discoveries taking place at Diamond. In the meantime, however, if you have any questions about Diamond or the research taking place there, the email address is podcast@diamond.ac.uk, or you can listen to previous editions of this program online at diamond.ac.uk/podcast or nakedscientist.com/diamond, you can also subscribe to the podcast on itunes. Thank you this month to Gianfelice Cinque, Emma Anderson, Frank Martin, Sarah Bucknall and Paula Salgado. I’m Meera Senthilingham, and until next time, goodbye.