Imaging the Invisible

Ben - The optical microscope revolutionised science.  It gave us access to an unprecedented world too small to see with the human eye.  It's still a relatively simple design, essentially a light source and a series of lenses that magnify the image that you're looking at.  There are also some very simple ways that we can adapt it to get increased resolution, a very tight depth of field and even three-dimensional images. Confocal microscopy was invented by renowned scientist Professor Marvin Minsky back in the 1950s and it's now being put to excellent use by, amongst others, Dr. Abigail Woodfin from the Centre for Microvascular Research at Queen Mary University of London.  Abigail, first of all, could you just tell us a bit more?  What is confocal microscopy?

Abigail -   Okay, so confocal microscopy refers to the technique of looking at three-dimensional objects through a series of two-dimensional slices, much like an MRI machine would look through your body during a scan.  After you've taken the series of slices down through a three-dimensional object, they're reconstructed to rebuild the 3D object that you're looking at.  And that enables you to get much better resolution than if you were looking at the entire object in one go.

Ben -   So this is very much like computerised tomography.  So for example, when you take a series of x-rays and then put them together, you get that wonderful view that you can sort of flip-book through the image and see an entire 3D aspect rather than just having to look at the one plane, the one single two-dimensional image.  How does that actually work?

Abigail -   So it's used in conjunction with fluorescence - illumination of the objects you're looking at.  So what we do is we use antibodies which are generated to specifically bind one particular protein and the antibodies have a coloured fluorescent tag bound onto them.  So when you add these antibodies to your cells or tissues, or whatever you're interested in, they bind to your particular protein, and you then use laser excitation to see the location of these fluorescent antibodies.  The objects we're looking at are so small that they are essentially transparent in the absence of using these coloured tags to mark the particular structures.

Ben -   What sort of structures are you actually marking out with these tags?

Abigail -   We're specifically interested in the blood vessels and how white blood cells or leukocytes react with blood vessel walls during inflammation.  So we would use antibodies which bind to proteins within the blood vessel wall such as one called PKAM and we also use cells where a gene for green fluorescent protein from jelly fish has been inserted into the white blood cells so the white blood cells themselves also have a green fluorescent colour.

Ben -   So there's lots of tricks you can employ to make sure that you're only seeing the bits that you want to see.  Under your microscope or in the images that you get, what does the rest of the surrounding tissue actually look like?  Is it completely invisible?

Abigail -   It depends.  When you're looking with transmitted light or say, a normal white light, which has all of the different wavelengths of light in, then you can see sort of some textures and some structure, you can see blood flowing through blood vessels, and you can see the white blood cells as transparent spheres rolling along the inside of blood vessel walls.  However, when you're looking with the laser excitation to look at the fluorescent tags which you've got in your tissue then only the fluorescent tags will be picked up.  Everything else will be negative or won't have any colour.

Ben -   So, quite often with advanced microscopy techniques, what we need to do is take the sample that we want to look at and then we freeze it or we section it, or we prepare it, we stain it, and ultimately it means that it certainly can't be part of living organism.  This sounds like if you're seeing blood flowing through things that you can actually use this with something in situ while it's still alive.  And so, rather than having to try and take a moment frozen in time and use that to guess what's happening, you can actually watch the processes taking place inside blood vessels.

Abigail -   Yes, that's exactly right.  Confocal microscopy has existed for some time, but the time it took to take one of these 3D images was such that it was not really possible to look at dynamic cellular interactions and also, we needed the ability to add the fluorescent tags into living cells.  So, the work we've been doing has involved fluorescently tagging the living cells and looking at the interactions with white blood cells with blood vessel walls in living tissues.

Ben -   So if in order to see this in 3D, you need to take the series of slices, it's going to limit you for how many essentially frames per second you can take.  What is the temporal resolution?  Are you seeing this in close to the 24 frames per second that we see on cinema screens or are we looking at something at a little bit more time lapse.

Abigail -   It's a little bit more time lapse.  For the work we've been doing most recently, I've been taking one of these 3D images every minute.  I could increase that slightly to doing say, two per minute, but for the size of tissue structure that I want to look at that's my sort of temporal limit of resolution.

Ben -   Still, that's a phenomenal step forward in what we're actually able to see and create videos of these incredible processes happening.  What's it actually told us so far about physiology?  What's it led us to learn?

Abigail -   So the study of the interaction of white blood cells with blood vessel walls has been going on for some years, but has been limited by our ability to really look at these dynamic interactions in real time, in vivo, or on living tissues.  So, now that we've managed to get this increased spatial and temporal resolution, we've been able to actually watch the process of white blood cells migrating through blood vessel walls and into the surrounding tissues. 

And answer, questions that have not been able to be answered previously using the sort of snapshot images of exactly what route the white blood cells take through the vessel wall, how long they take to do it, do they move in just one particular direction, or do they sort of change directions.  And we've managed to identify that the white blood cell migration can exhibit a sort of multidirectional behaviour so that, they go out with the blood vessel wall, they can also in some cases return back into the blood flow.

Ben -   So it's obviously opening some doors that were not open before, what do you think will be the next step?  How are we going to make this better and take it further?

Abigail -   I would say, improving the temporal resolution would enable you to look at faster processes.  The things I look at are sufficiently slow cellular interactions that this temporal resolution is sufficient.  But faster temporal resolution would enable you to look at faster processes.  Higher spatial resolution would enable you to see the smaller structures and interactions. 

I think also, increasing the quality of the fluorescent tagging would enable you to see different things.  So for example, the protein PKAM in blood vessel walls I mentioned earlier is what we use as our blood vessel marker, and that's the best marker that we have found.  But if we could develop ways of fluorescently tagging a whole host of different proteins within the blood vessel wall then that would enable us to deduce things about the functions of those proteins as well.

Ben -   And that will give us a whole new multilaminar way of looking at these things, I suppose.  Well thank you very much, Abigail.  That's Dr. Abigail Woodfin from Queen Mary University of London.

Embryonic nerve cells transplanted into a recipient brain survive, wire themselves up and can even correct a metabolic disorder in vulnerable individuals, scientists have discovered.

Successful brain repair in the future will almost certainly depend upon the implantation, within the injured or diseased nervous system, of healthy cells that can replace those damaged by degeneration.

But the fate of these fresh cells, when added to an abnormal brain, and whether they can functionally wire themselves up to and support existing nerve circuits, is poorly understood.

Now scientists have proved that these cells can do this by using cell transplantation techniques to remedy an obesity-triggering metabolic disorder in mice.

Writing in Science, Harvard scientist Jeffrey Macklis and his colleagues transplanted into the obese mice, which lack the receptor in their brains for a satiety-signal called leptin, 15,000 nerve cells collected from 13 day old mouse embryos.

The cells, which also synthesised a glowing-green pigment to discriminate them from the hosts' brain cells, were implanted into a region of the brain known as the medial hypothalamus, which controls appetite.

Five months later, the researchers looked for signs that the donor neurones were still present in the brain and were speaking electrically with their host brain neighbours.

The implanted cells, they found, had wired themselves in and were functional. They also found that, compared with control animals and mice that had received grafts from other brain regions, the treated animals were 30% lighter and had more normal blood glucose measurements.

This, say the scientists, is "proof of concept for cell mediated repair of a neuronal circuit controlling a complex phenotype."

The world's oldest tackle, together with evidence of deep-sea fishing 40,000 years ago, has been unearthed in East Timor.

Early human migrants, including those who first set foot in Australia 50,000 years ago, were clearly competent mariners. The journey from Eurasia into the northern part of the Australian continent would have involved crossing a 1500km wide deep-water archipelago, negotiatable only by boat and good seamanship. But archaeological evidence of these seafaring feats is sparse. 

Now, however, Australian National University scientist Sue O'Connor and her colleagues, working at a site in East Timor called Jerimalai, have discovered evidence that early human inhabitants of the coastline were catching and consuming fish from the open ocean as far back as 42,000 years ago.

Bones belonging to over 20 different fish species were recovered from an excavation at the site, 50% of them belonging to so-called pelagic (open water) dwellers like tuna. To catch these, the Jerimalai settlers would have to have ventured out to sea armed, most probably, with nets.

But even more exciting is the discovery, also amongst the finds, of two primitive fishhooks carved from pieces of trochus shell and dating from about 24,000 years ago. This is the earliest evidence of fishhook manufacture yet described.

As the team point out in their description of the findings, published this week in Science, "Capturing fish such as tuna requires high levels of planning and complex maritime technology. The evidence implies that the inhabitants were fishing in the deep sea."

Ben - Also this week, a study published in the Lancet has confirmed the safety of the widely used class of drugs known as statins.  These are the most commonly used drugs worldwide for heart disease and are taken by millions of people globally.  A randomised heart protection study back in the mid-1990s found the drugs to be highly effective against heart disease but subsequent epidemiological studies did raise some fears of an increased risk of cancer associated with taking them.  Now Richard Bulbulia from the University of Oxford has followed up the participants in that study from the '90s to have a look at any long term effects.  Richard, thank you ever so much for joining us.

Richard -   Good evening, Ben.

Ben -   What were they really looking at back in the '90s?  Were they just confirming that statins did work and did do what we think they should?

Richard -   Observational studies made it clear, 30 or 40 years ago, that people with higher levels of bad cholesterol had increased risk of vascular disease and to test whether this association was causal, people did large, randomised trials such as the Heart Protection Study which lowered cholesterol and consequently lowered vascular risk by around one quarter.  But the same epidemiological studies that highlighted the relationship between cholesterol and vascular risk also showed an increased risk of certain cancers and other causes of non-vascular death with lower cholesterol levels, and those of us who sort of responsibly interpreted that data suggested that these findings were due to something called reverse causality whereby it's the disease such as the cancer which causes the low cholesterol rather than the converse.  But the concerns were out there and they substantially delayed the widespread use of statins. 

Now, the Heart Protection Study which reported its main results in 2001 had reassurance in that there were no excess risks of cancer or other non-vascular deaths associated with taking simvastatin 40 mg for around 5 years.  But that 5-year period was really too short to reliably address the prevalent concerns about the risks and safety of lowering cholesterol in many millions of people.  And for that reason, we carried on following up all 17,000 surviving Heart Protection Study participants for a further 6 years.

Ben -   So what have you actually been doing to follow them up or are you just looking at medical records and incidences of different diseases or are you continuing to investigate more deeply what the lifestyle factors are?

Richard -   We followed up the survivors in two ways.  We asked them to complete postal questionnaires and the vast majority of the participants did so, and on these postal questionnaires, they told us certain things about their statin use after the trial period, whether or not they've been to a hospital, had any clinical events, and also indicated whether or not they'd be happy to receive a subsequent questionnaire the following year.  That questionnaire procedure was augmented by accessing national registries for cancer incidents and they're death certification for people who either had cancers or died during the follow up period.

Ben -   Surely, after the original trial, the people who had been taking placebo, as it was a controlled trial, they must've then been offered the statins.  So surely, actually, the conditions have changed.  How do you take account for that?

Richard -   That's a very important point.  At the end of the trial, it was clear that everybody in the Heart Protection Study would benefit from at least discussing whether or not they should take the statin with their GP and all were encouraged to do so.  Gratifyingly, over the 6-year period, more and more people in both treatment groups have began taking the statin therapy, so by the end of the trial, the average use of statins was around 75% in both original treatment groups.  So, our long term follow up results, which were in the Lancet this week, actually assessed the effect of the initial 5-year randomisation to either simvastatin or placebo over an 11-year period.

Ben -   I guess the fact that people did start taking them after, also means that you could actually stratify your results and show that people have been taking it for this long, you see the following effects, but if people have been taking it for twice as long, then either you see more effects or you still don't see any effect which should help you to be able to say the concerns about cancer were in fact not actually applicable.

Richard -   That's correct and there were three main findings in our study. The first two connected are looking at benefits.   I mean, it's important to remember that statins are incredibly effective form of treatment. During the randomised phase of the trial, the absolute benefits of statin therapy increased as treatment continued.  With year on year reductions of around one quarter, after the first in trial year, and second, the absolute benefits that those originally allocated simvastatin accrued during the in trial period persisted in the post trial period.  That is to say, the people who were originally on placebo and then switched to statin therapy after the trial had closed, never caught up with the original simvastatin group.  And those two findings do show that starting statins early and continuing them long term is necessary to maximise the reductions in major vascular events. 

And following on from that clear message, it's also very reassuring to note that over an 11-year period, there was no suggestion of an emergence of hazard on cancer either globally or in certain specific subtypes of cancer or indeed other forms of non-vascular disease and specifically non-vascular death emerging in this large cohort of trial participants.

Dave - Alien, or non-native species, can have serious effects on the landscape and indigenous wildlife.  Recent research in Australia has found that hikers could be partly to blame...During just one season in a national park there, hikers were found to carry up to two million plant seeds just on their socks!

James Bullock, from Wallingford's Centre for Ecology and Hydrology, was one of the authors of the study, which was based on some of his earlier work in Dorset, Planet Earth podcast presenter Sue Nelson joined him at a picturesque section of the River Thames in Oxfordshire to see what they could find....

James -   Riverbanks are actually one of the most invaded habitats we have in Britain, so the sort of species you might see along here are Himalayan Balsam or Japanese Knot Weed, all of which cause problems with riverbank stability affecting native biodiversity.  What you will see also this time of year is the chestnut leaf miner, a lot of chestnut trees their leaves will be removed by mining of this moth caterpillar.  Actually, we can see over here, just next to us, are Canada geese which have been established in this country for a long time and cause a lot of problems, not just affecting native bird species but by pooing in waterways cause a fertilisation effect which can effect what's growing in the water as well.

Sue -   So the effects then of invasive species are quite wide and varied, it's not just a threat to biodiversity in some cases?

James -   No, they have quite a wide range of effects.  They can effect biodiversity directly, but they can also affect aspects of the natural world which are of more direct impact on humans, such as bank erosion, pollution of waterways, causing problems with grazing lands, so you have invasive species spreading across grazing areas which affects the ability of animals to graze on them.

Sue -   Now the Australian study found that different parts of a hiker's clothing could spread different numbers and types of seeds.

James -   There are some seeds which have hairs or bristles on them which are probably evolved to allow their dispersal by animals; we come along with our socks or our trousers, almost like an animal skin and it forms a nice material for these seeds to attach to.  So, the more woolly the clothing you're wearing and the more bristly the seed the more seeds will get dispersed by people.

Sue -   How did this come out of research from Dorset?

James -   We were interested in quite a different aspect of dispersal in Dorset.  We were working on a species called Wild Cabbage there, which is quite a rare species restricted to our coastline and it forms quite discreet populations along the coastline which have been sitting there for probably hundreds of years.  So, we were interested in the reverse side of the question - what limits this species to grow where it grows and what are the possibilities for its dispersal? 

Along the coastline of Britain we have loads and loads of footpaths and one possibility we thought of was that hikers could take the seeds of the cabbage around.  So we did an experiment where we found that seeds rather than socks or trousers would stick into the mud on hikers boots and could be transported very long distances, the natural dispersal by wind could take seeds a maximum of about 200 metres, but hikers could take the seeds over five kilometres.

Sue -   Is that why you want to know?  Is that why you do this sort of research in order to predict possibly, or can you even predict when you've got millions of seeds capable of sticking to somebody's hiking socks what species are going to be transported where?

James -   Yes.  The prediction is the key here.  What we want to understand for alien species or non-native species is what species is transported, how far they're transported and what are the mechanisms of their transportation.  And the reason we do that is not simply understanding, but then we can use this sort of information in models of spread of these species to work out how we might limit that spread, so a lot of work on alien species so far has been saying, ok, we find out where the populations are growing and we go and try and kill them in some way. 

A much more efficient and effective method would be to prevent the movement of these species in the first place and so that's what we're working towards - understanding the whole process of spread so we can look at the crunch points and limit that spread by especially targeting dispersal.

Sue -   How would you advise hikers, how on earth are they going to limit the spread of seeds?

James -   Yes, that is a big question and it could sound a bit over the top telling people to be very careful about what they're transporting.  But certainly in the study in Australia, this is a highly protected area which is under threat from these European plant species coming in, so in that case, the recommendation has been to put signs up for walkers, to educate walkers, to say before you walk into these remote areas just clean your trousers and socks off, just pick the seeds off before you spread them into these pristine areas.  So, I think in very specific circumstances, where we're trying to protect particular areas, there is something that can be done.

Bruce -  Well the ultrasonic waves, similar form to acoustic waves, so we know they're travelling in air, but they also travel in liquids and solids.  And so, dealing with bits of metal then ultrasonic waves travel really very nicely in those bits of metal.  So, we have a series of sources which emit ultrasound into the metal structure and they're reflected from all of the interior structure which obviously includes cracks.  We can then pickup with our microphones, ultrasonic microphones, actually they're small electric elements but doesn't really matter there.  We can pick up that reflected energy, process it and from that, produce an image of the interior of the component.

Ben -   A lot of the materials that we might be looking at to try and find cracks are actually very highly, very precisely made materials in the first place.  Often, they can be a single metallic crystal.  Does that actually affect the way that the ultrasound would travel through?

Bruce -   Yes.  The crystal structure is utterly paramount to how waves travel in so that the single crystals are an isotropic in their material properties for which as far as ultrasound is concerned means that waves travelling in different directions go at different speeds, and you can imagine that if we're not very careful, that could create all sorts of confusions we have to compensate and take account of the fact that the velocities are different and the speeds of sounds are different in different directions.  And if we do that then we can produce images as if the velocity was uniform.

Ben -   So essentially, you use some kind of algorithm or some kind of computer modelling to compensate for how you know the sound would travel in a pristine example and that therefore means that you can compare your example to pristine, and easily see if there's something wrong.

Bruce -   Yes, we need to have a model of the structure we're trying to test in order to correctly image it, and one of the big challenges is when you go to a component and try to make a measurement where there's uncertainty in that model.  And so, one of the things we have been working on is what we call autofocus techniques where your camera automatically adjusts to put things in focus whereas we have been working on ways of trying to automatically extract this picture of what the structure is like.  So this model without knowing it beforehand or say, it's the particular principle orientation direction is unknown.  But we can extract that from the data itself if we know something about the structure like, for example, how thick it is.  

In terms of detecting cracks, the real challenge is, somewhat different to the medical challenge where you've got this very rich image. The interior of the piece of metal is you'd have thought relatively simple and it is, but there's a lot of scattering from other things within the material like grain boundaries, although they don't occur in the single crystal example that you just talked about but in most metals, there are many grain boundaries, all reflecting ultrasound and there may or may not be cracks and so, the challenge is to achieve that high resolution imaging performance of the crack surrounded by these other scatterers which basically set the signal to noise ratio of your measurement or the best possible measurement that you can make.

Ben -   And I assume that signal to noise is ultimately what will limit the resolution with which we can view this and the actual minimum size at which we can first detect that crack.

Bruce -   Yes, and that's always the challenge, to detect the smallest possible crack and that's where we're always trying to push the limits of what's possible there and that's what I'm interested in as a researcher.