The view through a confocal microscope
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.