 Our next speaker is another presidential award winner and is now part of the intramural program of the NIVIV to show how versatile the institute has become. Harry Schroff will be our guest speaker and he'll be speaking about new technologies for high spatial and temporal resolution imaging of cells and organisms. Harry, it's great to have you. Thank you. All right. So before I get started here, I just want to thank Rod for letting me be part of this 10th anniversary celebration. It's a great honor to be here and especially in the company of such great speakers. So I'm a microscopist by training and I try to think about ways of improving microscopes that are commercially available. Most of my work is not on sort of large organisms like humans or mice, although I seem to be sort of moving in that direction because I started out studying single cells. I'm going to tell you a bit about our work on the C. elegans and I've been also starting to work on zebrafish a bit. So half of my lab works on mechanisms for improving the spatial resolution of microscopes, in particular getting past the diffraction limit of light, which is about 250 nanometers in size. And although I have lots of pretty pictures from that side of the lab, I thought instead I'd tell you sort of a different story. I tell you a bit about imaging noninvasively and in particular how to avoid self-torture. So one of the things that I learned early in my tenure as a microscopist is that it's very easy to fry a single cell and here you see a fiberglass cultured on a single cover slip and you're looking at it in bright field modality, but I was also shining a laser on this cell and you can sort of see it curling up and eventually if I were to keep showing you the movie, the cell would lift off the cover slip and sort of die a watery death. Can people hear me or? All right, here we go. So the reason for this is that most microscopes that you can buy are remarkably inefficient. They dose the sample volumetrically, they dose the entire volume of the cell, even though we're recording this image with a CCD camera, which is a two-dimensional detector. So I'm going to tell you about a microscopy technique that we and others have developed that is far better and lets you image effectively much, much longer. So hold that thought in your mind because now I want to sort of switch gears and motivate the importance of this from a biological perspective. Another one of the goals of my lab other than improving microscopy is to better understand neurodevelopment in the brain. And so if you think about the human brain, it's of course this fantastically complicated organ, you know, billions of neurons, orders of magnitude more synapses and despite this large number of parts, the brain somehow manages to wire correctly a huge amount of the time. How this precise connectivity happens in vivo is still not very well understood, but one thing should be clear from the sort of mismatching numbers. This kind of complexity is somehow coded for by about 25,000 gene products, even though there are trillions of sort of functional parts that make up the nervous system. Merely cataloging the genes and their functions is not going to be enough to sort of understand how this thing wires. What you really want is sort of a movie of how the entire brain forms at subcellular resolution. But unfortunately with today's technology we can't do this with the human brain. So if you jump down a few orders of magnitude and complexity and think about a much simpler organism, the C. elegans worm, this is a beast that has only 302 neurons, about 5,000 chemical synapses, most of which have been mapped at the EM level of resolution and the sort of gene number here is about 20,000. This is a simple enough system, it's transparent, its lineage has been worked out. It's something where you could really ask with a microscope how these genes act in vivo to direct wiring. You can ask where are the neurons, how did they get there, where are the axons. If you want to look for general principles, I would argue the worm embryo is a good place to start. And for those of you that think that the worm embryo is too far removed from the human, I would just point out that there have been two arguably three Nobel Prizes that have involved this worm. One for sort of programmed cell death, one kind of involving GFP and then I should also say that technologies like RNA interference are also very easy to deploy in this organism. So what I'm really after is sort of a four dimensional atlas of neurodevelopment, sort of a dynamic Google map for the worm where I can optically reconstruct where are all the cells at every point in development, both in space and in time, where are all the processes. And if you had such an atlas of cell motions and cell processes, you could then map on to this atlas transcription factor expression dynamics. And I should say this is a collaboration with a neuroscientist at Yale and a development of biologists at Sloan Kettering. So this is sort of the goal, how do we get to this goal? Well given the fact that we have these awesome tools like green fluorescent protein and given the great genetic control we have in C. elegans, you can actually label all the neurons in the worm. And the problem is that you label typically all the neurons and that creates a contrast problem. So the things you need is a way of selectively marking sparse subsets of neurons, otherwise you have all of them light up and you can't distinguish one from another. Even if you have worm lines, genetically modified worm lines that give you a few transcription factors expressed in a few select neurons, you don't always know which neurons those are. And so what you need to be able to do for this atlas is identify which cell is which. And this is actually not a crazy idea because you can track nine out of tens rounds of cell division already just by following the pattern of cell divisions in the worm embryo. And you can do this computationally. So this is a computationally derived lineage tree. And then the reason that I'm interested in this problem is that you need a way of imaging this fast volumetrically and without killing the worm and that's quite a difficult thing. So why is this sort of a challenge? If you look at sort of a cartoon of the C. elegans every one cross section, it's about the size of a large cultured cell. So it's about 40 to 50 microns laterally and about 20 or 30 microns thick. And what I'm after is an atlas that gets you the position of all the cells to subcellular resolution. So what I'd like to do is sort of carve this embryo up optically into diffraction limited voxels of less than a micron in size. And I'd like to do this over the 14 hour developmental time period of the worm. So how might you illuminate the worm to get this sort of information back in your fluorescence microscope? One thing you can do is simply illuminate the entire volume of the worm. That's called wide field imaging or epifluorescence imaging. So if you shine a laser beam throughout the volume, you get fluorescence everywhere through the volume and if the worms are very thin this might be the way to go. There's no better way for very thin samples. It's very fast because all you have to do is move the plane of focus through the volume and build up a volumetric stack. But you have the problem that out of focus is blurred. There's no optical sectioning in this illumination strategy. And you also have a tremendous problem with out of focus bleaching and damage. So you might be visualizing a plane down here but if you're illuminating the entire stack then you're bleaching the fluorescence that occurs up here. Sort of another thing you might think about doing is using a confocal microscope and just to review this, the idea is you have a laser beam, you focus it into the sample and then you scan this laser beam around in the sample and for every position of the laser beam you record the fluorescence signal. You use a confocal pinhole to reject the out of focus light except in the vicinity of focus. And this is sort of a workhorse tool. You can buy one. It's prevalent everywhere. And unlike the wide field microscope it gives you optical sectioning and it can give you near diffraction limited resolution. The problem is that if you look at what this looks like in the context of the War Memorial, it, like the wide field microscope, also bleaches out of focus. So you also have the only useful information you gain at any instant of time is from the vicinity of focus. But you have this sort of cone of this hourglass shape of excitation that wastes the fluorescence up and below the focal plane. This is also intrinsically slow because it's a point scanning technique. So you record one piece of information here but then you have to scan this excitation this way up and down into and out of the page. And in order to make that fast you usually have to take the laser and turn the intensity up and that frequently fries your sample. So conceptually anyway you can do much better if you just illuminate one plane at a time. You use a light sheet to illuminate only the plane that you're detecting. And then to build up a volume you can scan this light sheet in one direction that's very fast. If you tailor the light sheet the right way you can minimize out of focus bleaching and damage. The idea of using light sheets in microscopy is actually nothing new. The Germans were doing it in 1903, 1904 and investigating colloidal suspensions. But this technique has undergone a renaissance in the last decade or so, especially in the context of developmental biology. And they sort of undergo, they go by this acronym, selective plane illumination microscope techniques or SPIM techniques. Just to give you an idea of what these microscopes have looked like historically, some of the early work was done in fish. And so you take your fish and embed it in an agarose cylinder, put that agarose cylinder in a water filled chamber and then come in from one side with a light sheet. You detect the fluorescence in this perpendicular detection and you scan the light sheet very fast in this direction and build up this optically sectioned volume. And so you can, this is movie is from almost, you know, from quite a while ago but it's still pretty illustrative. If you were to do the same imaging where you illuminate the entire volume you have all this out of focus blur. But if you do this light sheet based volumetric imaging you can get very optically clear images, but in this case this fish embryo. And if you have the time to rotate the sample around you can really fill in this volume very beautifully and with a minimum of damage to the sample. So this technology has been around for years but there are very few of these actually around and it has not yet been deployed in the market. And part of the reason for this is that this is really a pain in the ass to build. So as a tool developer as a microscopist I started thinking about ways of making this sort of easier to use and the solution we came up with is something we called inverted microscope based spin or eye spin. So the idea that we had is to just implement this geometry in place of the bright field illumination pillar of a conventional epifluorescence microscope. So you have these two perpendicular objectives. In my case the worm embryo sits over here on a glass cover so that the focus of both objectives. And then you bring in the light sheet via this objective and you stand it in this direction and the fluorescence gets sent to a camera. The advantage of this geometry is that you can use conventional sample mounting protocols like glass cover slips and you can investigate the sample before you do the spin imaging using the lower conventional optical microscope train. So you can look at it at different magnifications. If you want you can do photoactivation. You can do lots of different things using this flexible approach. So we sort of built this system and then we started to prototype it on C. elegans embryos and I'm going to show you just a few movies. This is one example of a C. elegans embryo from the two stage stage eventually all the way to action. And what is displayed here are the positions of the nuclei. The nuclei have been marked with the GFP histone marker. And using this light sheet microscope we can collect about 25,000 volumes over the course of embryogenesis. We can image every two seconds over about 14 hours without frying this worm. And this was a good control for us because all of the cell divisions have been studied in great detail in the 80s. And we detect no difference using this imaging modality in the pattern of sort of stereo type cell divisions. The embryo is all hatch on a dime. This is about 30 times faster than a confocal spinning disk which is sort of the state of the art fast optical microscope you might buy commercially. One of the things you notice that is a little bit of an imaging nightmare is that after the muscles form the embryo starts to twitch pretty rapidly. So you have to image super fast to be able to still resolve these individual cells but we can do that for the most part we can track these individual cells although there is a bit of motion blur. And if I kind of advance this movie over here what you find repeatedly is that eventually the worm hatches out of the egg shell and goes on to make more worms. So it is indeed not invasive. And so this was sort of a good proof of concept for us. What I'm really after though is the neurons. And so as a proof of concept another proof of concept we built a worm strain that had only a few neurons highlighted. And again I've sort of taken this four dimensional movie and in the interest of time sort of broken it up into four different time segments. In this particular movie there are about four neurons that you can kind of see over here. They start out at the anterior end of the embryo and if I advance this movie you see that they kind of crawl from the anterior end to the posterior end. And once the embryo starts to twitch all hell breaks loose but you can still even after the muscles twitch observe sort of by eye the motion of these individual cells a little later you can see that two of these cells form axons which are these sort of long structures over here. And by maybe if I pause the movie I can even point out that there are growth cones at the very end of these axons they're a little clearer maybe in this movie over here. So you can actually see sub micron type structures in this microscope and you can actually follow and track these cells using this non-invasive microscope. And I also want to point out that this is a pretty dynamic system. So the neurons start out in a morphology that is nowhere near where they end up. And I would argue that you really want to do this kind of dynamic imaging to see how the neuro development happens instead of fixing an embryo and looking at many thousands of fixed embryos it's much easier to see what's going on if you have one embryo and can follow the sort of developmental progression throughout time. Now when we built this strain we didn't actually know what the identity of these neurons were. And in compiling this atlas that's sort of the necessary components. So just as just to show that that's possible you can do multiple color imaging where you label all the nuclei and rad let's say the neurons in green and then you can if you can correlate the position of the nuclei with the neurons then you can so for example this particular cell over here it's moving a little fast and we bring it back. This guy over here you can sort of see and correlate where the nucleus is and the cytoplasmic signal. So you can build up these lineage trees and actually identify these neurons as these particular names which really don't mean much to me but at least in principle you can do this sort of analysis for all of the neurons in this worm and then identify each one from the data itself. Now given this kind of data you can do sort of cell biological studies. You can ask how long are these neurites as a function of time? Where are they in relationship to another? I'm just gonna illustrate one piece of information that we learned from these movies. If you take one of these neurons this so-called ALA neuron what you find if you look at wormatlas.org which documents where all these things end up in the adult worm you find that this neuron sort of kinks back and then goes towards the tail of the embryo although it projects first the other way and we can actually see this in our movies. So I've marked over here this ALA neuron at about seven hours post fertilization and if you look a little bit further at about seven hours 15 minutes you find these kind of outgrowts of this neuron. If you look a little bit further you find these outgrowts bifurcate you find this forked structure over here. This forked structure persists for a little while and then it commits and then one of the forks disappears and this neuron goes always in the other direction. So at least in principle you can use this system to study neurodevelopmental events in vivo as they happen. All right, I'm gonna finish just by discussing sort of where we're going with this technology. One of the things we learned in the course of doing this is that although imaging every two seconds an entire volume of a worm is pretty fast it's actually not quite fast enough all the time. And so we're trying to push the speed further. We've also discovered that there's a way of actually increasing the isotropic resolution of this microscope. All of the movies that I showed you were always X, Y views as opposed to X, Z views. And that's because this microscope as in like any single view microscope has worse resolution along the optical axis than perpendicular to the axis. There are less angles that give you resolution along the optic axis. One solution to this is to rapidly collect two perpendicular views and this 90 degree objective geometry actually gives you those. So what we've done is we now mount a camera on each arm of this selective plane illumination microscope, record the views from each property to the direction and then we can fuse those and get a volumetric time series that is more isotropic. So here's an example of that. We're collecting each volume now in less than a second, both views. And then you can see the X, Y and the X, Z views. What I like about this movie is that it's actually quite difficult to say which direction is which. So performing this multi-view imaging really does even out the isotropy of the movie. And again, this is not so damaging to the worm that they don't hatch on time. All the stellar divisions also seem to happen on time. These are two areas that we need help on. So the neural strain that we built is one of only a few that has just a few neurons on. We need to sort of basically develop lines that have all of the 222 neurons marked sparsely. One way that we've thought about doing this is to employ photoactivatable markers that label all the neurons and then go in specifically with a different color laser and activate just a few neurons. The other thing we need to do to compile all of this volumetric data into a digital atlas is to straighten the worm embryos. Because although the embryos themselves have a stereotype developmental program, the twitching, of course, is stochastic. So if you wanna compare one embryo to another embryo, you have to align it along the common body active. So if you have ideas about this, please email us, we're happy to talk about this. And then finally, I'll just say that you can use this system to image many other phenomena besides worms. So you can understand things like the zebrafish, lateral line, we've started to image interactions between mouse, egg, and sperm. So we'd like to observe the events that happen after the sperm binds to the egg and penetrates the zone of lucid. You can investigate microtubular dynamics, influenza, infection of cells, vesicle transport, anything that you need to require volumetric images pretty fast and without killing the organism, this microscope is excellent at doing. So with that, I think I'll just stop by thanking Richard Liepman and Hank, even my bosses who hired me, various support both inside the NIH and outside and in my lab. The vast majority of this work was done by Yichong Wu, who unfortunately couldn't be here, but fortunately is having a baby. And then Ali Rizek, a post-bac who also helped him. So thank you. Time for one question for Dr. Schwarz. All right, just clear as mud. It's exciting. Yes, please. So I can't sustain these imaging rates with this information because I fry the embryo. So it's over because you put in energy throughout the volume in that thing and you rely on these tricks to get rid of the cyclones. It turns out for the embryo, it's too much. Yeah, that was one of the first things we tried. Right. Thank you very much.