Developing PALM microscopy
Transcript of Part 1: Developing PALM microscopy
00:00:14.16 Hi, I'm Eric Betzig. Hi, and I'm Harald Hess 00:00:18.11 We'd like to present today, a little story behind a new kind of microscope that we invented 00:00:24.01 which can take resolution from normal microscope resolution to super resolution, 00:00:29.07 and also give you a little story behind how we came to the idea and it came to pass. 00:00:35.06 Harald and I have been friends for pretty much over 20 years. 00:00:39.05 I first met him when I went to Bell Labs and joined him 00:00:43.03 in the semi-conductor research department. 00:00:44.13 I went to Bell to continue my work from my graduate thesis 00:00:49.17 on the development of the first super-resolution optical microscope 00:00:53.03 called the near-field optical microscope. With that microscope 00:00:57.03 you were able to, for example, on fixed cells, go from resolution like this in the conventional sense, 00:01:02.17 to what you see here in the near-field sense. 00:01:05.17 But probably what is most germane for this talk, is with the microscope 00:01:10.04 I was also able to see, for the first time, single molecules under ambient conditions, 00:01:16.10 and make standard observations in imaging of these molecules, 00:01:19.05 with a resolution, well not a resolution, but a localization precision down to about 12 nm. 00:01:26.06 Meanwhile, I was also at Bell Labs, and I was focusing on scan-probe microscopy, but particularly at low temperatures. 00:01:34.03 This was an exciting field at the time. I was trying a few different variations of 00:01:38.07 scan-probe microscopes to sense 00:01:39.29 tunneling current, magnetic field, or electrical field 00:01:43.01 and actually had a lot of fun with that, but sooner or later Eric and I decided 00:01:48.11 to join forces and we combined his near-field technology, 00:01:54.18 which sort of puts light in a very small diffractive area together with my low-temperature system. 00:02:00.01 We focused initially on a system called quantum wells, where you have these little luminescent centers, which are supposed to glow. 00:02:07.13 Another way to represent that data, is with this little block that you see right here, 00:02:12.08 where you see X and Y down here, which is real space 00:02:16.07 and the vertical scale up here is now spectral. 00:02:19.28 So you can see that spreading the data out in the spectral dimension, really helps us to see this individual luminescent centers 00:02:29.15 and was key for this particular experiment. 00:02:33.07 So while I had a lot of fun with Harald, and doing my other experiments with near-field while I was at Bell, 00:02:40.02 eventually I got pretty fed up with the whole thing in part because of the physical limitations of the near-field technique 00:02:47.24 and in part because it engendered a really big academic bandwagon 00:02:52.09 with many people getting into it and the hype greatly exceeding the reality. 00:02:57.27 I got to the point where I said, I really want to try something new. I'm really sick of the whole structure of academic science. 00:03:06.01 So I left, and did my first mid-life crisis, and while I was thinking a light bulb popped on and I thought, 00:03:14.19 you could actually combine the ideas of my single molecule experiment 00:03:19.09 with the quantum experiment that Harald and I did, to potentially create a molecular resolution optical microscope. 00:03:26.03 So the idea would be to consider a bunch of molecules here, which are initially not resolved, because a bunch of fuzzy spots overlap 00:03:34.03 but again, if they have some feature by which they differ from one another, you can identify, like they glow different colors 00:03:39.26 or they blink, or they have different polarizations or whatever, then if you measure those parameters, 00:03:47.01 you can isolate the molecules, just like we did with the exciton recombination sites 00:03:54.06 you can isolate these points in this multi-dimensional space, and once they are isolated 00:03:58.00 then you can find the centers of these fuzzy balls to much better than the diameter of the fuzzy balls 00:04:02.21 like I did in the near field technique 00:04:04.29 and then you are able to project those center positions back to spatial coordinates 00:04:09.02 and basically get a super resolution map of where all of the molecules are located 00:04:12.27 So to do that with the technology at the time 00:04:18.06 was going to be very difficult because to be able to see many molecules in one diffraction limited region, 00:04:23.19 was going to require a very high resolution in that third dimension 00:04:27.09 you might be able to do it spectrally but it would have been a heroic experiment 00:04:31.05 and I was pretty fed up with things at that time. 00:04:34.00 So I went on a completely different tack and started work for my dad's machine tool company in Michigan 00:04:39.00 Actually a year or two later, I also left Bell Laboratories 00:04:43.28 I sort of felt the field of scanning probe microscopy was maturing 00:04:47.23 and I thought there might be other larger opportunities, 00:04:50.03 particularly in these small little start-ups, which were forming out in California. 00:04:54.01 At that point I joined up with this company called PhaseMetrics, 00:04:56.03 which does tests and measurement equipment for the hard disk drive industry 00:05:00.08 and I thought some of my nanotechnology imaging experience could both benefit and get new ideas for myself. 00:05:07.07 So these are some sample machines, which check discs or read/write heads. 00:05:11.03 That company later on got bought-out by another one, KLA-Tencor 00:05:15.16 So that was all a lot of fun and now we even had the opportunity to explore a new concept, 00:05:20.21 and actually it came up with a nice idea, which got funding and was ready to launch 00:05:25.22 but it was going to be launched in San Jose and I was faced with a decision, 00:05:29.15 either move and join this little project, or go back to a bit more research mode, and I was talking with Eric 00:05:38.11 and I decided in the end that it would be fun to take the more adventuresome path 00:05:44.00 and search for something new, but it wasn't quite clear what. 00:05:47.25 So while I was doing my searching, I was trying to think of where I wanted to go 00:05:54.10 and what I wanted to do to get back into perhaps science, because although 00:05:59.04 I learned after seven years at my dad's company, A: that I'm a really bad salesman, 00:06:03.21 but B: that I didn't like the academic structure of science, but I really loved the science itself. 00:06:11.20 So I started thinking about different ideas, and at the same time... 00:06:19.10 I was also contemplating, along with Eric, and we actually had many trips to National Parks, 00:06:23.18 trying to figure out where are the opportunities in science, where are the new challenges, where are the untrodden paths? 00:06:29.14 We sort of were overcome sometimes with just the feeling of insignificance 00:06:34.07 compared to the vastness of what is out there. 00:06:37.22 Eventually, although I didn't want to do microscopy, I wanted to try something new and different 00:06:46.10 in 2003, I first read a paper about green fluorescent protein, which of course has since, even by that time, 00:06:56.08 was in the process of completely transforming cell biology and many other fields of biology 00:07:00.23 Honest to goodness, I was probably the last man on earth to learn about GFP, 00:07:05.21 but I immediately realized that this would be transformative not only to biology, but to biological microscopy 00:07:11.26 because of what we might be able to do with it. 00:07:14.07 So in my job searching, I had also contacted a lot of other friends 00:07:18.21 and one of the places, which I visited was Tallahassee, Florida 00:07:24.01 and there, I thought might be an interesting place to see whether Eric's idea could possibly fly. 00:07:31.15 Particularly, at that place, there is a laboratory called the Magnet Laboratory, where it had a network of colleagues 00:07:38.24 and one person there, Michael Davidson, was remarkable. 00:07:42.21 He actually has a wonderful website, very comprehensive, and was writing very important reviews of all the major developments in the field. 00:07:53.08 In particular, while we were there, he pointed out there's not just the green fluorescent protein, 00:07:59.22 there is a whole new class of optical highlighters, or photoactivatable PA-FPs. 00:08:05.15 Those proteins, fluorescent proteins, are essentially dark, or maybe off in a different spectral range 00:08:11.05 and when you normally look at it, you see nothing, but if you shine blue light you can effectively turn them on. 00:08:18.29 This was magical. As soon as Harold and I left Tallahassee, it became obvious to us 00:08:26.28 that this was really the missing link to make that idea that I had published after I first left Bell work. 00:08:33.15 So the thought is that rather than bathing the entire specimen with blue light until it all glows, 00:08:39.14 is just turn on the blue or purple light for a very brief period of time, so only a few molecules turn on at once 00:08:47.08 then since they are isolated from one another, we can find their centers, and plot those, 00:08:51.04 and then they burn out and bleach and we turn on a new set of molecules by pulsing 00:08:55.07 the light on again, and repeat this process for many frames until you determine the coordinates of every molecule inside of the sample. 00:09:02.18 So instead of that original quantum well experiment, where we separated the exciton 00:09:08.01 recombination sites and terms of X, Y, and wavelength now it's in terms of X, Y, and time. 00:09:15.15 Just in case you didn't quite get Eric's explanation, let me just restate it in plain english. 00:09:22.16 Basically, scoot over just a little bit, right there you see a lot of molecules and they are normally very densely packed, 00:09:29.12 impossible to resolve them clearly, but if you put in a little bit of blue light, you can turn on 00:09:34.12 a very small subset and they are far enough apart, that you can see each one glow independently 00:09:41.03 localize it's center and you repeat this until you exhaust all of the molecules and you can then resolve the complete 00:09:49.15 super-resolution image whereas if they all glowed at once you would just see a massive blur that looks like this. 00:09:54.29 Once we realized that this was possible, we immediately set off to a quite place, Sedona Arizona, 00:10:04.28 and wrote up our ideas in a patent and started scheming how can we make this microscopy happen fast, 00:10:11.26 it was an idea that was very ripe, and potentially very powerful at the time. 00:10:15.04 With-in about a month or two, we were actually out in my living room, assembling, collecting parts, and 00:10:24.21 assembling the microscope itself, we were able to sort of bypass the complete funding procedure 00:10:29.01 with venture capital and were able to move at lightning speeds, so within literally a few months, 00:10:32.28 in the summer of 2005, this was existing in the living room. 00:10:36.22 But the one missing piece still was as physicists we didn't know the first thing 00:10:42.14 about how to do real biology so we needed to collaborate with good biologists. 00:10:46.15 So I had been set to give an interview talk at NIH at about the same time, 00:10:52.15 and so when I was there I begged to be able to speak to Jennifer Lippincott-Schwartz and George Patterson 00:10:57.27 who were the inventors of the original photoactivated GFP, and so we clued them in on 00:11:05.03 what the idea was, swore them to secrecy, and asked them if they wanted to collaborate 00:11:09.06 and they were very receptive and very helpful, and Jennifer offered us not only her help, but lab space, 00:11:16.06 and some equipment money and so forth, and so soon we were off and running, not just with the instrument from Harald's lab, 00:11:23.08 but we were able to cart that to Bethesda and get started very quickly. 00:11:27.25 So here again is Harald's movie, but now shown with real data, so maybe you will finally really understand it this time, 00:11:36.13 so the frame on the left shows single molecule frames from lysosomes, 00:11:42.24 that have been cut through to a very thin section and then turning on the blue light 00:11:47.15 very briefly, in small bursts, to turn on small amounts of molecules, which are clearly isolated from one another. 00:11:54.14 If you summed up all of those frames, you get the frame in the middle, 00:11:57.23 which then represents what you would see in a normal optical microscope. 00:12:01.22 But if instead you find the centers of all of those molecules from the frames at the left, 00:12:06.01 and plot them over on the right, then you end up getting a super-resolution image. 00:12:12.03 You can see that a little bit better here, where you see in the middle on the left, 00:12:17.13 a diffraction limited image of these lysosomes, and on the right, the PALM image of the lysosomes. 00:12:24.22 And to really appreciate exactly how much the resolution has improved, if you zoom in here, 00:12:29.12 you go from this type of resolution here to this type of resolution here. 00:12:33.28 So this became the basis for our first PALM Science paper back in 2006. 00:12:41.01 I though we would conclude by just trying to put together some thoughts, which I think were very helpful to us in succeeding. 00:12:52.29 I think both of us have a little bit of aversion for doing the mainstream, and so we actively sought out 00:12:59.21 areas which were not very fashionable at all, and tried to avoid those areas very explicitly. 00:13:07.12 I think for both of us it was actually very valuable to seek out a diversity of experiences. 00:13:13.01 We sort of bounced through multiple fields and just experiencing new problem sets 00:13:17.19 from the outside, not just from the immediate research, but from the outside. 00:13:22.10 I think it was very helpful, and actually very liberating for us and made the whole thing a lot of fun. 00:13:27.08 Just to reiterate what Harold just said, I think one of the key lessons is to not necessarily jump on those bandwagons 00:13:35.20 like I said, but forge your own path. Most people who are probably looking at this video, have been in science for awhile, 00:13:44.10 and if you are a young guy you are trying to figure out what you are really going to do for your career. 00:13:48.29 You've probably invested a lot of time and effort to get to this point. 00:13:52.07 I think it's a mistake too many people make to try and go the safe route 00:13:59.10 from a funding perspective and whatever, to go into the fields that are already fairly mature. 00:14:04.12 The thing to do is to really try, in my opinion, to strike your own path, 00:14:09.05 but you have to really have the courage of your convictions to tune out what other people are saying, 00:14:14.25 and to not be upset when you don't get the first grant or two and to try to be 00:14:20.13 a little bit scared, because the adrenaline pump also helps in being productive. 00:14:26.17 But really whatever you do, you should do the thing that you love doing, because nothing worthwhile was ever done without passion.