Discovery Talk: 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.

This Talk

Speakers: Harald Hess, Eric Betzig

Audience:

Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: September 2010

Educator Resources for this Talk

Talk Overview

Harald Hess and Eric Betzig talk about developing PALM microscopy and discuss the research article titled “Imaging intracellular fluorescent proteins at nanometer resolution.” During their 20-year friendship, Betzig and Hess worked together and separately, in academia and industry, before eventually joining forces to develop the first super-high-resolution PALM microscope. They tell us the story of this journey and emphasize how their unusual and varied backgrounds provided the skills to complete the project. Eric Betzig was awarded the 2014 Nobel Prize in Chemistry for his role in developing super-resolution microscopy.

Speaker Bio

Harald Hess

Harald Hess

Harald Hess is a Group Leader at The Howard Hughes Medical Institute’s Janelia Farm Research Campus. Together with Eric Betzig and individually, Harald Hess continues to develop new imaging methods that can be used to peer more and more deeply into tissues and cells. Continue Reading

Eric Betzig

Eric Betzig

Eric Betzig is a Group Leader at The Howard Hughes Medical Institute’s Janelia Farm Research Campus. Together with Harald Hess and individually, Eric Betzig continues to develop new imaging methods that can be used to peer more and more deeply into tissues and cells. Eric Betzig was awarded the 2014 Nobel Prize in Chemistry for his… Continue Reading