What Can You Learn From Single Molecules, Even When Trapped Without Optical Forces?
Moerner, William E.
Thirty years ago, tiny individual organic molecules were first detected optically. One inspiration for this came from Ashkin and Chu, et al. who in 1986 used a focused laser beam to create “optical tweezers.” Tweezers grab single small dielectric particles or cells using optical forces and are heavily used today in force spectroscopy to study single biomotors, protein unfolding, etc. But can we grab much smaller single molecules without optical forces? Yes, a closed-loop feedback device called an “ABEL” trap can suppress Brownian motion in solution, even for a single dye ~1 nm in size. Holding a single fluorescent molecule or a photosynthetic protein for a long time allows multiple simultaneous measurements, enabling mechanisms of biological photoprotection, enzymes, aggregation, etc. to be explored in aqueous solution, molecule by molecule. Readings: Allison H. Squires, Adam E. Cohen, and W. E. Moerner, “Anti-Brownian Traps,” in G. C. K. Roberts, A. Watts, European Biophysical Societies (eds.), Encyclopedia of Biophysics. Springer, Berlin, Heidelberg, 2018. (DOI: 10.1007/978-3-642-35943-9_486-1) W. E. Moerner, Yoav Shechtman, and Quan Wang, “Single-molecule spectroscopy and imaging over the decades,” Introductory Article, Faraday Discuss. 184, 9-36 (2015) (DOI:10.1039/c5fd00149h)
 Good morning everybody and welcome to this first Agorah talk. As you may know Agorah was the main square in ancient Greek cities where people came together to exchange ideas, to discuss and so on. And that's the main purpose of this event here too, to exchange ideas, to discuss things with the Nobel laureates. And I have to introduce myself. My name is Günther Werth from the University of Mainz in Germany. But more important I introduce our speaker here, Professor William Mörner from Stanford University. And he received this Nobel Prize not in physics but in chemistry, but he's a trained physicist. He received the Nobel Prize in 2014 together with two colleagues, Erik Betzig and Stefan Professor Heller. Professor Heller I think is present at this meeting and he will give a talk later on this morning I suppose. Anyway, he received the Nobel Prize for what's called surpassing the limitations of the light microscope. What are these limitations? What we learned in first year optics when we talk about resolution. What is the minimum distance of two objects which can be resolved by visible light? And that is law by the optician Abbey in Germany said the minimum distance of two objects should be the wavelength of the light. The wavelength of the light is 500 nanometers about, a couple of 100 nanometers. The distance of two atoms in a molecule is the order of a few nanometers. So a factor of 100 about smaller than the wavelength of the light. There's no way to detect single atoms in a molecule unless we have a clever idea. And this clever ideas, one of these clever ideas came by Professor Mörner and in this talk he will explain what he did and what the prospects for the future are I suppose. So we'll listen to him within 15 minutes, 10, 15 minutes and later on it's your turn to discuss, ask questions to Professor Mörner and exchange ideas. Okay, go ahead. Well thanks so much for the introduction. I'm so happy to see you all here today and I might ask the question, you may be asking the question why am I here talking about molecules at a physics meeting. But I just want you to remember that molecules follow physics as well. And so the important thing to realize about my entire talk is that it's going to be an intermixing of different fields. And I intend to want to show you that it's the interaction and interdisciplinary nature that makes some things very, very exciting these days. So let's go back to that great year in 1905 which was already mentioned upstairs. Albert Einstein not only worked on the photoelectric effect but he also worked on Brownian motion. And he said that bodies of myoscopically visible size suspended in liquids must as a result of thermal molecular motions, perform motions of such a magnitude that these motions can easily be detected by a microscope. And so I'd like to show you a little video that illustrates what this is all about and mostly the topic of my presentation today. This is, ignore these little electrodes that appear here. This movie shows you that at room temperature molecules are jiggling all around. These happen to be actually tiny beads, 200 nanometer beads, fluorescent beads. But it's this jiggling, this Brownian motion that Einstein was thinking about and is the topic kind of of what I'm going to describe. How do we suppress this Brownian motion for single molecules? This is a very critical process on the nanoscale in cells. So it's worth knowing about. So now if you move forward into the mid-1980s, Art Ashken, and he just received a Nobel Prize last year but could not be here. So this is a little shout out for art. He along in this paper described a trap, a single beam gradient force optical trap for dielectric particles. And you see even Stephen Shue's who is here as well is on this paper. This was a great experiment that involved realizing that when you focus a light beam down, then the gradients that are produced at that focus generate restoring forces that draw a dielectric sphere into the center of the laser beam, including along the z direction because there's radiation pressure pushing, but then there's the gradient force from the focus since it's right after the focus pulling the object backward. When those two balance, you get z as well as x, y, and y trapping. So a beautiful idea. This tight optical focus was called optical tweezers and it depends upon the gradient of the intensity as I just said, but also on the polarizability of that sphere. That sphere polarizability scales as the volume, so please remember that. This idea works down to about 200 nanometers because if you try to go smaller, you need too much intensity to try to trap the object if you're not at resonance. So it has many implications for many fields. Now I was inspired by that work, but also inspired by other wonderful experiments to detect individual molecules. And here's some of those other wonderful inspirations, STM and AFM of single atoms on surfaces, single cooled ions and vacuum electromagnetic traps, Weinlein, Phillips, and all at this meeting, the great pioneers, and then even ion channel currents. This another single system that was being studied in the mid-80s, but no one had detected a single molecule optically. So in 1989, we did that. We detected a single molecule optically in my lab at IBM Research. And this experiment, now there's no tweezers from here on, there's no optical forces from here on. I just want to talk to you now about light and the molecules and what we can do with them. And in order to detect the single molecules, we had to pump them with lasers, we had to do spectroscopy of how they absorbed light, we wanted to pick the molecules correctly, and we had to do very high signal to noise as you might expect. I mean, that's always the bread and butter of a great experiment where you have to do something that hasn't been done before, you have to optimize signal to noise. These pictures appear to be three dimensional, but it's really 2D images of the light coming from the molecules that I'm showing here, where the ZX is just how bright the molecule is. So that's all very well and good, but what I'd really want to tell you about in a moment is going to be how we trap those molecules using the same ideas of pumping molecules and collecting their light. Think about that single molecule regime for a minute, if you will, with me. What do we see when we're at this single molecule level? So quite often we want to observe something, for example, the motion of a protein. Sometimes the protein is fluorescent, sometimes it's not, so we have to often attach some fluorescent label, some covalently attached small molecule that acts as the light source that tells us where that protein is. And here in this schematic, let's suppose we're interested in a cell, so the surface proteins can be labeled this way, the transmembrane proteins can be labeled this way. So these single molecule experiments simply require pumping that molecule, that fluorescent label, and collecting the emitted light, which is shifted to long wavelengths. And let's just look at how this can appear in a real experiment. In a 12 by 12 micron field, we're looking at the surface of the cell, just like that picture, and these beautiful little dots are now nanoscale objects, single molecules, that are dancing on the surface of the cell because they're anchored in the membrane and that represents a viscous medium and they're diffusing around, okay, within that two-dimensional membrane. They're also slowly disappearing. And finally, if you noticed in that picture, each of the little single molecule images was a disk, it wasn't infinitely small. That's due to this diffraction limit that it was just mentioned. A single emitter appears to be a few hundred nanometers in size. So by now, you may have realized, I'm not talking about super resolution today, you'll hear about that from Stefan Hell. What I want to do is to trap those individual molecules in analogy to what Art Aschkin did earlier, but now by a different technique, not using those optical forces. Let's once again figure out how, explain what the problem really is. Here's a little sort of illustration of what's going on. If you are working in solution, this Brownian motion makes the molecules move away from a region of interest. If you're looking here with a laser and you want to do a detailed experiment on the molecule, they run off to you quickly. And X and Y, you only get a burst of fluorescence if you don't do something to stop that Brownian motion so you can get lots of information. So what we do is to measure the positions of the molecule. We want to use microscopy to see where it is so we track it. And then we use the information from the position to generate a force, a feedback force that we push against the molecule whenever it jumps out due to Brownian motion, we push it back with these external forces. And we do this in a closed loop feedback configuration. So now what happens? Now if you don't have the trap on, then you get that motion. But when the trap is on, notice that the molecule stopped in the center and its emission gets very, very constant here. Sorry. You can see that it's not really just a burst anymore, but it's sort of a continuous emission because we've held it where we can look at it in detail. And so this is the trap that I'm talking about. We call it the anti-Brownian electrokinetic trap or the Able trap. And now it gives us a way to sort of grab these single objects and measure them and study them for a long time to see what they're doing, how they're behaving. So it's worth a second to tell you a little bit about how it works. So a little bit more detail, but you guys are all ready for this, I'm sure. The molecules are placed in a microfluidic geometry in solution, of course, and we're going to trap in the center of this. The Z motion is just suppressed because it's a thin layer. And so here's a schematic of a molecule being put into the trap. We illuminate it with a laser. And now what we're going to do is actually move the laser beam around. We're going to move it in this pattern, which is the pattern that is a knight will move on a chess table. It's equivalent to confocal microscopy, but it's just a more useful pattern of motion of the laser produced by some deflectors that move the beam in directions. Then we collect the emitted light coming back from the molecule, like I mentioned, because that's going to give us, with the motion of the laser, that position information that we need. The position information is then processed by a field programmable gate array of FPGA. And then we will put the forces on that are generated by the computer. When it knows which direction to put the force on, then the computer will put voltages on to drive the molecule. The molecule can be moved by electrophoresis, a process that we can talk about, or it can be moved by electroosmosis, another process. The molecule may be charged or not. It doesn't matter, both of them work. And then finally, we want to ask a lot of questions about this molecule. So we put on the detection side a whole bunch of additional optics and components, a second detector to measure the two polarizations, a filter, a dispersing device, a spectrometer to say what's the spectrum of the emitted light, and then we use the time information to get lifetime. So we can measure four variables, four variables simultaneously for each single molecule. Let's end the trap before it goes out of the trap. Great. So there's the molecule being trapped. It's jiggling around the center. I'm not trying to localize it very precisely. All I want to do is just to make sure we can measure it so it doesn't have to be very precisely localized. That means this trap doesn't have to be a super strong trap. Well, so you might want to know what we get out of this. So very quickly, from those two detectors, we get every photon from that single molecule. Notice that they seem to be many and then less and then maybe photobleached. So if you look at what's really going on here, you might see that there's a high brightness level and then a lower one. And this effect is actually really fascinating. Why would one molecule have a constant emission and then change somehow to another configuration and give you a different, relatively constant emission? Fascinating sort of issue. It's showing that there are what we call states, states of these molecules inside the trap. And so for each state, we can measure information. For example, the excited state lifetime. We can measure during one time interval, what's the excited state lifetime? And then another interval, what's the other excited state lifetime? By processing every photon. We can measure the spectra of the emitted light and look at the spectra of the different states. And finally, we can get other information from the motion of the molecule in the trap. Even though we're trapping it, it's still jiggling around. And that jiggling gives you information about the diffusion coefficient of the molecule, even though we're not allowing it to diffuse and the electrokinetic mobility of the molecule, which gives information about its radius and its charge. So now just think about this. All of a sudden, we've got six different things we can measure for each molecule that's in the trap. So this can be applied to many different systems. And because my time is short, I believe, I'm not sure exactly the timing, I'm okay. So you can apply this idea to many different interesting situations. For example, in photosynthesis, okay, this important process that we're seeing a lot of going on outside. In photosynthesis, there are these antenna proteins that absorb the light and then transfer it down to the reaction center. Those turn out to be pigment protein complexes. They're often composed of many little pigments, like chlorophyll and so on, that are inside a protein. If you take one of those, one of those structures, let's say this one, this one's called alloflecosionin and put it in the trap, then you see fascinating dynamics. These states that I was talking about, changes in brightness, lifetime, and spectrum, et cetera. The light harvesting complex 2 from bacteria, the light harvesting complex LHC2, which is from green plants, all of these have been studied at the single molecule in the able trap. You can look at multi-subunit enzymes and count the number of ATP that are bound to an enzyme. You can look at electron transfer events. So redox processes of individual proteins, conformational dynamics of G protein coupled receptors, this very important set of receptors that are used in our bodies for many applications. And then finally, this able trap can trap a single fluorophore, a single one nanometer sized object. That's because there's no optical forces involved. All we have to do is to collect the photons fast enough and apply the forces fast enough. And so there are further utility that comes from measuring D and mu is to look at oligomers to see whether you have one protein or let's say a dimer or a trimer or whatever. You can look at these distributions of different multibarits just by using that information about D and mu from the trap molecules. So I have a detailed example for one specific case. I'm not going to go through it just because that's not what we want to do here. I want to answer questions and have discussion. So the summary of this is that there are many arenas and new things that can be done by looking at the single molecules or nanoparticles trapped in solution. We can look at mechanisms for these individual states of complex nano emitters, biologically induced conformational changes, proteins, enzymes, receptors, DNA, RNA and so on. And non-aquia solvents can be considered for catalysis. Aligomer distributions can be very important for these diseases. Amaloid diseases that you may have heard of, Huntington's, Parkinson's and so on, are based on different kinds of aggregates and you can measure the shapes and sizes. Let me say sizes, not so much shapes of those. So to finish up, I was asked by Bill Phillips who's giving a talk at the same time. To remind you all, we have this different topic now. There's this wonderful new set of SI units that have just come out very fairly recently in May 20th. And we, the three of us are very excited to talk to you about the new SI unit system. And many of the units are defined precisely to an exact number. An important one for us is that, remembering one molecule, okay, we're studying one molecule here, right? And I like to view that as one guacamole, one guacamole, right? Because it's one over Avogadro's number of moles, right? And this is still precisely correct in the new system. We can talk about that later. So thank you very much. Thank you.
 Good morning everybody and welcome to this first Agorah talk.  As you may know Agorah was the main square in ancient Greek cities where people came  together to exchange ideas, to discuss and so on.  And that's the main purpose of this event here too, to exchange ideas, to discuss things  with the Nobel laureates.  And I have to introduce myself.  My name is Günther Werth from the University of Mainz in Germany.  But more important I introduce our speaker here, Professor William Mörner from Stanford  University.  And he received this Nobel Prize not in physics but in chemistry, but he's a trained physicist.  He received the Nobel Prize in 2014 together with two colleagues, Erik Betzig and Stefan  Professor Heller.  Professor Heller I think is present at this meeting and he will give a talk later on this  morning I suppose.  Anyway, he received the Nobel Prize for what's called surpassing the limitations of the light  microscope.  What are these limitations?  What we learned in first year optics when we talk about resolution.  What is the minimum distance of two objects which can be resolved by visible light?  And that is law by the optician Abbey in Germany said the minimum distance of two objects should  be the wavelength of the light.  The wavelength of the light is 500 nanometers about, a couple of 100 nanometers.  The distance of two atoms in a molecule is the order of a few nanometers.  So a factor of 100 about smaller than the wavelength of the light.  There's no way to detect single atoms in a molecule unless we have a clever idea.  And this clever ideas, one of these clever ideas came by Professor Mörner and in this  talk he will explain what he did and what the prospects for the future are I suppose.  So we'll listen to him within 15 minutes, 10, 15 minutes and later on it's your turn  to discuss, ask questions to Professor Mörner and exchange ideas.  Okay, go ahead.  Well thanks so much for the introduction.  I'm so happy to see you all here today and I might ask the question, you may be asking  the question why am I here talking about molecules at a physics meeting.  But I just want you to remember that molecules follow physics as well.  And so the important thing to realize about my entire talk is that it's going to be an  intermixing of different fields.  And I intend to want to show you that it's the interaction and interdisciplinary nature  that makes some things very, very exciting these days.  So let's go back to that great year in 1905 which was already mentioned upstairs.  Albert Einstein not only worked on the photoelectric effect but he also worked on Brownian motion.  And he said that bodies of myoscopically visible size suspended in liquids must as a result  of thermal molecular motions, perform motions of such a magnitude that these motions can  easily be detected by a microscope.  And so I'd like to show you a little video that illustrates what this is all about and  mostly the topic of my presentation today.  This is, ignore these little electrodes that appear here.  This movie shows you that at room temperature molecules are jiggling all around.  These happen to be actually tiny beads, 200 nanometer beads, fluorescent beads.  But it's this jiggling, this Brownian motion that Einstein was thinking about and is the  topic kind of of what I'm going to describe.  How do we suppress this Brownian motion for single molecules?  This is a very critical process on the nanoscale in cells.  So it's worth knowing about.  So now if you move forward into the mid-1980s, Art Ashken, and he just received a Nobel Prize  last year but could not be here.  So this is a little shout out for art.  He along in this paper described a trap, a single beam gradient force optical trap for  dielectric particles.  And you see even Stephen Shue's who is here as well is on this paper.  This was a great experiment that involved realizing that when you focus a light beam  down, then the gradients that are produced at that focus generate restoring forces that  draw a dielectric sphere into the center of the laser beam, including along the z direction  because there's radiation pressure pushing, but then there's the gradient force from the  focus since it's right after the focus pulling the object backward.  When those two balance, you get z as well as x, y, and y trapping.  So a beautiful idea.  This tight optical focus was called optical tweezers and it depends upon the gradient  of the intensity as I just said, but also on the polarizability of that sphere.  That sphere polarizability scales as the volume, so please remember that.  This idea works down to about 200 nanometers because if you try to go smaller, you need  too much intensity to try to trap the object if you're not at resonance.  So it has many implications for many fields.  Now I was inspired by that work, but also inspired by other wonderful experiments to  detect individual molecules.  And here's some of those other wonderful inspirations, STM and AFM of single atoms on surfaces,  single cooled ions and vacuum electromagnetic traps, Weinlein, Phillips, and all at this  meeting, the great pioneers, and then even ion channel currents.  This another single system that was being studied in the mid-80s, but no one had detected  a single molecule optically.  So in 1989, we did that.  We detected a single molecule optically in my lab at IBM Research.  And this experiment, now there's no tweezers from here on, there's no optical forces from  here on.  I just want to talk to you now about light and the molecules and what we can do with  them.  And in order to detect the single molecules, we had to pump them with lasers, we had to  do spectroscopy of how they absorbed light, we wanted to pick the molecules correctly,  and we had to do very high signal to noise as you might expect.  I mean, that's always the bread and butter of a great experiment where you have to do  something that hasn't been done before, you have to optimize signal to noise.  These pictures appear to be three dimensional, but it's really 2D images of the light coming  from the molecules that I'm showing here, where the ZX is just how bright the molecule  is.  So that's all very well and good, but what I'd really want to tell you about in a moment  is going to be how we trap those molecules using the same ideas of pumping molecules  and collecting their light.  Think about that single molecule regime for a minute, if you will, with me.  What do we see when we're at this single molecule level?  So quite often we want to observe something, for example, the motion of a protein.  Sometimes the protein is fluorescent, sometimes it's not, so we have to often attach some  fluorescent label, some covalently attached small molecule that acts as the light source  that tells us where that protein is.  And here in this schematic, let's suppose we're interested in a cell, so the surface  proteins can be labeled this way, the transmembrane proteins can be labeled this way.  So these single molecule experiments simply require pumping that molecule, that fluorescent  label, and collecting the emitted light, which is shifted to long wavelengths.  And let's just look at how this can appear in a real experiment.  In a 12 by 12 micron field, we're looking at the surface of the cell, just like that  picture, and these beautiful little dots are now nanoscale objects, single molecules, that  are dancing on the surface of the cell because they're anchored in the membrane and that  represents a viscous medium and they're diffusing around, okay, within that two-dimensional membrane.  They're also slowly disappearing.  And finally, if you noticed in that picture, each of the little single molecule images  was a disk, it wasn't infinitely small.  That's due to this diffraction limit that it was just mentioned.  A single emitter appears to be a few hundred nanometers in size.  So by now, you may have realized, I'm not talking about super resolution today, you'll  hear about that from Stefan Hell.  What I want to do is to trap those individual molecules in analogy to what Art Aschkin did  earlier, but now by a different technique, not using those optical forces.  Let's once again figure out how, explain what the problem really is.  Here's a little sort of illustration of what's going on.  If you are working in solution, this Brownian motion makes the molecules move away from  a region of interest.  If you're looking here with a laser and you want to do a detailed experiment on the molecule,  they run off to you quickly.  And X and Y, you only get a burst of fluorescence if you don't do something to stop that Brownian  motion so you can get lots of information.  So what we do is to measure the positions of the molecule.  We want to use microscopy to see where it is so we track it.  And then we use the information from the position to generate a force, a feedback force that  we push against the molecule whenever it jumps out due to Brownian motion, we push it back  with these external forces.  And we do this in a closed loop feedback configuration.  So now what happens?  Now if you don't have the trap on, then you get that motion.  But when the trap is on, notice that the molecule stopped in the center and its emission gets  very, very constant here.  Sorry.  You can see that it's not really just a burst anymore, but it's sort of a continuous emission  because we've held it where we can look at it in detail.  And so this is the trap that I'm talking about.  We call it the anti-Brownian electrokinetic trap or the Able trap.  And now it gives us a way to sort of grab these single objects and measure them and study  them for a long time to see what they're doing, how they're behaving.  So it's worth a second to tell you a little bit about how it works.  So a little bit more detail, but you guys are all ready for this, I'm sure.  The molecules are placed in a microfluidic geometry in solution, of course, and we're  going to trap in the center of this.  The Z motion is just suppressed because it's a thin layer.  And so here's a schematic of a molecule being put into the trap.  We illuminate it with a laser.  And now what we're going to do is actually move the laser beam around.  We're going to move it in this pattern, which is the pattern that is a knight will move  on a chess table.  It's equivalent to confocal microscopy, but it's just a more useful pattern of motion  of the laser produced by some deflectors that move the beam in directions.  Then we collect the emitted light coming back from the molecule, like I mentioned, because  that's going to give us, with the motion of the laser, that position information that  we need.  The position information is then processed by a field programmable gate array of FPGA.  And then we will put the forces on that are generated by the computer.  When it knows which direction to put the force on, then the computer will put voltages on  to drive the molecule.  The molecule can be moved by electrophoresis, a process that we can talk about, or it can  be moved by electroosmosis, another process.  The molecule may be charged or not.  It doesn't matter, both of them work.  And then finally, we want to ask a lot of questions about this molecule.  So we put on the detection side a whole bunch of additional optics and components, a second  detector to measure the two polarizations, a filter, a dispersing device, a spectrometer  to say what's the spectrum of the emitted light, and then we use the time information  to get lifetime.  So we can measure four variables, four variables simultaneously for each single molecule.  Let's end the trap before it goes out of the trap.  Great.  So there's the molecule being trapped.  It's jiggling around the center.  I'm not trying to localize it very precisely.  All I want to do is just to make sure we can measure it so it doesn't have to be very  precisely localized.  That means this trap doesn't have to be a super strong trap.  Well, so you might want to know what we get out of this.  So very quickly, from those two detectors, we get every photon from that single molecule.  Notice that they seem to be many and then less and then maybe photobleached.  So if you look at what's really going on here, you might see that there's a high brightness  level and then a lower one.  And this effect is actually really fascinating.  Why would one molecule have a constant emission and then change somehow to another configuration  and give you a different, relatively constant emission?  Fascinating sort of issue.  It's showing that there are what we call states, states of these molecules inside the trap.  And so for each state, we can measure information.  For example, the excited state lifetime.  We can measure during one time interval, what's the excited state lifetime?  And then another interval, what's the other excited state lifetime?  By processing every photon.  We can measure the spectra of the emitted light and look at the spectra of the different  states.  And finally, we can get other information from the motion of the molecule in the trap.  Even though we're trapping it, it's still jiggling around.  And that jiggling gives you information about the diffusion coefficient of the molecule,  even though we're not allowing it to diffuse and the electrokinetic mobility of the molecule,  which gives information about its radius and its charge.  So now just think about this.  All of a sudden, we've got six different things we can measure for each molecule that's in  the trap.  So this can be applied to many different systems.  And because my time is short, I believe, I'm not sure exactly the timing, I'm okay.  So you can apply this idea to many different interesting situations.  For example, in photosynthesis, okay, this important process that we're seeing a lot  of going on outside.  In photosynthesis, there are these antenna proteins that absorb the light and then transfer  it down to the reaction center.  Those turn out to be pigment protein complexes.  They're often composed of many little pigments, like chlorophyll and so on, that are inside  a protein.  If you take one of those, one of those structures, let's say this one, this one's called alloflecosionin  and put it in the trap, then you see fascinating dynamics.  These states that I was talking about, changes in brightness, lifetime, and spectrum, et cetera.  The light harvesting complex 2 from bacteria, the light harvesting complex LHC2, which  is from green plants, all of these have been studied at the single molecule in the able  trap.  You can look at multi-subunit enzymes and count the number of ATP that are bound to  an enzyme.  You can look at electron transfer events.  So redox processes of individual proteins, conformational dynamics of G protein coupled  receptors, this very important set of receptors that are used in our bodies for many applications.  And then finally, this able trap can trap a single fluorophore, a single one nanometer  sized object.  That's because there's no optical forces involved.  All we have to do is to collect the photons fast enough and apply the forces fast enough.  And so there are further utility that comes from measuring D and mu is to look at oligomers  to see whether you have one protein or let's say a dimer or a trimer or whatever.  You can look at these distributions of different multibarits just by using that information  about D and mu from the trap molecules.  So I have a detailed example for one specific case.  I'm not going to go through it just because that's not what we want to do here.  I want to answer questions and have discussion.  So the summary of this is that there are many arenas and new things that can be done by  looking at the single molecules or nanoparticles trapped in solution.  We can look at mechanisms for these individual states of complex nano emitters, biologically  induced conformational changes, proteins, enzymes, receptors, DNA, RNA and so on.  And non-aquia solvents can be considered for catalysis.  Aligomer distributions can be very important for these diseases.  Amaloid diseases that you may have heard of, Huntington's, Parkinson's and so on, are  based on different kinds of aggregates and you can measure the shapes and sizes.  Let me say sizes, not so much shapes of those.  So to finish up, I was asked by Bill Phillips who's giving a talk at the same time.  To remind you all, we have this different topic now.  There's this wonderful new set of SI units that have just come out very fairly recently  in May 20th.  And we, the three of us are very excited to talk to you about the new SI unit system.  And many of the units are defined precisely to an exact number.  An important one for us is that, remembering one molecule, okay, we're studying one molecule  here, right?  And I like to view that as one guacamole, one guacamole, right?  Because it's one over Avogadro's number of moles, right?  And this is still precisely correct in the new system.  We can talk about that later.  So thank you very much.  Thank you.
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Good morning everybody and welcome to this first Agorah talk. As you may know Agorah was the main square in ancient Greek cities where people came together to exchange ideas, to discuss and so on. And that's the main purpose of this event here too, to exchange ideas, to discuss things with the Nobel laureates. And I have to introduce myself. My name is Günther Werth from the University of Mainz in Germany. But more important I introduce our speaker here, Professor William Mörner from Stanford University. And he received this Nobel Prize not in physics but in chemistry, but he's a trained physicist. He received the Nobel Prize in 2014 together with two colleagues, Erik Betzig and Stefan Professor Heller. Professor Heller I think is present at this meeting and he will give a talk later on this morning I suppose. Anyway, he received the Nobel Prize for what's called surpassing the limitations of the light microscope. What are these limitations? What we learned in first year optics when we talk about resolution. What is the minimum distance of two objects which can be resolved by visible light? And that is law by the optician Abbey in Germany said the minimum distance of two objects should be the wavelength of the light. The wavelength of the light is 500 nanometers about, a couple of 100 nanometers. The distance of two atoms in a molecule is the order of a few nanometers. So a factor of 100 about smaller than the wavelength of the light. There's no way to detect single atoms in a molecule unless we have a clever idea. And this clever ideas, one of these clever ideas came by Professor Mörner and in this talk he will explain what he did and what the prospects for the future are I suppose. So we'll listen to him within 15 minutes, 10, 15 minutes and later on it's your turn to discuss, ask questions to Professor Mörner and exchange ideas. Okay, go ahead. Well thanks so much for the introduction. I'm so happy to see you all here today and I might ask the question, you may be asking the question why am I here talking about molecules at a physics meeting. But I just want you to remember that molecules follow physics as well. And so the important thing to realize about my entire talk is that it's going to be an intermixing of different fields. And I intend to want to show you that it's the interaction and interdisciplinary nature that makes some things very, very exciting these days. So let's go back to that great year in 1905 which was already mentioned upstairs. Albert Einstein not only worked on the photoelectric effect but he also worked on Brownian motion. And he said that bodies of myoscopically visible size suspended in liquids must as a result of thermal molecular motions, perform motions of such a magnitude that these motions can easily be detected by a microscope. And so I'd like to show you a little video that illustrates what this is all about and mostly the topic of my presentation today. This is, ignore these little electrodes that appear here. This movie shows you that at room temperature molecules are jiggling all around. These happen to be actually tiny beads, 200 nanometer beads, fluorescent beads. But it's this jiggling, this Brownian motion that Einstein was thinking about and is the topic kind of of what I'm going to describe. How do we suppress this Brownian motion for single molecules? This is a very critical process on the nanoscale in cells. So it's worth knowing about. So now if you move forward into the mid-1980s, Art Ashken, and he just received a Nobel Prize last year but could not be here. So this is a little shout out for art. He along in this paper described a trap, a single beam gradient force optical trap for dielectric particles. And you see even Stephen Shue's who is here as well is on this paper. This was a great experiment that involved realizing that when you focus a light beam down, then the gradients that are produced at that focus generate restoring forces that draw a dielectric sphere into the center of the laser beam, including along the z direction because there's radiation pressure pushing, but then there's the gradient force from the focus since it's right after the focus pulling the object backward. When those two balance, you get z as well as x, y, and y trapping. So a beautiful idea. This tight optical focus was called optical tweezers and it depends upon the gradient of the intensity as I just said, but also on the polarizability of that sphere. That sphere polarizability scales as the volume, so please remember that. This idea works down to about 200 nanometers because if you try to go smaller, you need too much intensity to try to trap the object if you're not at resonance. So it has many implications for many fields. Now I was inspired by that work, but also inspired by other wonderful experiments to detect individual molecules. And here's some of those other wonderful inspirations, STM and AFM of single atoms on surfaces, single cooled ions and vacuum electromagnetic traps, Weinlein, Phillips, and all at this meeting, the great pioneers, and then even ion channel currents. This another single system that was being studied in the mid-80s, but no one had detected a single molecule optically. So in 1989, we did that. We detected a single molecule optically in my lab at IBM Research. And this experiment, now there's no tweezers from here on, there's no optical forces from here on. I just want to talk to you now about light and the molecules and what we can do with them. And in order to detect the single molecules, we had to pump them with lasers, we had to do spectroscopy of how they absorbed light, we wanted to pick the molecules correctly, and we had to do very high signal to noise as you might expect. I mean, that's always the bread and butter of a great experiment where you have to do something that hasn't been done before, you have to optimize signal to noise. These pictures appear to be three dimensional, but it's really 2D images of the light coming from the molecules that I'm showing here, where the ZX is just how bright the molecule is. So that's all very well and good, but what I'd really want to tell you about in a moment is going to be how we trap those molecules using the same ideas of pumping molecules and collecting their light. Think about that single molecule regime for a minute, if you will, with me. What do we see when we're at this single molecule level? So quite often we want to observe something, for example, the motion of a protein. Sometimes the protein is fluorescent, sometimes it's not, so we have to often attach some fluorescent label, some covalently attached small molecule that acts as the light source that tells us where that protein is. And here in this schematic, let's suppose we're interested in a cell, so the surface proteins can be labeled this way, the transmembrane proteins can be labeled this way. So these single molecule experiments simply require pumping that molecule, that fluorescent label, and collecting the emitted light, which is shifted to long wavelengths. And let's just look at how this can appear in a real experiment. In a 12 by 12 micron field, we're looking at the surface of the cell, just like that picture, and these beautiful little dots are now nanoscale objects, single molecules, that are dancing on the surface of the cell because they're anchored in the membrane and that represents a viscous medium and they're diffusing around, okay, within that two-dimensional membrane. They're also slowly disappearing. And finally, if you noticed in that picture, each of the little single molecule images was a disk, it wasn't infinitely small. That's due to this diffraction limit that it was just mentioned. A single emitter appears to be a few hundred nanometers in size. So by now, you may have realized, I'm not talking about super resolution today, you'll hear about that from Stefan Hell. What I want to do is to trap those individual molecules in analogy to what Art Aschkin did earlier, but now by a different technique, not using those optical forces. Let's once again figure out how, explain what the problem really is. Here's a little sort of illustration of what's going on. If you are working in solution, this Brownian motion makes the molecules move away from a region of interest. If you're looking here with a laser and you want to do a detailed experiment on the molecule, they run off to you quickly. And X and Y, you only get a burst of fluorescence if you don't do something to stop that Brownian motion so you can get lots of information. So what we do is to measure the positions of the molecule. We want to use microscopy to see where it is so we track it. And then we use the information from the position to generate a force, a feedback force that we push against the molecule whenever it jumps out due to Brownian motion, we push it back with these external forces. And we do this in a closed loop feedback configuration. So now what happens? Now if you don't have the trap on, then you get that motion. But when the trap is on, notice that the molecule stopped in the center and its emission gets very, very constant here. Sorry. You can see that it's not really just a burst anymore, but it's sort of a continuous emission because we've held it where we can look at it in detail. And so this is the trap that I'm talking about. We call it the anti-Brownian electrokinetic trap or the Able trap. And now it gives us a way to sort of grab these single objects and measure them and study them for a long time to see what they're doing, how they're behaving. So it's worth a second to tell you a little bit about how it works. So a little bit more detail, but you guys are all ready for this, I'm sure. The molecules are placed in a microfluidic geometry in solution, of course, and we're going to trap in the center of this. The Z motion is just suppressed because it's a thin layer. And so here's a schematic of a molecule being put into the trap. We illuminate it with a laser. And now what we're going to do is actually move the laser beam around. We're going to move it in this pattern, which is the pattern that is a knight will move on a chess table. It's equivalent to confocal microscopy, but it's just a more useful pattern of motion of the laser produced by some deflectors that move the beam in directions. Then we collect the emitted light coming back from the molecule, like I mentioned, because that's going to give us, with the motion of the laser, that position information that we need. The position information is then processed by a field programmable gate array of FPGA. And then we will put the forces on that are generated by the computer. When it knows which direction to put the force on, then the computer will put voltages on to drive the molecule. The molecule can be moved by electrophoresis, a process that we can talk about, or it can be moved by electroosmosis, another process. The molecule may be charged or not. It doesn't matter, both of them work. And then finally, we want to ask a lot of questions about this molecule. So we put on the detection side a whole bunch of additional optics and components, a second detector to measure the two polarizations, a filter, a dispersing device, a spectrometer to say what's the spectrum of the emitted light, and then we use the time information to get lifetime. So we can measure four variables, four variables simultaneously for each single molecule. Let's end the trap before it goes out of the trap. Great. So there's the molecule being trapped. It's jiggling around the center. I'm not trying to localize it very precisely. All I want to do is just to make sure we can measure it so it doesn't have to be very precisely localized. That means this trap doesn't have to be a super strong trap. Well, so you might want to know what we get out of this. So very quickly, from those two detectors, we get every photon from that single molecule. Notice that they seem to be many and then less and then maybe photobleached. So if you look at what's really going on here, you might see that there's a high brightness level and then a lower one. And this effect is actually really fascinating. Why would one molecule have a constant emission and then change somehow to another configuration and give you a different, relatively constant emission? Fascinating sort of issue. It's showing that there are what we call states, states of these molecules inside the trap. And so for each state, we can measure information. For example, the excited state lifetime. We can measure during one time interval, what's the excited state lifetime? And then another interval, what's the other excited state lifetime? By processing every photon. We can measure the spectra of the emitted light and look at the spectra of the different states. And finally, we can get other information from the motion of the molecule in the trap. Even though we're trapping it, it's still jiggling around. And that jiggling gives you information about the diffusion coefficient of the molecule, even though we're not allowing it to diffuse and the electrokinetic mobility of the molecule, which gives information about its radius and its charge. So now just think about this. All of a sudden, we've got six different things we can measure for each molecule that's in the trap. So this can be applied to many different systems. And because my time is short, I believe, I'm not sure exactly the timing, I'm okay. So you can apply this idea to many different interesting situations. For example, in photosynthesis, okay, this important process that we're seeing a lot of going on outside. In photosynthesis, there are these antenna proteins that absorb the light and then transfer it down to the reaction center. Those turn out to be pigment protein complexes. They're often composed of many little pigments, like chlorophyll and so on, that are inside a protein. If you take one of those, one of those structures, let's say this one, this one's called alloflecosionin and put it in the trap, then you see fascinating dynamics. These states that I was talking about, changes in brightness, lifetime, and spectrum, et cetera. The light harvesting complex 2 from bacteria, the light harvesting complex LHC2, which is from green plants, all of these have been studied at the single molecule in the able trap. You can look at multi-subunit enzymes and count the number of ATP that are bound to an enzyme. You can look at electron transfer events. So redox processes of individual proteins, conformational dynamics of G protein coupled receptors, this very important set of receptors that are used in our bodies for many applications. And then finally, this able trap can trap a single fluorophore, a single one nanometer sized object. That's because there's no optical forces involved. All we have to do is to collect the photons fast enough and apply the forces fast enough. And so there are further utility that comes from measuring D and mu is to look at oligomers to see whether you have one protein or let's say a dimer or a trimer or whatever. You can look at these distributions of different multibarits just by using that information about D and mu from the trap molecules. So I have a detailed example for one specific case. I'm not going to go through it just because that's not what we want to do here. I want to answer questions and have discussion. So the summary of this is that there are many arenas and new things that can be done by looking at the single molecules or nanoparticles trapped in solution. We can look at mechanisms for these individual states of complex nano emitters, biologically induced conformational changes, proteins, enzymes, receptors, DNA, RNA and so on. And non-aquia solvents can be considered for catalysis. Aligomer distributions can be very important for these diseases. Amaloid diseases that you may have heard of, Huntington's, Parkinson's and so on, are based on different kinds of aggregates and you can measure the shapes and sizes. Let me say sizes, not so much shapes of those. So to finish up, I was asked by Bill Phillips who's giving a talk at the same time. To remind you all, we have this different topic now. There's this wonderful new set of SI units that have just come out very fairly recently in May 20th. And we, the three of us are very excited to talk to you about the new SI unit system. And many of the units are defined precisely to an exact number. An important one for us is that, remembering one molecule, okay, we're studying one molecule here, right? And I like to view that as one guacamole, one guacamole, right? Because it's one over Avogadro's number of moles, right? And this is still precisely correct in the new system. We can talk about that later. So thank you very much. Thank you.
ideas, one of these clever ideas came by is that it's going to be an intermixing of nanometer beads, fluorescent beads. But it's this very critical process on the nanoscale in cells. dielectric particles. And you see even Stephen sphere into the center of the laser beam, optical focus was called optical tweezers and it to go smaller, you need too much intensity to try no one had detected a single molecule optically. here on, there's no optical forces from here on. now nanoscale objects, single molecules, that are nanometers in size. So by now, you may have this Brownian motion makes the molecules move generate a force, a feedback force that we push grab these single objects and measure them and study them for a long time to see what they're solution, of course, and we're going to trap in polarizations, a filter, a dispersing device, a we can measure it so it doesn't have to be very proteins, conformational dynamics of G protein protein or let's say a dimer or a trimer or different multibarits just by using that in solution. We can look at mechanisms for these guacamole, right? Because it's one over