Reaching Molecular Size Resolution in Lens-Based Microscopy: the Diffraction Limit Blown Away
This is a modal window.
The media could not be loaded, either because the server or network failed or because the format is not supported.
Formal Metadata
| Title |
| |
| Title of Series | ||
| Number of Parts | 340 | |
| Author | ||
| License | CC Attribution - NonCommercial - NoDerivatives 4.0 International: You are free to use, copy, distribute and transmit the work or content in unchanged form for any legal and non-commercial purpose as long as the work is attributed to the author in the manner specified by the author or licensor. | |
| Identifiers | 10.5446/45088 (DOI) | |
| Publisher | ||
| Release Date | ||
| Language |
Content Metadata
| Subject Area | ||
| Genre | ||
| Abstract |
|
00:00
NobeliumSetzen <Verfahrenstechnik>SchmerzschwelleZunderbeständigkeitCommon landBearing (mechanical)OperonReaction mechanismInitiation (chemistry)Nobelpreis für ChemieMoleculeSchmerzschwelleIce frontPlant breedingLactitolBase (chemistry)ProteinfaltungAreaHope, ArkansasLibrary (computing)Crystallographic defectStorage tankWine tasting descriptorsSunscreenPondPressurePhysical chemistryAageLecture/Conference
04:24
NobeliumInsulinTransition metalInjection (medicine)MoleculeSample (material)Nuclear Overhauser effectNoble gasSeparator (milk)MoleculeBleachFluorescenceFunctional groupCoordination numberSeparation processWeaknessÜbergangszustandZunderbeständigkeitSea levelShear strengthMolecular beamSeleniteEmission spectrumStimulantWursthülleRacial segregationWine tasting descriptorsEpitaxieSystemic therapyStratotypSolutionSurface finishingThermoformingPondBerylliumInfiltrationsanästhesieBlock (periodic table)Hope, ArkansasExciter (effect)Essenz <Lebensmittel>Crystallographic defectElimination reactionElectronic cigarettePaste (rheology)DyeingElectron donor
11:55
Sample (material)FluorescenceNobeliumInsulinTau proteinLymphangioleiomyomatosisZunderbeständigkeitFluorescenceHydrophobic effectMoleculeElectronic cigaretteStratotypExciter (effect)Emission spectrumCalculus (medicine)Sample (material)Breed standardMaßanalyseColourantWine tasting descriptorsPhysical chemistryStop codonSolutionSetzen <Verfahrenstechnik>Molecular beamElectron donorInfiltrationsanästhesieOrganisches MolekülEssenz <Lebensmittel>PondController (control theory)WursthüllePaste (rheology)ElectronCollisionOctane ratingStorage tankCrystallographic defectPan (magazine)Injection (medicine)Computer animation
19:26
NobeliumProteinZunderbeständigkeitRibosomeProtein subunitMoleculeCycloalkaneCell (biology)TinLebensmittelfarbstoffKernproteineMolekulardynamikProteinfaltung11 (number)Combine harvesterMoleculeKernproteineWursthülleCell (biology)Sea levelNobeliumNuclear poreStratotypZunderbeständigkeitWine tasting descriptorsFunctional groupProteinPhysical chemistryEmission spectrumPaste (rheology)ChemistryElectronic cigaretteElectron donorAqueous solutionMultiprotein complexSunscreenBreed standardSense DistrictIsotopenmarkierungBearing (mechanical)Crystallographic defectNucleolusFlux (metallurgy)Forkhead-GenColourantStream bedEssenz <Lebensmittel>InfiltrationsanästhesieAngelic acidProteinfaltungStuffingGesundheitsstörung
26:57
NobeliumFlux (metallurgy)Computer animationLecture/Conference
27:30
NobeliumNobeliumStockfishBrown adipose tissueActive siteLecture/ConferenceMeeting/Interview
28:15
NobeliumInclusion (mineral)InsulinLecture/ConferenceMeeting/Interview
28:57
NobeliumSea levelSolutionCell (biology)MoleculeSample (material)PondFlux (metallurgy)Lecture/ConferenceMeeting/Interview
30:19
NobeliumMoleculeDeterrence (legal)IsotopenmarkierungFluorescenceChemistrySea levelPhysical chemistryAntibodies (film)PharmacyGreen fluorescent proteinClick chemistryElectronic cigaretteWine tasting descriptorsProteinInsulinLecture/ConferenceMeeting/Interview
31:43
NobeliumÜbergangszustandSeparator (milk)ElektronentransferMoleculeIngredientElectronCrystallographic defectChemistryIsotopenmarkierungLecture/ConferenceMeeting/Interview
32:43
NobeliumCrystallographic defectBearing (mechanical)GesundheitsstörungReflexionsspektrumFluorescencePhysical chemistryPharmaceutical drugEssenz <Lebensmittel>Process (computing)ThermoformingPlant breedingStuffingLeft-wing politicsLecture/Conference
35:33
NobeliumEmission spectrumWursthülleWalkingMoleculeBleachPatentMatchLecture/Conference
37:04
NobeliumReflexionsspektrumSeparator (milk)Emission spectrumModul <Membranverfahren>FluorescenceLecture/Conference
38:13
NobeliumGeneHorizontaler GentransferIronNobeliumLecture/Conference
38:47
NobeliumSolutionReflexionsspektrumZunderbeständigkeitPhysical chemistryElectronic cigaretteLecture/Conference
40:11
NobeliumElutionPhysical chemistryData conversionElectronic cigaretteLecture/Conference
Transcript: English(auto-generated)
00:14
I want to warm welcome everybody to this session. It's a great pleasure to stand here in front of you students from all over the world today.
00:23
It's such an honor. And I hope the Lindau Week started well. We usually say that the problems of today will be solved by the laureates of tomorrow. And I hope we will be able to welcome some of you to Stockholm in some coming years. That would be really nice.
00:41
And remember, I saw you first. So now we will meet one of them who we had the pleasure to welcome to Stockholm. Someone with a great passion for microscopy. Professor Stefan Held from the Max Planck Institute for Biophysical Chemistry in Gottingen. And Professor Held was awarded the Nobel Prize in Chemistry in 2014.
01:02
Together with Eric Betzig and William Murner for the development of super-resolved fluorescence microscopy. And he has himself witnessed about the importance to meet with peers when you're a student. He said meeting with Isidor Rabi was a very important one.
01:21
So you should really grab the moment this week because the laureates are here for you. So take the possibility and grab them and talk to them because a meeting can actually change your life. So now I am very, very curious to listen to Professor Held's presentation about microscopy. He got the prize because he increased the resolution at tenfold.
01:44
And he has no respect really for diffraction barriers. So I'm really curious for this. So please welcome up Professor Held. Thank you very much, Anna. So as a matter of fact, I'm going to speak about the recent developments of my lab.
02:04
And of course they have something to do with the diffraction bearing and blowing it away entirely. Now the 20th century, of course, the 20th century resolution was limited to about 200 nanometers as it is shown here. And the so-called confocal microscope was state of the art. But the development of that microscopy showed that there is physics, so to speak, that allows you to overcome the diffraction barrier.
02:26
And in practice attain a spatial resolution that is substantially higher. As it is shown here in this view graph by a factor of ten. So 200 nanometers diffraction barrier initially. And the resolution was then about 20 nanometers.
02:41
And this development shared the Nobel Prize in Chemistry in 2014 as referred. And this is the official poster of the Nobel Foundation. And you see a sketch of the STAT microscope and its basic principles of operation. And a sketch of the Palm Storm microscope which has many things in common with STAT microscopy.
03:03
Many things are substantially different. And of course you may ask, what do they have in common, which I think is interesting. Well, one thing they have in common is the fact that in principle, in principle, they can attain a spatial resolution that is at the molecular scale.
03:21
But, well, if I may add, in practice they don't. In practice the resolution is usually limited to about 20 nanometers. And only there are some exceptions where you can get higher with either of the two concepts. But not as a, say, in a standard way. So the Nobel Foundation actually put it quite right in the poster that you can see here.
03:45
So there microscopes cross the threshold as you see, but as I said, did not attain the ultimate limit. And since I'm passionate about breaking a diffraction barrier, since I knew that in the end, you know, you should be able to get down to, say, the size of the molecule.
04:03
There's no fundamental reason. So why not going after that and do that? So the title of my talk, as a matter of fact, is Attaining Molecular Size. So one nanometer, two nanometer spatial resolution in three dimensions. Now in order to get there, of course, you have to understand what actually broke the diffraction barrier and the mechanisms for that.
04:24
And I'm trying to speak simplistically, but a simple way is often the most profound way. Do you feel this is, at least most of us believe, that if it's simple, then it's probably true. Now, the role of a lens in a microscope is of course to focus the light down to a point.
04:42
If you could, of course. So if you could focus the light down to a point like here, to a single molecule, then you could single out that molecule, detect the light from there, of course, and then you could separate at molecular scale resolution. But in reality, there is diffraction, and we get a spot of light that has a minimum size of half the wavelengths in diameter
05:00
and about a wavelength along the optic axis. And as a matter of fact, what you get is that you, say, illuminate more molecules at the same time. And therefore, there will be more molecules in general, several molecules, scattering the light back to the detector. And of course, at the detector, you not only have a blob from one molecule,
05:21
you have a blob for many, say, molecules, a backscattered light for many molecules, diffracted. And of course, no detector could tell the diffracted signals from those molecules apart. So what is the consequence of this?
05:42
The consequence of this is that light is very bad at separating features. Just that, separating features. If you want to separate features by light, you have a problem, that's clear. So you have to find another way to separate features. And the solution to this problem, this is the general solution to this problem and the basis for all the so-called super-resolution techniques.
06:01
You use a state transition to separate features. So in other words, of course, if you have those many molecules there that scatter light back, just make sure that some don't. So put the molecules into a state that is dark, shown here in dark color, in black color, where the molecules are not capable. Of scattering light back.
06:20
And then, of course, you get the signal from that single molecule, and then the whole system behaves as though just that molecule would have been illuminated by light. And so you need to have an on-off transition. And that works well for fluorescence. It's hard to do in reflection, I've not figured out how to do that. But in fluorescence, it's very obvious. W.E. just gave a talk. I mean, you excite molecules, you turn them on, so to speak, by the absorption of a photon, the green photon here.
06:45
Then you have backscattered emission, which is fluorescence measured at the detector. But you can turn them off by applying a beam of light that makes sure that in the case a molecule gets to the excited state, there's always one photon for sure that keeps the molecule back down to the ground state, sends it down to the ground state, stimulates the photon, goes straight on.
07:02
We're not interested in light that goes straight on. We're just interested in backscattered photons. And then it's off for the backscattering, and then we are capable of doing just that. So this was the principle of stat microscopy. And that donut-shaped beam that is shown in red has two functions. One, to turn the molecules off. And the second function is to clearly state where the molecule is on, where the molecule is off.
07:25
And at the donut zero in the center, the molecule is still capable of emitting, whereas in the rest, it is not. Now the concept of POM is related in as much as it also uses an off-transition for separation. But in this case, it's based on W.E.'s discovery of being able to detect single molecules.
07:41
So just one molecule is turned on, could be thermally, could be by light, by anything, and at a random position in the diffraction region. So again, I'm showing this inevitable diffraction blob of light in green, and that single molecule, of course. And if it's done stochastically in space, so to speak, without a pattern of light that determines where it is,
08:01
you have to know where the molecule is, where the molecule is located. And then, for that, you can do the following. You have a camera that is a pixelated detector that provides a coordinate reference. And if the molecule emits many photons in a bunch, produces a pattern of light at the camera, then you can find out, so to speak, the position of the molecule,
08:20
but just by calculating the centroid of emission, because you know the centroid of emission kind of relates to the position of the molecule. Now, the precision with which you can do that depends on the number of the photons in the pattern. It's very clear. The more photons you have on the pattern, the better you can calculate the centroid. And so the higher the precision will be in order to find out the position of the molecule. The separation is already done by the on-off transition, so that's sorted out.
08:43
You just need to deal with or find out the position of the molecule. Now, this is interesting to compare it now with that. It's very obvious that you always need many, many, many, many, many photons for defining a coordinate. It's very clear. With one photon, you could never define a coordinate,
09:01
because within the diffraction zone, it would go either here or there or there, and there's no way to define a coordinate. So in POM, STORM, it is done by having those many photons that define the coordinate of emission, but how is it done instead? Well, in essence, the same thing. I mean, you have a pattern of light, a donut beam, that defines the coordinate, which is the center where there is no light.
09:22
Donut center is very well defined, and the beam is, of course, the baddest defined position. So in the end, it boils down to the same thing, having many photons for defining a coordinate. The better you can define a coordinate, the better you can localize the molecule. But you see now the strengths and the weaknesses of the two concepts. A definite strength of POM, STORM is the fact that you deal already with single molecules.
09:44
It doesn't give you molecular resolution, but you deal already with single molecules. That's good if you want to have high spatial resolution down to the molecular level. But the weakness of the POM, STORM concept is that the pattern of light where you determine actually the position is done with the feeble fluorescence, fragile fluorescence.
10:03
The molecule blinks, goes on and off, they did bleaches. You have all these problems, and usually you don't get the high number of detected photons that you require in order to get a very, very high precision. There's other issues coming there as well. But that's different instead, of course, because instead, of course, you do the localization,
10:23
so to speak, the definition of a coordinate with the photons from the laser. Resilience photons from lasers, plenty of photons from lasers. You can define that zero with arbitrary precision in space. And here you have the few photons, many but limited photons from the dye, and now you see which is better at what.
10:41
It's very obvious that you come up with a method that combines, so to speak, the single molecule features of POM and, of course, the coordinate definition of STAT using a donut. Then you have combined the strengths of the two, and then you should get down to the spatial resolution that is at the size of a molecule.
11:01
To make it clear what people think of localization when they talk about localization, they say typically I'm having a normal epi fluorescence microscope, I have bright field illumination like this, and the star now is a molecule. And then, of course, what happens, I have a camera, and the picture on the camera is actually the fraction pattern that is produced
11:22
by the molecule on the camera, as I just explained, and they define as localization, as calculating the centroid of that position. And, of course, the more photons you have, as I said already, the better will be the precision, and you know where the molecule is located. So the positioning, the definition of the coin entirely relies on the emission of the fluorescence emission.
11:43
But now we do it differently. So instead of doing epi fluorescence illumination, we don't use a camera. We have just a single point detector that is shown in here, and we define a coordinate with a doughnut. This is shown here in green. Now this little doughnut uses light for exciting molecules,
12:01
for interrogating if the molecule is at a certain position, and now you can imagine that if the doughnut is bright enough, of course, the zero of the doughnut will be very well defined. And if the molecule is right in the zero of that doughnut, it will not emit light. That's very clear because there is no light at that very, very point. Of course, if it's slightly off, then of course it will emit light because it's slightly off.
12:21
And so you see the doughnut zero efficiently defines a chord in sample space. The chord is not defined by the fluorescence emission, just by the fact that we have a bright doughnut beam, but we can, of course, interrogate the position of the molecule just by seeing how well the molecule is off. And then, of course, this is very useful. Now since my last name is Hal, I thought I have to introduce a demon.
12:45
And this is a little demon. And what this demon does, demons are always very clever. The demon kind of knows where the molecule would go if the molecule moves. And this little demon can act actually on the position of the doughnut, can shift the doughnut around.
13:01
And you see what happens. Let's assume the molecule will move and we would have to find out its position. You see, it moves. But the demon knows where the molecule goes, positions the doughnut very quickly such that the doughnut's center always coincides with the position of the molecule. So why am I showing you this Gedang experiment, this thought experiment?
13:21
Because this thought experiment tells you, you can precisely define or find out, so to speak, the position of the molecule without requiring a single fluorescence emission. Because if the doughnut zero coincides with the position of the molecule, there won't be any fluorescence emission. So this is quite opposite to what is done in the normal localization,
13:42
you cry many, many, many, many, many emissions, 10,000 or so, to get down to the highest position. Here, without any single emission, emission is fragile, bleaching, and the rest of it, and then the molecule blinking, and a finite rate, and so on. And this is something which is, of course, very viable if it's possible.
14:02
Well, this is not viable because it's a demon and there is no demon. But you can approach a situation by having an electronics, of course, which detects the fluorescence emission if the doughnut is slightly astray, it goes slightly away from the position of the molecule. That gives you information about the actual position of the molecule that's fed in into a controller,
14:22
and you can kind of mimic the situation where the course, say, finding out of the position of the molecule is done by the doughnut, because the doughnut already gives or injects a position into the sample space, it injects a position into the sample space, and then the emission is only used for relating the position of the molecule
14:40
to that injected position of the doughnut. And so you need only a few emissions in order to find out where the molecule is, because the majority of the localization is already done with the doughnut by injecting the core into the doughnut. Again, quite different to Palm Storm, where every time, for every emission, you have to find out where the molecule is.
15:01
And so you may ask, how do you do it in practice? So I'm speaking a bit about details here. Well, it's very simple in the end. If you think about the principles, let's assume the molecule, the little star, is located between that purple and orange point. So we don't know where it is within that line,
15:20
but it's somewhere on that line. Now this little doughnut, of course, in the center of the beam has a quadratic intensity dependence, what we would assume. And then if you move, of course, that zero across the molecule, then at a position where the zero, doughnut zero, coincides with the position of the molecule, then you won't have, of course, any fluorescence emission.
15:42
I'm showing this again. It's quite obvious now the zero moves across the body. Now there is no fluorescence. And so you know where the molecule is located, and then you know exactly where it is. Now, in reality, you don't have to scan the zero over that molecule. It may take too much time. If you know that this is a quadratic function,
16:01
of course, that you have there, then it's enough to measure the fluorescence from the end point. So the end zero and the end one, and do a simple seventh grade, so to speak, algebraic solution of a quadratic equation, and then find out that the position of the molecule is given by this distance, l, between the points where you anticipate the molecule to be,
16:21
divided by unity, plus the square root of the ratio of the two fluorescence numbers end points, like 50 photons on the left, 30 photons on the right. So you divide them square root, and then you can calculate the position. Of course, with a certain position that is just given by the number of photons that you have.
16:40
Now, one striking thing, you get a position without wavelength dependence. You say, okay, I studied physics, I've learned. I mean, if you do focused light, so it's always depending on the way. No, there's no wavelength dependence. So it really means the diffraction barrier is gone. So this is surprising if you think about it. Not really. I mean, the molecule is a point.
17:00
So it's a point, full stop. But a zero, sorry, is a point again. If you just match two points, where should the dependence on the wavelength in first order come from? So it's gone. Wavelength dependence is gone. But of course, the precision will depend on the number of fluorescent photons that you have. And so, if you calculate the standard deviation,
17:21
of course, it gives you the resolution. It scales with a distance L, that's clear. So the better you know where the molecule is, the higher the precision, divided by the square root of the number of photons that you detect. So the big N is the sum of the two emission points. But this is good news. Why? Because it scales with the distance
17:41
of the zero to the molecule. And the closer, the better you are of a demon, of course. The closer the zero is to the position of the molecule, the higher the precision will be. This scales linearly, whereas the dependence on the photon numbers, like in Palm Storm, just scales with the square root, which is highly damped.
18:01
So it's very effective to scale down the L, meaning that once you know roughly where it is, don't wait for more photons. Just bring the zero close to the molecule, and then, of course, the precision goes up. So in essence, of course, what you can do, you can always bring the molecule, or bring the zero close to the molecule, and your precision goes up, because it scales linearly with the distance.
18:21
Now in 2D, of course, it is not much different. You just have to take more points. In this particular case, we use four points, but you can use other types of arrangements, no doubt. And then you measure the fraction that is emitted if you place the donut to the four points, shown in different colors. And then, say, the estimator looks a bit different. It's not just that simple equation.
18:42
But in the end, the physics stays the same. You have a linear dependence on the distance between the zero and the position of the molecule, and, of course, this square root dependence of the number of photons, and the linear dependence always wins, because, as I said, the closer you bring the zero to the molecule,
19:01
the higher the precision will be. Now to just show you how this outperforms the classical, say, calculation of diffraction pattern localization. So I'm showing you the total number of photons for a distance L of 50, for example. Say, you see, with 10 photons, you get precision, something of 10.
19:20
For a camera and a realistic precision, you would require 29 times more photons, or 290 photons. And it shows that you can be 29 times faster, of course, when it comes to... And this is really useful, because if you're faster, you can track molecules quicker by, for instance, emission. Or you can save photons and so on. But again, as I said, the key is that
19:41
you bring the zero close to the position of the molecule. You can compensate that for increasing intensity. It won't glitch, because you're still at a minimum. You remain at a lower level. There's no optical nonlinearity in there. It's all linear. And then, of course, you can do either imaging, nanoscopy or something, or you can do tracking,
20:03
as the little demon did. So I'm first showing you the tracking, because I thought it's good to show a movie, and then in case you have fallen asleep, the movie always wakes you up. So here, a little movie that shows actually the movement of a molecule, of a labeled molecule, in a living, say, bacterium.
20:23
Okay, now I'm seeing wiggling stuff. This is not impressive, but if I'm telling you, this is way faster than anything you can do with a camera. Then, I think, for the experts of you, well, here you could have probably taken 20 localization at this resolution. Here you see, we're talking something like 8,000 or so.
20:42
So you can really effectively use this concept to track the movement of single molecules. But I'm not a movement guy. I'm interested in imaging. And now I'm showing you images. Now, at the time the Nobel Prize was awarded, in 2014, an object like this, where you have these fluorescent molecules
21:01
at, say, distance of 11 nanometers, arranged like that, you could not have resolved it. So this is showing a palm storm, say, attempt. The image of those molecules failed. So the combination now of, say, turning molecules on and off, of course, this is very important. We have to look at a single molecule at a time. And the finding out their position with the donut
21:23
which we call min-flux, so that's the name of the acronym, the name of the concept, tells them apart. You see, there. I'll give you another example. Now the molecules are six nanometers apart. Imagine, six nanometers. There's a fraction, fraction, fraction of a wavelength. Palm storm, of course, doesn't take it apart.
21:41
Min-flux takes it apart. And don't forget, this is done just with focused, visible light with regular objective lenses. Now imagine, when I was a student, I said, okay, listen, guys. At some point, we'll have something of a resolution at a molecular scale using focused, visible light. Say, oh, you're totally nuts now.
22:00
We know you're nuts, but this would be totally crazy because there's no way of doing that. I'm happy to come back. Why people thought about that for many, many years, that it wouldn't be possible, maybe a factor of two, three, maybe four, okay, five, but never, never at a molecular scale. I think it's psychologically very, very interesting. So molecular size resolution is essentially reached.
22:23
And now you may say, okay, these are little things, but is this useful? Can you do some useful images? So I'm showing you useful images that are, and the use of it, so to speak, in biology. So these are, again, nuclear pore complexes because they are very good standards when it comes to spatial resolution, and this is something that biologists would call a real image.
22:42
So these are the little places where proteins go in and out of the cell nucleus. And here, if you analyze the resolution, it's definitely below three nanometers, and zooming in, now this is basically what you see in individual molecules. Zooming in here, again, I'm zooming in here, and now I have the fun, of course, to compare it with that.
23:02
This is the Nobel Prize winning method, which was not bad, given the fact that initially we had just this thing on the right, but now min-flux is better by another factor. And it really shows that we are done more or less, conceptually it's very clear, but we're down at the molecular scale resolution.
23:22
And this is interesting because it can also be done in living cells. This is now from a living cell, nuclear pore complex under aqueous condition, mammalian cell, as a matter of fact. So here, the molecule that was used to turn on the individual molecules was a fluorescent protein, and you see the spatial resolution.
23:42
It's really impressive to me, it's impressive to me. Anyway, it also can be done in 3D. So we use a donor that was used actually in the first stat paper. This is just to show you that we have completed this quest and have a Z resolution that is as good as an X and Y. So you can have unilateral three-dimensional resolution
24:02
that is at a molecular scale, that is as the size of the protein, as the size of the molecule, as the size of a linker. You can have multicolor, this is just an example of multicolor, multicolor 3D. This is important for biologists, because they can relate, of course, the position of the proteins with respect to each other. And of course, here I'm showing you
24:22
an important protein from the synapse. So this is a 3D rendition of the distribution of individual molecules that are attached to proteins in the synapse. So this is going to be exciting, because we have now the option to image in a cell molecules
24:40
and molecular complexes in 3D, basically at a size of that, say, chemistry that labels the protein. We never see the protein itself. This is impossible, because it's fluorescent microscopy. But at the size of that protein level, there's a molecular size, so that's conceptually the end of the story, if I may say that. The resolution of, say,
25:01
one to three nanometers in 3D, molecular movements much faster. I estimated 10 microseconds for one nanometer precision in 10 nanometer range. So one can imagine not only seeing, say, molecular maps of synapses, vesicles, nuclei, viruses and so on, but also perhaps protein folding,
25:21
molecular dynamics, things that people think it's not possible to access because it needs x-ray diffraction and something that doesn't really see the movement. I'm very confident this can be worked out. And this is just the beginning, because now the engineers can come and make it better and better and better. And in 10 years from now, you have mind-boggling images, you say, oh my goodness, the concept is clear, the physics is a closed matter.
25:42
And speaking about that, of course, I'm acknowledging the people who really did the hard work on those experiments and have a substantial contribution to the development of this concept. I don't have a large group anymore, so these are really outstanding people, I can tell you. And two of them are Lindau alumni, actually three of them are Lindau alumni,
26:01
as a matter of fact. And the final insight I'm giving to you, often when I'm talking to physicists, they say, yeah, but you must use some optical nonlinearity. There is no optical nonlinearity in this. It's a molecular scale resolution, no optical nonlinearity. As a matter of fact, the notion that you can overcome
26:20
the diffraction barrier with optical nonlinearity has led to the thinking, oh, you need an optical nonlinearity to overcome the diffraction barrier. And this has led physicists astray for many years and has blocked the fresh view of the problem. And only if you have a fresh view of the problem that does away with the thinking of the past, you can really do something new.
26:42
This is the message I would like to give you now. If you want to create something new, don't use the language of the past. Do away with that. Your peers won't like it because they always want to translate it in a language they have learned. But this is a very good example that you have to do away with the language in order to create something new. Thank you very much.
27:08
Wow, this sounds like a dream for life science, new discoveries. Can't you go back to the picture that was really impressive with the confocal, the stead and the min-flux.
27:26
This is, it's like a dream coming true, sort of. So this is your Nobel Prize. So speak, yeah. I'm a little bit, almost this goose bumping here.
27:40
So you increased the resolution 10 times and now you have increased it 10 times more. So 100 times. Yeah, yeah, absolutely, yeah. I just need to ask you. So when you got this call from Stockholm saying, hello, Professor Hell, we have a small prize for you. Did you see this coming or were you thinking you were done here?
28:02
Well, I haven't spoken about this in public but the truth of the matter is yes. We worked already on that concept and the Nobel Prize was a kind of delay because it was distracting. And so my psychology at the time I got the call was, wait a moment, I still have more work to do.
28:20
There is still more that is coming and so why are you calling now? And so this was, so I must say, I mean, I can say now I was a bit irritated but I didn't know how to handle the situation because I couldn't have said, okay, now, well, listen, there's more to come. I mean, you cannot speak about ongoing projects.
28:41
But yes, I knew and at the time I went there and the king gave me, of course, the medal and the scroll. I knew there was more to come. I said, you will never regret that you've given the prize to these three individuals because this is a groundbreaking development for sure. It's not just that step. It is more than that.
29:01
It's the solution to the problem. So 3D living cells at the molecular level. Well, I'm a little bit shocked here. So I'm sure there are tons of questions. So go ahead. Yes. Yeah. So for this min-flux, of course, you have to see individual molecules.
29:23
So right now this is all, say, within a short distance from the cover slips or something like five microns or something. But I'm very confident I actually have ongoing projects that go in the direction of looking deep down, further deep down. Why? Because contrary to the typical Palm Storm setting where you have a camera and you have a so-called wide field detection
29:42
where all the stray light falls on the camera, here you have a confocalized detector. So you can kind of just detect layers deep, specifically layers deep in the sample. So I'm quite confident this will work out much deeper in the sample than any of this, say, single-molecule methods will.
30:01
So I cannot tell it now, but maybe when I give a talk, maybe in two years or three, I'm quite confident that high net micron will be possible. But who knows? I mean, this is engineering, but the concept as such is much better at looking deep into something than the other concept.
30:33
Now, taking the specific molecule works by fluorescent labeling. So this has been sorted out by other people.
30:40
There are plenty of ways of labeling. So there are so-called fluorescent proteins. There is direct chemical labeling. There is now even more fancy chemistry like so-called click chemistry and so on. Of course, there's antibodies, but antibodies are very large compared to the resolution we have, so they're not useful anymore. You can use fragments.
31:01
You can use so-called nanobodies. These are cameloid antibodies. They're much smaller. So this is a science by itself to make the fluorescent labeling. And it becomes very, very important now because the physics resolution is at that level. And so you have to sort out. You have to sort out labeling.
31:20
So I'm now redirecting a new institute in Heidelberg. And so the first thing I did, I hired or helped hiring, of course, the best labeling chemist there is right now, and he came, fortunately, because this is actually a bottleneck now. The physics is understood. And the labeling, of course, determines what you can see in the end.
31:45
No, no. So the separation is done by an on-off transition that could be done by anything that goes from, say, a transient off-state to a long-lasting, a transient on-state to a long-lasting off-state. You can imagine all kind of things.
32:00
Cis-trans ionization, electron transfer, all kind of, say, switching, which is a chemistry issue. And that signals out the molecule from the rest within the diffraction barrier, very clear. And you need to have the contrast, of course. But that's a chemistry issue. And then, of course, you need to suppress. The signal comes from the unlabeled molecules,
32:22
but again, say, a concept that has this pinhole, and not a camera that detects everything, is very good at suppressing the signal from the surroundings. We've seen it. And this is also part of the technical ingredient that helps us detecting those molecules so well, because we detect them actually at a minimum, so to speak,
32:41
and so we need to suppress the surroundings. So over there, in the back, with the black. I have no idea. Well, it's very interesting how I got to this problem.
33:02
So I never want to do optics. I never want to do microscopy. And I have to correct Anna. I'm sorry, Anna. I was actually never really interested in microscopy. The only thing that was interesting, I wanted to sort out the diffraction barrier problem. And I was not motivated by helping life sciences. Don't forget that. If I had been a microscopist,
33:21
so I would have done the same, sorry, boring stuff, most other microscopists did, which was the physics of the 19th century. So I got fascinated by the problem. I wanted to find out, is there a physics, a viable physics, that allows me to show this picture that you see on the left? And Jan says yes. For instance, clearly yes.
33:41
And we're down there. But I was never interested in microscopy per se. I had curiosity. Yeah. No, actually, I gave it in other talks, of course. So I kind of ventured into microscopy because I wanted to find Ph.D. diseases that helps me getting a job.
34:03
So someone do something applied against my own inclination. And I did that. And for my Ph.D. season, it was totally boring. I had to inspect computer chips for Siemens with a light microscope. And then I thought, okay, this is all silly and this is not why I studied physics and I wanted to do something more profound. And then I realized, okay, maybe there's still something cool
34:20
that I could sort out in the microscopy business. And I got the idea of breaking the fraction barrier. And I was not interested in doing it for fluorescence. I was not interested per se in doing it for the life sciences. It just happened that I figured out it's easier to do it for fluorescence. If I could have done it for reflection or anything else, I would definitely have done it for reflection.
34:42
I had no interest in the life sciences. And if I may say that, I mean, at that time, not now, don't get me wrong, at that time, I looked down on biologists. Because biologists, for me, this is the reason why I didn't study medicine. I thought, okay, they memorize things, they learn things by heart,
35:01
they do not understand concepts, they cannot do mathematics. So these are kind of not clear thinkers. That was my thought at that time. So I didn't ask, why should I do something for those? No way. I was so arrogant, if you ask me. Of course, I have a different view now. So don't quote me.
35:21
But this was my thinking at that time. I'm a life scientist. So you sort of resolved my problem as a PhD student. I'm very, very happy with this interdisciplinary. I think sometimes you are used to, have to cross the borders of disciplines. So now we have so many questions.
35:42
In the white shirt. What is the main limitation of photo numbers? So I can tell you. For stat, in principle, one photon, one emission is enough if you define, of course, the position with the donut.
36:02
One photon is enough. Usually this is not the case because there are other limits. The donut zero is not perfect. Then there is, of course, bleaching of the molecule and so on. So you need, usually you need four. But in principle, one emission is absolutely enough. So in this min-flux case, a few emissions are absolutely enough. Of course, if you are close to the zero,
36:22
this is zero to the position of the molecule. So it's clear that the concept that uses a coordinate definition, that injects a coordinate, clearly can do with a minimal number of emissions because the position is determined by the pattern. This is a fundamental insight. I'm sorry that I had. And I was surprised to see that the field didn't really adapt at this view.
36:46
As a matter of fact, I wrote about it in the Science View 2007. It gets cited, but not for this reason, although this was actually one of the things that was in there when comparing PALM and STAT, that you inject the position, you target actually position, and this really gives you an edge when it comes to finding out where the molecule is.
37:05
So we take two more questions, and we have one over there. Based on reflection? Well, if you can turn reflection on and off, because you need that, say, modulation of the signaling,
37:25
so to speak, for separation. This is the key for this business. If you figure that out, you can do it for reflection. So I haven't figured out how that can be done properly. Again, under some limiting circumstances, I can imagine that, that you can convey that, but it's not as fundamental as it is
37:43
and not as widely applicable as it is for fluorescence, where, of course, you can easily modulate fluorescence emission because the states have lifetimes that allow you to do some things. The fluorescence state is about a nanosecond, and in the dark states, there are microseconds to milliseconds even, and so there's time to do something with them
38:02
and play with them and measure with them and create that on-off contrast. The quicker the state is, if it's just a transient state or something, the harder it is. If it's a scattering or so, it's difficult because it's so short-lived. Since your PhD, what drives you now here to the Nobel Prize?
38:25
So what is driving you now? Which curiosity are driving you now? Which are you passionate to discover? Which are your open questions now? Yeah, I think maybe it's a personality defect, so I still enjoy this, and I have a lot of fun. I mean, getting there and showing this image,
38:46
of course, I'm thinking about the next thing. Do you beg your own limit? Maybe go even higher resolution? Well, I think it's basically down. You can do it better. You can do it faster, 10 times faster. You can do better in 3D, more useful here and there,
39:01
but the principle is very clear. So that's engineering. That can be sorted out by throwing money on it because there are clever people that sort out the difficulties over time, and you can help them. But I'm interested in seeing what else is there, and there might be something else.
39:21
I'm not saying this is the end, per se, of the field, but clearly this is a quite accomplished solution for sorting out. You're down at the molecular scale, so where do you want to go? Again, doing it for reflection would be one thing, or doing it for fast scattering, or doing it for, I don't know,
39:43
going to ultrasound, or I don't know, I mean, Steven Chu is talking about ultrasound, maybe there is something to be done there. So I think this is the right thing to do. But just making it now slightly better in 3D is the usual trait, which has to be done because biologists come and say, oh, I want to see that, and so you're twice too slow.
40:01
So, okay, that's useful and gives you time to think. But that is sorted out by engineering and by throwing money on it. So I'm very happy that you are having fun and that you keep your passion for physics while you are still helping the life scientists anyway, and I'm looking forward to see what will happen with this in the future.
40:22
So I'm a little bit afraid, or I'm hoping, sort of, that I will be able to welcome you maybe back. I don't know. And you will have an afternoon. Yeah, so I very much look forward to that. I've always had very, very nice conversations. So if you're interested in this and I want to get more about, say,
40:42
how we got there and why it's like this and not like that, please come and see me in the afternoon. Exactly. So all of you who had questions that you were not able to get, you come to Stefan, either outside or in the afternoon. Thank you so much, Stefan. Thank you.