We have all been brought up with the notion I think that the resolution of a light microscope that uses regular lenses and focused visible light is fundamentally limited by diffraction.
Now for that reason, the electron microscope was invented and of course it's getting probe microscopes and there is no doubt about the fact that as a result of the higher spatial resolution these microscopes have attained,
they have fundamentally changed our understanding of the world.
However, it's also important to realize that if you want to apply this microscope to certain things, for example to living cells, they are fundamentally limited in the way they operate.
Because if you want to look inside a cell, inside a live cell in particular, inside live tissue, there is no alternative to using focused visible light.
Because focused light is the only way to gain information from inside an intact cell and get this information outside without disturbing the fundamental functions of a living cell or organism.
Now for that reason it made a lot of sense to re-address the resolution issue and to ask is there really no way to overcome the limits set by diffraction to come up with a light microscope that has a spatial resolution with sort of an electron microscope.
Now today we know that this is possible, at least for fluorescent imaging. This is a subfield of microscopy which uses fluorescence tags in order to see the objects of interest.
But as I will explain towards the end, it's not fundamentally limited to fluorescent imaging because the underlying physical principles are significantly more general.
Now I'm going to explain it in a very simple way. Now we all know that in a light microscope the job of a lens is to focus the light down to a point.
But the lens will fail in doing so simply because light propagates as a wave and so what we will get here is a blob of diffraction intensity, diffraction maximum that is at least about 200 nanometers wide and about 500 nanometers along the optic axis.
And so if you're having several features, several objects falling within that distance, falling within that region, they will be illuminated at the same time and they will give off light at the same time and so no detector will ever be able to separate a signal if they are of the same kind.
Now the man who realized this problem first with all the consequences was this man, Ernst Abe, who lived at the end of the 19th century as we all know.
And he coined this diffraction resolution barrier in the equation which is still named after him.
It's basically saying that in order to be separated in a light microscope, two features of the same kind have to be further away than the wavelength divided by twice the numerical aperture of the objective lens.
And of course you can find this equation basically in any textbook of physics or optics and also cell and molecular biology because in these fields the light microscope plays a preeminent role as I indicated already.
Now you may know or not know that Ernst Abe has had many contributions to optics but for some reason this diffraction resolution barrier is regarded as his most scientific legacy.
Because if you happen to travel to Jena where he lived and worked, you will find this memorial erected in his honor and this equation written in stone.
And this is what we want to beat now. We would like to show that we will find a way to discern features of the same kind that are closer than the distance d.
Now if you don't believe in this, you probably still have to believe in something. And what I believed in that in a fluorescence microscope if you fully explore the properties of fluorophore, the spectral properties of fluorophore, there must be features, there must be transitions out there in that fluorophore that will allow you to get sharper images.
So why was this unusual at that time, early 90s? It was unusual because at that time people thought the role of a fluorophore in a fluorescence microscope is to work as a label.
You attach the fluorophore, for example, to a protein and you know exactly where the protein is located. Or maybe you can use the fluorophore, for example, to measure ion concentration such as calcium concentration or pH.
But here the notion was different. Perceive the fluorophore as a vehicle to break the diffraction barrier.
So there must be transitions out there in a dye that allows you to get a spatial resolution beyond the diffraction barrier.
Now philosophy is fine, but in the end you have to come up with something that really works.
And the first concept that worked with a focus in light microscope is the concept of Stead Microscope, which I am briefly explaining how this microscope works.
Again, we have a lens and the role of the lens is, of course, to focus the light down to a point.
And we have a laser, for example, an excitation, a light source providing light, this green light showing here, bouncing of this dichloric mirror.
And the lens, of course, will focus the light down to this focal region in order to excite molecules, fluorescent molecules, from the ground state to the fluorescent state.
Usually they will dwell a bit in that lower lying vibration level of the first electronic excited state about nanosecond.
Then they come down by emitting a fluorescent photon.
And that fluorescent photon, of course, is collected by the same object that goes this way and measured here in the detector.
So this is the process of generating fluorescence.
This is a simplified energy diagram of an organic fluorophore.
Now, the lens, of course, as I said, will not succeed in focusing the light any sharper than to about an nanometer.
So all the molecules falling within that distance will be excited more or less at the same time, statistically.
And then they will come down to the ground state by emitting fluorescent photons more or less at the same time.
So the objective lens, of course, will register the light that is emitted here from this region.
And then it will go to the detector.
And what we will get?
We will get here a signal that is the sum of the signal generator of all these features.
How do we get an image in this case?
Well, we have to scan that spot across the specimen like this.
And then we get point by point the signal generated here at the detector.
And then we get an image.
That's clear.
But it's also clear.
You won't be able to discern features that are closer than this distance.
It will not be possible to discern what is lying within that distance simply because they emit at about the same time.
Now, if this is the problem, we have to find a solution for it.
Now, you can say, well, one solution would be to make that region of fluorescence generation or excitation smaller.
But clearly, the lens is not able to do a better job than that.
So if the lens is not able to do a better job than this, there's only one thing that could do the job.
It must be somehow in the dye.
There must be a feature out there in the dye, a transition out there in the dye.
It will allow us in the end to generate the signal for fewer molecules than are just here covered by the excitation light.
Now, if you trained as a physicist and you ask, what could that be?
Then I think if you think a bit about it, you think, oh, there must be such a thing.
It's called simulated emission.
Because just as we can excite the dye to generate fluorescence, we can also de-excite the dye.
By pushing the molecule down to the ground state and hence stop the spontaneous emission of light, stop the fluorescence.
And this is very simple.
We need photons that are redshifted in wavelengths so that they are not able to excite the molecule from the ground state up to the excited state.
But according to very basic laws of molecular physics, they will notice those molecules that happen to be located here in a nanosecond-lift fluorescence state,
produce a copy photon of themselves, a simulated photon that goes this way.
We don't care about it.
It has the same wavelength, same direction.
But the net result is the molecule will instantaneously go down to the ground state and will not be able to emit spontaneously anymore because it has been quenched.
Now, if you're worried about the fact that you may generate, of course, an excitation from here to here, don't worry.
Because the state into which the molecule is dumped here, this higher vibration level, is a very quickly decaying state.
It decays within less than a picosecond.
So once the molecule is down here, you can safely rely on the fact that it will not be able to press because it will instantly fall down to the lowest vibrational state at room temperature.
And now you can imagine what we do.
We use that beam of light, but phase modify in such a way, put it in a phase plate, not to make a spot in here, but to make a ring, a donut.
And now, as you can see, we can stop molecules lying here at the outer part of the focus, and they cannot emit if they are hit by a stimulating photon because they are shut off.
Now, that must improve the spatial resolution a bit because we see now the signal from fewer photons as a result of the implementation of the red beam.
But that wouldn't be much in this scheme because only a few molecules are stopped.
So what we want to do, we want to stop more so that fewer molecules in here are able to emit light.
How could we do that?
Well, one option would be to make the ring smaller, but Abbe would say, no way.
You cannot make smaller rings than spot.
There's no way.
That's also limited by diffraction.
But you don't need to do that.
The only thing we need to do is we need to stop the fluorophore.
And to stop the fluorophore, the only thing we have to do is we have to put in enough red light, enough red photons so that we can be sure that in the case the molecule gets excited,
there's always a photon out there that will kick down the molecule by stimulating the emission.
So we have to crank up the intensity such that we can be sure that the dye will always be quenched in the case of getting excited.
In fact, if you calculate or measure the probability of a molecule to emit or to reside in that fluorescent state,
if you flood it with stimulating light, with red photons, the population of this state will decay exponentially.
That's clear.
With the intensity of the beam of clutch, you will empty that state.
The probability of populating this state will decrease substantially.
And so we can say if the intensity of the red beam is beyond a certain threshold,
we have basically switched off the dye because the dye is not able to assume this state because once it's in here, it's instantly kicked down.
And now it's clear what we want to do.
We increase the intensity of the red beam in such a way that even in the weaker parts, where it's weaker in percentage, say 20% of the maximum,
it's still beyond the threshold.
It's still beyond the threshold.
So we increase it further up and more and more molecules will be shut off.
You see, only these are now allowed to emit.
And now we get a signal specifically from this region.
So the job of the stimulating beam and the only role of the stimulating emission beam, which we call the state beam, is to switch off the dye.
This allows the population of this state.
All the molecules here are flooded with excitation light.
No way out.
But only these here are allowed to emit.
And that's the trick.
And now we must get sharper pictures.
It's very clear because we get the signal more selective in space.
And here it is in this view graph.
Here we can separate these two from those three because at the time, all of them were excited.
Only this one were allowed to emit.
And only these were allowed to emit here.
Here's nothing.
We don't know if there is something because we just scanned.
But here there are three.
Here we can separate them.
And now clearly, you must get a higher spatial resolution.
So this is the basic principle.
Does it work?
That was the big question.
The answer is yes, it does.
I would like to give you a few examples.
Here's one example.
On the left-hand side, you see a recording of a so-called confocal microscope.
That's a high-end, say, diffraction-limited microscope.
These are single molecules dispersed on a surface.
Confocal standard on the left, stat on the right.
Now you can see these two molecules are clearly discerned.
Here they are not.
Why?
Here, they gave off light at the same time.
But here, at a time this one was emitting, this one was stopped.
And so we could see and separate the signals.
So we could see them separately.
Now, this works for a single molecule.
And the point I'm making here, that's nice about the physics of it.
This is just by applying the appropriate molecular transition.
On-off transition.
We need not any, say, information about distribution of molecules,
density of molecules, or whatever.
Just we apply the right set of beams to do the right transitions.
And here, we've got the sharpest.
Okay.
Now, this first single molecule, these are sort of more complicated features.
Confocal on the left, stat on the right.
It's a kind of 2D crystal of polymer beams.
Again, just by molecular transitions.
Then, next step was show that you can do this, say, in a useful way.
For example, apply to distribution of proteins in a cell.
Because this is where, for example,
imaging has so many advantages.
Because we can tack proteins very specifically in a cell
and then see these specific proteins.
Now, this is a normal diffraction-limited confocal recording
of a plasma membrane patch of molecules.
And I see there is almost no structure.
Here, you see the details quite clearly.
By the way, there's a so-called SNARE protein, SNAP-25,
which plays an important role in the process
against SNAP-25, which plays an important role
for synaptic vesicle fusion.
So, for neurobiologists, this is very important
because this defines the region, for example,
where so-called vesicles fuse with pre-synaptic membrane in a cell.
Okay.
Again, just by molecular transitions.
Raw data, if you like.
You can do computational improvement, if you like,
but there's no need to.
Because the physics does the job.
Okay.
Now, the next step in the development
was to show that you learn something specifically.
And so, we teamed up, actually, with a colleague
in the institute that I work in, Reinhard Jan.
And Reinhard is a neurobiologist
who is interested in finding out how the synapse works in neurons.
And now what you see in here is a sketch of a neuron.
At the time we started a study,
people knew, of course, that neurons communicate
by having synaptic vesicle fuse with the membrane in here
and then it spills neurotransmitter into the synaptic cleft.
And this fusion is mediated by proteins.
Now, the question was, what happens with the proteins
that do the job of fusion?
Do they stay at its place when the fusion takes place
or are they smeared out?
This is important to understand the recycling machinery of this protein
because the proteins have to be used again.
Now, with confocal microscopy, one couldn't tell.
Because this could be due to diffraction, this blur,
or, of course, it could also be due to the fact
that the proteins are smeared out.
Now, I said to him, why don't you do electron microscopy?
And I said, oh, that's hard.
Because it's very hard to stain proteins specifically
in an electron microscopy preparation.
It's very difficult.
You don't get the statistics and so on.
This is why this was a question for many, many years
despite the fact that electron microscopy was out there.
And so we did it with that.
And we could discern now the protein of the patches,
so to speak, of individual synaptic vesicles
and come up with a conclusion, of course,
after doing many, many controls,
that the synaptic vesicle protein, synaptic tagline 1,
which is investigated here, remains clustered
after exitosis, at least in large parts.
And this gave insights into how this and the neurons work.
Just another example done here with another biologist
colleague, Stefan Ziegliis, now at the University of Berlin.
And at that time, so he wanted to find out
if a particular protein, which has a German name,
Brookpilot, which is important in Drosophila,
so the fruit fly, in the pre-synaptic active zone
formation.
That's a detail.
He wanted to know how that protein is arranged in space.
Again, standard resolution, he couldn't tell.
With the high spatial resolution of that,
he saw for the first time that this protein, the end,
the C-terminal end, forms baskets.
And that helped arrange this pre-synaptic active zone.
So this was the inside that was generated in here.
So here I'm showing you more recent data
done in two color recordings, where we also
labeled the end terminal and to the other end of the protein.
And just to give you an idea, so you see very clearly
this basket shape.
And this model, well, it probably
wouldn't have been there without the STAT investigations.
But, well, if you're a physicist, of course,
you're interested just in the resolution gain.
So this is what you have in raw data with STAT,
and this you have is the normal confocal microscope.
And this is nice to see that the physics works out that way.
You get this information.
OK.
Now, physics, I think, is sort of obvious.
But what about making it simple, this methodology?
Because then, of course, you can disseminate it widely.
This was very important in order to make it more accessible.
Of course, it made it more applicable.
So you put a lot of effort in that.
Initially, I should say, and those of you
who have some notion of quantum optics know that, oh,
this is a relatively complex laser system.
We used those laser systems initially
because we thought we have to have the right wavelengths
for the excitation.
We have to have the right wavelengths
for the stimulated emission.
We have to play with whether it's polished,
and the pulse is short or long, and so on.
All good reasons to use that.
And of course, another reason was it gave nice images.
But at the end of the day, these systems
easily cost something like 300,000 or 500,000 euros,
at least at that time.
And so we had to do something in that regard.
Now, fortunately, we were helped by, I would say,
major development in laser physics
that took place during the last five years.
And one of them is the generation of supercontinua,
which has implications for other things, of course.
But here, these supercontinuum sources
were actually quite handy to get the right wavelengths
for excitation, say, visible light, green light,
and doing, say, red-shifted light for switching off
of the dye by stimulated emission.
And so we set up such a stat microscope, basically,
for a student's lab course.
And so this is a gain in resolution
that you get with your lab course kind of setup
for the Schrodinger article.
OK.
Now, this is actually taken by a student at that time.
Now, this is just two-color recordings
from something not so nice, the tumor cells, diffraction
limited, and now sub-diffraction.
And if the room was a bit dimmer,
you could see the so-called clapturing-coated bits in here.
So these are very important structures.
And now you can correlate, of course,
this protein's better in space and learn something
about the role.
Now, it has also become commercially available.
The reason why I'm showing this is the following.
So this is a standard commercial confocal microscope
that biologists used to put a sample, push a button,
and then they get a three-dimensional picture,
for example, just by scanning.
Now, what we've done in here, or the company did,
to be precise, they added, say, a beam of light
that does the turning off of the fluorescence
by stimulated emission.
And that produces sharper pictures.
So in the end, of course, the biologists
can use the system as before.
Of course, it's clear that the wavelengths
for stimulating the dye down to the ground,
say, for keeping the dye dark, have
to be tuned to the spectrum of the dye.
And the more, of course, wavelengths you have available,
the more dyes you can use.
With simulated emission, as we all know,
is a very fundamental phenomenon that
takes place basically with any flow form.
Now, a strength of the method is, of course,
to record dynamics.
And I would like to show some applications
through the dynamic changes.
Because if it's an electron microscope,
it's very hard to observe dynamics.
And it's not really viable with live cells,
because the vacuum requirements are very strict conditions.
But here, we see a stretch of a dendrite.
And these are the so-called spine, spine necks and spines.
So these are the so-called postsynaptic side.
So you can imagine there's another neuron here.
And it gives a signal to this neuron.
And then it goes to the axon, for example.
Now, what we see with this microscope
is now changes over time.
So 0 times 0, 140, 160, 180 seconds.
And one can see how these postsynaptic sites are formed.
Now, why are these changes in morphology so important?
This is taken out of a hippocampal tissue slice
of a wreck.
It's important because it's known
that these morphological changes are critical when
we learn something for long-term memory formation.
And we hope, actually, that the STEDT microscope,
in particular, will play an important role
in deciphering the role of these morphological changes,
for example, when learning and helping us find out
what's going on when we memorize, for example.
Now, important, of course, for such a method
that is able to penetrate sort of deeply into the tissue.
And this is a sort of technical slide
showing that here it's 10 microns deep in the brain.
This is 25 microns deep in the brain still.
The resolution is still there.
It's better than 68 because this is just a profile.
And then it's 65 microns deep down,
150 microns deep down in a living brain slice.
And this is quite OK.
But we have not used things like active or adaptive optics
in here.
And I think once we do that, it will be even better.
Now, another remark that I would like to make here.
This is the STEDT on a yellow fluorescent protein.
So this is a genetically encoded protein
that is produced by the living organism.
Now, this is dynamics on the second scale.
But what about faster dynamics?
Switch gears a bit.
Now, this is beats on a glycerofil, recorded normally.
And then switch on STEDT.
And you see, all of a sudden, you
see individual particles moving in here,
whereas in the normal microscope we don't.
So this shows that we can pass the diffraction barrier
and at the same time be fast.
The requirement is, of course, that we
get enough photons per second.
So if it's bright enough, we can do that.
Here, it's 80 frames per second.
So actually, display here is about 2 and 1 by 2 times slower
than the actual recording.
Now, this has implications if you study, for example,
such things as diffusion or particle behavior and so on.
Colloidal crystals, for example, formation.
Now, to switch back to biology, we
thought that, OK, if you have such a system that is able
to image beyond refraction barrier
and you want to, say, apply, for example, neuropathology,
an important application is to image, for the first time,
the movement of individual synaptic vesicles
in a living axon, for example.
Now, as I mentioned already, so neurons
have these little containers.
The vesicles are about 30, 40 nanometers in size.
But it's hard to follow the movement
of individual synaptic vesicles if they are densely packed.
So this is a confocal snapshot.
You see almost nothing.
So it's packed.
But now we do it with that.
And now we see the movement of this individual synaptic vesicles
recording at 28 frames per second.
So it's sort of real time recording.
Now, this is not random motion.
This motion really makes sense.
You can change this motion.
And the person who was really critical in this study,
it only has taken the study further
to learn about how these synaptic vesicles move about.
And he found out, for example, they move differently
if they are freshly incorporated later on, for example.
OK.
So this is the implications of different fields.
So I'm coming back to the basic physics, of course.
Now, it's obvious that we can go beyond the refraction barrier.
But the question comes up now.
What is the resolution gain that we can have?
Is there a new barrier?
Or what does this mean, actually?
So what are the limits?
So what is the obtainable resolution?
Now, to explain this in a very simplistic general manner,
I'm coming back to the initial slide.
Now, I think I was quite clear about the fact
that the gain resolution is due to the fact
that we turn off, say, the signaling capability of some
of the features in here.
And so we can find the signaling capability, the fluorescence
capability, to a very small dimension.
That dimension is, of course, smaller than Abhis diffraction
barrier.
And so if we increase the intensity
further and further and further, so the peak intensity
of the doughnut in here, then the resolution D cannot be given
by Abhis equation anymore.
And it's also clear that the ratio I, which
is the intensity in here, over IS over that threshold
intensity, must be somehow in the denominator.
Because if it gets larger and larger, D must get smaller.
So it must be here.
Now, if you do the calculation right, you will find out
it's not just the ratio I over S is the square root
of the ratio I over S. I can't explain this at the end.
Now, this equation is OK, but just about OK.
Because if we stop the red beam, if we
stop that simulating beam here, then we
would have a normal microscope.
And we would have normal, a normal Abhis resolution.
But if the beam is stopped, then I
would be 0, and the square root of 0 would be 0.
And of course, we would divide by 0,
as the equation wouldn't hold anymore.
We can easily fix that.
If you're an experiment physicist,
you just put a unit in here, and everything is fine.
And so this equation is really fine.
It explains the situation quite well.
So if I is 0, you have a normal resolution.
If I becomes large over S, if this number becomes large,
5, 10, 100, and so on, then D becomes very small.
And so it also means it can become very small, obviously.
But you can also tune it.
That's good.
Because it means that if the resolution need not
be that large, why wouldn't you see
you have something at 5,000 resolution
if the structure is 50 nanometer?
You can grab, of course, the signal from many molecules,
as it is shown in here.
And then you can be faster.
Because if you turn off many, then you
have to scan very densely.
You have to do the thing, the time on-off switching
many times, and that goes, of course,
at the expense of recording time.
So you can play, of course, and tune
the speed of recording with resolution.
You can adjust them according to the needs that you have.
OK, so here, poor spatial resolution and the resolution
becomes higher because with density, it's growing up.
So by tuning.
But now, from the more basic viewpoint,
one might ask, what is the contribution of this concept
to the art of microscopy per se if you view it perspective?
Well, I think there are two of them.
First of all, it's the first concept
that broke the diffraction barrier
by playing an on-off game, making sure
that all the molecules are flooded with, say,
excitation light are capable of emitting.
But then, of course, it also made abundantly clear
that you can really break the diffraction barrier.
Now, if you have two molecules falling very close
at molecular distances, five-dynamic distance,
can you separate the two?
Yes, in principle, of course we can.
If you switch this one off, you see the signal of that one.
By switching that one off, you see the signal of that one,
although they are very, very close.
And this is how we can have, of course,
resolution at the molecular scale, shown in here.
Now, we turn this one off to see that one,
and then we turn this one off to see that one.
And so we can resolve conceptually
with molecular resolution, with focus visible light.
So in other words, the diffraction barrier
has not been shifted just to a new barrier,
but it has been truly broken.
And you can have molecular scale resolution
with visible light and regular lenses.
And this is actually reflected here in the equation,
because if I becomes large over the threshold IS,
then D goes virtually down to zero,
at least to the molecular dimensions.
Now, the question is, what does this actually mean?
I becomes large over IS.
What is the meaning of this?
Well, I think it's obvious.
I'm going back to this cartoon now, this on-off,
and this kind of exponential curve.
Well, it means that this curve becomes very steep.
If IS is small with respect to I,
small with respect to IS,
then this curve must be very, very steep.
In other words, if you have a perfect on-off switch,
if you switch perfectly,
we must be able to get a very, very high spatial resolution.
Now, the question is, can you produce such a sort of perfect
on-off switch in reality?
Now, with organic molecules, it's maybe not that easy,
because of course, if you increase the intensity,
one might may say that there may be additional transitions
going on and that would lead to photo bleaching and so on.
I wouldn't subscribe that entirely,
as you will see in a second,
but clearly there's a challenge.
But there are, of course, inorganic flow,
for many of you will be aware of that do the job already.
You can think of, of course, for example, of color centers.
And I'd like to show you an example here
with a very specific color centers
that have been, actually in the science of it,
have been very much pushed by Joerg Wachtrup
at the University of Stuttgart,
who pioneered the study of charged nitrogen vacancies
in diamond for various reasons.
And he found out in the mid-90s
that these are very, very photostable.
And because they are so photostable,
he thought, OK, let's try to do that stacked trick,
that on-off trick with these charged nitrogen
vacancies in diamond,
so with this color center fluorophores.
And here it is. This is not a cartoon anymore.
This is a measurement.
This is true on-off.
And you can do that as often as you like
because there's no chemical reaction going on.
They will destroy the fluorophore.
And that has, indeed, allowed us to demonstrate
something very sharp.
So here, 220 nanometers would be the standard resolution.
It's about 8 nanometers in size,
meaning that you can really discern,
in principle, things that are that close
because you turn them off if they're here,
but they are allowed to be on only if they're here.
And so it's a physical demonstration
that these kind of resolutions are not just a dream,
but they are attainable.
Now, to give you an example,
so these are color centers in diamond, bulk diamond.
This is normal recording.
You don't see them because they are too close.
But here with a spatial resolution,
in this case, 20 nanometers, you can separate them.
You can record them many times as you like
because there's no photo reaction going on
that will destroy them.
And so this is just a confocal comparison.
It goes on like this.
You can collect as many photons basically as you want,
as long as the system is stable, of course.
Now, that's nice because there is no bleaching
and you can have constant resolution to any nanometers.
As a side remark to the biologists view,
if you make little diamonds out of those spot diamonds
and attach them to a proton,
you have a wonderful fluorophore,
you can take as many pictures as you want.
There will be no degradation.
So this is a very fascinating prospect.
I think that will come to fruition sooner than later.
But if you get so many photons, you can say,
well, I'm collecting here plenty, plenty photons,
and then it must get a very crisp image.
And because I'm getting a very crisp image,
and since these defects, these color centers
are separated already, I know exactly
at which position they have emitted
because these are individual emitters.
And so I can easily calculate the centroid, of course,
and that's the position of the emission.
This is obvious.
This is not breaking of the diffraction barrier, of course.
We know this point emitters,
and so we can calculate the center of gravity of emission.
Then we know the coordinates.
But the risk of sounding dramatic, too dramatic.
This is 1.5 angstrom with precision.
We can get the position,
and this is what you get the normal microscope
for nothing, you see nothing.
And it really tells you that something has happened.
Here you see the individual color centers
defect in the bulk diamond with atomic sort of precision.
Here you see nothing.
Okay, now, this is just physics.
That's the remark that I'm making here.
There are many implications.
They have interesting spin states and so on.
I don't want to dwell on that.
But I would like to mention something else at this point.
This on-off principle, which is underlying
the STAT principle, is by far more general
than using stimulated emission.
This is really important.
So I suggested initially, of course,
the first suggestion was to use stimulated emission.
And this was, of course, important.
Why?
Because stimulated emission, if you think about it,
is certainly the most fundamental way
of turning off a dye,
of switching the fluorescence capability of the dye.
And it's the most general way
of turning on and off the fluorescence capability of a dye.
Why?
Because it directly acts on the fluorescent state.
It directly disturbs the fluorescent state
kicking down to the ground state.
But this generality, or fundamentality,
comes at a price.
And the price that we pay is we have to make sure
that there is always a photon there.
We've seen that nanosecond lifetime of the fluorescent state.
That's the time in which you have to act.
There's always a photon that instantly kicks down
the molecular ground state.
So if the beam is too weak, the simulating beam,
the beam will miss the excited state photon.
And so the photon will, the excited state molecule,
the molecule will be able to emit.
And this is the reason why we have to put in a strong beam.
Because the lifetime of that excited state is very short.
The window of action is very, very short.
So in other words, it means, okay, if this is a bottleneck,
if this is a problem, there must be other on-off
transitions out there in a dye or in a material
that do not push us so hard in terms of time,
in terms of action.
If we switch something between long-lived states,
there is more time that is given to us.
And so we can apply beams that are less intense.
Because we don't need to put in the photons so quickly.
And so if you think about that, again,
if you just trained as a physicist,
then you'll say, oh, one option is to flip a spin.
These organic molecules, they have a so-called triplet state,
dark state, spin state.
This is the so-called singlet state
in which they are emitting, by the way.
And if you flip a spin and put them to a triplet state,
then they're not able to emit.
Because once in a triplet state,
they stay there for a microsecond,
they won't come down that easily to the ground state
because that spin-forbidden dipole transition,
in that sense, or as a dipole transition,
spin-forbidden, of course.
And so there's no way for the molecule
to come easily in here.
It stays there for some time.
It's a metastable state.
And now we can switch off, so to speak, to die
by parking in the triplet state
that works by strong excitation, of course.
And then there is always some probability of crossing
to the triplet state if there is enough spin-orbit coupling.
And so you can play the on-off game
with something that has a much longer lifetime,
say, microseconds, as opposed to nanoseconds.
And so the threshold of intensity
goes down by three orders of magnitude,
kilowatts, not megawatts per square centimeter.
And so this was the idea behind this concept
which I call ground-state emission.
They feed a ground state parking in triplet state
to make them dark.
Now, once you have realized that you can flip and spin,
then you know, well, maybe you can also flip an atom.
And if the states, of course, like cis-trans isomerization,
is what a chemist would, for example, say,
if one of the states, or both states,
are, in fact, long-lived states,
then there's enough time to produce that on-off difference
in state, this on-off disparity in states.
And there are even proteins that can be switched on and off,
for example, with a cis-trans transition, isomerization.
And, of course, these are very handy
because they're genetically encoded.
So when I realized this, in the generality of this,
I saw I cannot call this concept stat anymore
because there's nothing to do with stimulated emission.
One has to generalize this.
And so this has been generalized under the name RESOLVE.
This acronym, which stands for reversible,
cis-sachable, or switchable transitions between two states.
I worked hard on it to make it sound right,
and it almost sounds right.
But the net outcome is that, again, all these molecules,
in principle, would be flooded with excitation light
because we cannot make that spot of excitation light any smaller.
We're limited by diffraction.
But we turn these molecules off with a beam of light
that spares out some of the molecules that
give us a certain location.
And then, of course, only these molecules
will be allowed to emit.
But, again, it's not done with stimulated emission.
It's done by, say, a spin flip, or it's
done by cis-trans isomerization, or something.
And this operates, of course, at lower power.
So the same thing, the same story,
but at much, much, much lower light levels.
Now, initially, of course, I should say,
and if you look at the numbers, dates 95 and 2003 or so, 97,
it didn't work that well because the fluorescent protein that
were available at that time didn't switch off very often,
on and off very often.
But this is exactly what you have to do, of course,
if you play this trick because, as you can imagine,
if I'm moving such a beam that turns on and off
the fluorescent photon, I have to turn them
on and off many times.
And there is a thing called switching fatigue.
And if you can switch them, say, only a few times,
then the method will not work.
And so we had to go through the effort
of developing, say, switchable fluorescent protein,
reversible switchable fluorescent proteins.
And we did that in a department that I work in.
And so we came up very recently.
So this is very recent work that's
going to be published, actually, next week,
where we showed that you can turn off a specific protein
that we designed 1,200 times.
And this has allowed us to resolve, for example,
this dendrite in the living hippocampal organotypical
slides with high spatial resolution
you'll see it over time with this concept.
But the point here, at a million times lower intensities
than that was that.
So the break of diffraction barrier
is not an intensity game, per se.
It's an on-off game.
And if you can realize this on-off game
with much, much, much lower light levels, then you're done.
And this is, of course, very interesting.
We think this concept will be very, very important
in the future.
And these are just the first examples.
Now, as I said, a drawback of doing it like this,
defining where the molecule is on and off
with a beam of light that has a zero, such as a donut,
is that you turn the molecule on and off many times.
And molecules usually don't like to do that,
or that many of them don't like to do that.
In the interim, since we suggested this,
another concept came up, which runs under the name Palm
Storm.
Now, this concept, Palm Storm, I'm
briefly explaining this concept to you,
uses, say, cis-trans isomerization,
switching, or on-off switching, between metastasis as well.
So this was not you.
But there was something that was very new and different
to the concept that we had about at that time.
Instead of determining with a pattern beam of light
containing a zero at some point, where the molecules are on
and where they are off, where they have to be on,
and where they should be off, here, it is done randomly,
molecule by molecule.
You apply a beam of light, for example,
to molecules that are initially in the off state,
such that only one molecule within the 200 nanometer
region is turned to the on state,
for example, by cis-trans ionization.
So the rest will stay dark in here.
But that molecule is on.
And if that molecule, because it's on,
emits many photons in a bunch, then you
can find out where it's on.
Because initially, if it's just turning on stochastic in space,
you don't know where it's turned on.
So you have to find it out.
This can be done by grid detector, for example,
a camera.
So image it on a camera.
Then you find this position.
Then locate it, make a tick mark on its coordinate,
and know where it is.
Then you do the same thing with the next and the next.
And so you can reconstruct the sample, basically, in space.
This is why some people call it storms,
stochastic optical reconstruction.
But again, it's the on-off game that does the job.
Now, this concept has major strengths.
There is no doubt about the fact.
And one major strength is you need only one on-off cycle
to do the trick, so per molecule.
Disadvantages are here as well.
Now, the name, as it was suggested, of course,
this stands for photoactivation localization microscopy,
suggested that you need special fluorophores, just
this fluorophores.
But it's very important to understand the basics of something.
And if you understand the basics of something,
then you know actually what it implies.
And so since we knew that you can also
switch dyes by making this transition to the metastable
state here by a spin flip, for example, it
was clear that, well, you can, of course, use any dye.
For example, any rhodamine or so
that would normally produce light to fluorescence
if you're excited with excitation light very clearly.
But you can excite it so hard that most of the molecules
go to the triplet state.
But only one of them is left over.
And then you can locate that molecule.
You can do the same trick, the stochastic game.
So you can use also spin flip in order
to produce this type of stochastic imaging,
not just the special fluorophores.
And this was important because it generalized the method.
So basically means you can do it with any standard rhodamine
or typical fluorophore.
And this is just an example here.
This is three colors recording with,
these are basically rhodamine derivatives of most of them.
And this is currently the simplest way
of producing a sub diffraction resolution image with regular
folks in light microscope.
To some extent, it also works in living cells.
And of course, it has also disadvantages.
Imaging fast is not so easy.
It's also becoming what has become commercially available
and seems to become very, very popular.
Now, coming towards the end, of course,
you've seen several acronyms.
And the few of you perhaps who have followed the literature,
they're acquainted with the fact that many acronyms are there.
But if you do this as a science, of course,
then you ask yourself, why is the reason why we can do now
these pictures?
It's obvious that we can do the picture.
But 15 years back, we couldn't take these pictures.
So there must be a sort of ingredient in the image
formation that has led to this transition.
So the question is now, what is it?
Why can't we take these pictures?
Why can't we do them now?
We couldn't do them 15 years back.
Is the fact that we make donuts?
Of course not.
People have made donuts and all kinds of features
or focal shapes in an objective lens,
but they couldn't take sharper pictures.
Is it the fact that individual molecules are located?
In some cases, of course not.
People did that before and tracked
the position of individual molecules,
yet they couldn't take a picture.
So it must be something else.
There must be something else as a common enabling element that
allows us now to take the picture.
So what is it?
I think it's very clear by now.
It's the on-off game.
It's the on-off transition.
So we use a mechanism for preventing signal.
That's the whole thing.
No matter how we do it, either say,
in this mode where we determine the pattern of light,
where the molecules have to be off,
and they're allowed to emit on here, either with stimulated
emission, or with a spin flip, or cis-trans isomerization,
you can choose your own transition,
or you can do it stochastically, the molecule by molecule.
Then here, of course, the molecules
are not allowed to emit because they
are in a state which doesn't permit them to signal.
And so we can discern the features
because that all the molecules are allowed to be,
despite of the fact that all the molecules are flooded
with excitation light.
So with light, they will potentially produce signal.
That's the trick.
And this is why we can take the pictures now.
Now, there are currently two families.
So this one with deterministic and this one
which is stochastic, each one has its strengths and weaknesses,
and they differ in a way.
So here, you can have many molecules at the same time
and need only a few photons to generate a meaningful signal.
Because we know if there are a few photons coming in,
oh, OK, there's such a thing like a microtubule,
any feature in here.
Here, you can see only one molecule at a time
within this diffraction region.
And you need many photons, many photons
to get a meaningful signal because you
need to locate that position of that molecule,
and you need many photons for that.
So there's clearly a disadvantage
of the stochastic method.
The stochastic method has also a strong advantage.
You need only one on-off switching cycle
to produce an image, and so it works with any lousy
switchable molecules.
Here, you have good switchable molecules
that can be turned on and off many times.
And so each of the families has its strengths and weaknesses.
Now, with that, I would like to mention
that this is done with that.
And it was done with that, and it's a video recording.
And the reason for that is very simple.
This is a method that determines where the signal is generated,
and there can be many molecules in here.
This is why we are so fast.
So strength deterministic method is
that it's good at producing fast images.
There are many implications, including in the, say,
polymer sciences.
I didn't show it here.
This is biophysics, lipid formation, and so on.
I'm not going to dwell on that.
I'm finally acknowledging the people who
have contributed to this development,
PhD students, highly motivated postdocs, of course.
I would highlight Chef Agnakos, for example,
has many contributions, of course,
to developing the reversible, ritual proteins,
and the recent data that I've shown just now.
Now, with that, of course, I'm coming to my final slide.
This is our best equation.
It's very obvious that our best equation, if you will,
does no longer hold.
We are able now to certain features
that are closer than what is given by this equation.
So we can modify it.
That's not very difficult. We have
shown how we can do that.
We just plug in the square root factor.
And now, of course, we can account for the new situation.
The question that's coming now is,
what does this actually stand for?
What is actually the new ingredient?
And as I said, why can we take now these pictures?
And it's worth by spending a few thoughts on this.
Now, you may ask, why is it a square root, by the way?
Well, that's not very difficult to explain.
You've seen we have confined a state, say, the emissive state,
so it's a beam of light that has a zero.
Now, if you approximate that zero,
in first approximation, if you do a Taylor series,
it's a really parabola quadratic term.
And this must be the inverse function of it.
So this is the reason why this is a square root.
So this is not surprising.
Again, you see the resolution scales with the wavelength.
It still scales with the wavelength.
And it must scale with the wavelength,
because we work with diffracted beams.
We don't break diffraction.
Diffraction is still there.
But we break the barrier set by diffraction.
Because although the resolution scales with the wavelengths,
it is not limited by the wavelengths.
Because if this expression becomes large,
then D becomes arbitrarily small.
So the question is now, what does this expression stand for?
What does this I of S stand for if we make that large?
Well, I of S stands for that transition
between one state or the other.
Because the large we make,
the strong gradient of states will be induced
by this ratio I of I S.
The intensity induces a difference in state.
So if you ask me,
what broke the diffraction barrier in the end,
what is new that has been introduced in here?
It is, what has been introduced is the decision of states.
This is just about weight, but this is the states.
So putting in the states in the equation,
this is what changed matters.
And if you ask me, then why hasn't this happened before?
Or why did it take so much time?
My personal view, and this is just my personal view,
is people were somehow distracted by the fact
that there is waves and there is diffraction.
So we're constantly focused on diffraction.
Diffraction, there's waves, diffraction.
Because there is diffraction,
it will be very hard to get beyond the diffraction barrier
because of that.
And they didn't think about introducing the states.
Once you put in the states,
you don't have to worry about the waves anymore.
Also, the field optics, for example,
to make a special remark,
this is a method by which you can find, of course,
the wave propagation with a small tip.
That's about waves.
Not so very easy to do because it's about waves.
Or if you think about things like so-called metamaterials,
where you try to change the way the waves propagate
through a material, that's also very hard.
This is much simpler.
Use the thermal states.
Once you have a transition that allows you
to separate things with states,
you're done and you don't have to worry about the waves.
And this also tells us
that this is not fundamentally limited to fluorescence.
Any set of states that allow you to distinguish
a signal that they produce will work in the end.
And so, in essence, this is very general.
It means once we introduce the states of the material
into the resolution problem, we have one.
We don't have to worry about the waves anymore.
And this is the main message of the day.
Thank you very much.
Thank you.