Well good morning everybody.
It's a great pleasure to be here and I'm very happy to have this opportunity to talk to the young scientists.
Let me first give you a quick road map of what's going to happen.
I want to start out by telling you about the historical overview to give you the feeling of what happened in
the very first days of detecting single molecules.
It's a beautiful story of spectroscopy but also statistics so I hope you're awake now.
We're going to talk a little bit about mathematics in a few moments.
Then in the middle, I'm going to give you some things that you can use.
That you students and young scientists can use to tell your friends about molecules and show them about molecular fluorescents.
Then I'll talk about the super resolution idea and how it works and give you a few examples to wind up.
So first of all, back to the history.
Let's go back to the middle of the 1980's and at that time, many amazing things were happening
produced by many of the Nobel Laureates who are here actually.
People were looking at individual ions in a vacuum trap and looking at single quantum systems in various ways
and the question that arose, why not molecules?
Shouldn't we be able to look at a single molecule as well as these single ions?
Of course, there was a bit of a problem.
This great physicist, one of the founders of quantum mechanics, Erwin Schrödinger, wrote in 1952 that we never experiment
with just one electron atom or molecule.
He had this feeling of course but it's sort of one of the dangers of saying, "What can not be done," if you like. (laughs)
But there were plenty of other people who felt that detecting a single molecule optically might be very difficult.
So what I have to do first is to explain how we got to the point to believe that is was possible.
That requires talking a little bit about spectroscopy.
So think about this kind of a molecule, this is a terrylene molecule.
It has conjugated pi systems so it has absorption in the visible and at room temperature
you could measure a UV-vis spectrum of it, you would see absorption bands.
But let's cool it to low temperatures, in a solid, in a transparent host of para-Terphenyl.
What happens when you cool down, you get rid of all the phonons, of course, and the absorption lines get very, very narrow,
so narrow that I'm now showing those absorption lines here, this is the first electronic transition, there's not really
four of them, there are four different sites inside the crystal, so think of this as
the lowest electronic transition of the molecule and it's gotten very narrow.
We can't even see the width very well.
Let's expand the scale a little bit to try to do a more high resolution spectrum and here these lines appear to be resolved.
But they have this big width, this I've now switched to pentacene, it's another molecule that's similar to terrylene
and it has four sites but I'm only showing to of them.
- [William] This lowest electronic transition is more like a wave number or 30 GHz in width.
The question is, is that all there is?
Well it shouldn't be of course.
This molecule with all the phonones turned off and all vibrations turned off at this low temperature,
ought to have it's first transition to be narrow, really on the order of 10 MHz because of the excited state lifetime should be
the only thing limiting the width of the spectral line.
So what's going on?
Well what's going on here is something that is called Inhomogeneous Broadening.
It's really like this picture shown on the right.
There are very narrow absorption lines for the molecules because of what I just said
but there is a distribution of central frequencies coming from the different environments in the solid
that have slightly different stresses and strains.
These absorption lines have gotten so narrow that now you can see the perturbations or the shifts produced by these individual,
or different local environments.
Inhomogeneous Broadening prevents you from seeing the homogeneous widths underneath it.
So there were a number of techniques invented to measure the homogeneous width such as photon echoes.
Another one is spectral-hole burning.
A way of marking this inhomogeneous line or making a dip in the inhomogeneous line to see the homogeneous width.
There was a lot of beautiful research being done on these topics in the 70's and 80's.
I was at IBM research at this time where we were trying to use this spectral-hole burning idea as an optical storage scheme.
Well the beauty of that particular research lab and those environments in those days
is that we also could ask fundamental questions about this particular idea.
I was intrigued by the following question, is there some sort of a spectral roughness
on this inhomogeneous line that might represent some limit to signal-to-noise of writing data
in this inhomogeneously broadened line that might be coming from statistical number fluctuations?
So remember, this picture that had been measured of the inhomogeneous line, it sort of looked like
a smooth Gaussian and so we're sort of asking the question, is this a smooth Gaussian or not?
Now to understand what's going on here, I want to give a quick probability exercise for you this morning okay?
Suppose we have these 10 boxes and we're going to throw 50 balls at these 10 boxes and all of the boxes
are going to be equally likely for the balls to fall into the boxes.
Here's one possibility, what do you think?
Do you think this is a probable situation or not?
How many think this is a probable situation?
How many think this in an improbable situation?
Ah good, okay, you know, that in this kind of an experiment, what is more likely to happen
even though, they all have equal probability that you'll get different numbers in those different bins.
Basically, scattering according to the square root of N around the mean.
That's what I mean by number fluctuations.
It's the well known number fluctuation effect.
Who's amplitude is about the square root of N.
It rises from the fact that the molecules are independent when they enter these boxes.
So now, think of this horizontal axis here as a frequency axis or this spectral axis
that I was just showing you a few moments ago and everyone of these bins is a homogeneous width
in size and so now you'll see that the number of molecules that might fall into different
regions of frequency space if they're going to spread all over like this, should vary with frequency.
There should be some spectral roughness on the inhomogeneous line.
Here's a quick simulation.
Now I'm using a Gaussian shape probability which is smooth but in this simulation, you'd still see that there's this roughness
on this inhomogeneously broadened line.
In the mid 80's, no one had ever observed this roughness directly.
So we set out to measure it.
My postdoc Tom Carter and I, for pentacene in para-terphenyl at low temperatures and now you want
to think of this inhomogeneously broadened line as being spread out tremendously
so it's essentially horizontal on the scale of the stage and we used a laser technique, a powerful laser,
high resolution technique to ask whether there is any such spectral structure and here's what we saw.
It's amazing, there's this unbelievable structure that is, in fact, not noise.
See, I'm not calling it noise, it's a roughness.
If you measure it once, you see that shape.
If you measure it again, you see exactly the same shape.
That's because at low temperatures, this is static inhomogeneous broadening and this is coming from the effect I just described,
that every spectral window, even though the probability of molecules landing at different frequencies is uniform,
we see the number fluctuations.
So we call this Statistical Fine Structure,
where the average absorption, of course, scales as N, in fact, essentially everything you measure
in experiments normally scales as N or if there's no linear effects maybe N squared or others.
But having a spectral feature who's RMS size scales as the square root of N is pretty different,
pretty interesting, something to wake you up okay on a Thursday morning early.
Now, let me say that this was detected by using a special technique called FM spectroscopy.
I won't describe all the details of that but it is uniquely suited to measuring
the deviations of the absorption from the average value, that's what we needed here,
admitted by Gary Bjorklund, 1980 and also in this area was developed a lot by Jan Hall and his collaborators.
I want to emphasize one key thing here.
I'm scanning a tunable single frequency laser to measure this and the laser line width
is about 1 MHz, notice the scale here 400 MHz here.
Every feature here is fully resolved.
There is no limit produced by the measuring device.
The measuring device is narrower than every spectral feature.
These widths or the shapes, the ultimate narrowness of these things is being produced by the width of the molecules absorption.
So everything in resolved here.
Remember that because we're going to talk about resolution later in the spatial domain.
So this was interesting to observe and it also had another effect for me.
It made it clear that the single molecule limit was obtainable.
Why is that?
Well if the average absorption scales is N and let's say N is 1,000 in this case, then that means that the RMS amplitude
of these features is about square root of 1,000 or 32 which means that once you see this, you only have to work 32 times harder
to get to the single molecule limit.
Only 32 times harder.
Not 1,000 times harder.
Believe me, that's a lot better.
So we decided to push on with this method.
That sort of came out of the blue, out of right field or something at this
research that was going on on an optical storage scheme at this industrial lab.
Can we push it to the N equal to one limit?
Indeed, in 1989, Lothar Kador, my postdoc at the time, and I did push it to the single molecule limit
and measured single molecule absorption by detecting these tiny changes in the transmitted light.
For pentacene molecules as dopants in para-terphenyl.
Now, this was an important advance because we showed that it could be done and a useful model system we had found.
That method can be quantum limited and can be very, very sensitive but you can't
get a lot of power on the detector in these measurements.
Otherwise you'll broaden the absorption's and then you won't have as much sensitivity.
A year later, a very important advance occurred, Michel Orrit, in France at that time,
decided to detect the single molecule absorption also for pentacene in para-Terphenyl but by detecting the emitted light,
the fluorescents from the molecules.
This method, of course, could be difficult to do if you have backgrounds or scattered light.
You have to detect this tiny signal from the individual molecules but they showed
it could be done as very important advance in the field.
You got better signal-to-noise by this method so everyone switched to fluorescents detection afterwards.
So immediately the surprises started to occur.
Here's some examples of where we're scanning the laser and detecting the molecules.
You can see the molecular absorption's there.
But look what's going on.
There's something crazy.
These molecules are moving around in frequency space, they're moving back and forth in a digital sort of fashion.
When my postdoc Pat Ambrose saw this, he came running in and said, "The molecules "are jumping around." (laughs)
We go, "Wow, that's really interesting."
This is a surprise for us.
We're at low temperatures, liquid helium in a crystal and so forth for a very stable molecule why would this be happening?
Well, some theorists like Jim Skinner and others helped us to understand that this is coming
from some dynamics in the host crystal that are changing at that low temperature to shift the molecules back and forth.
Anyway, if I set the laser at one fixed frequency here some times the molecule will be in resonance and you'll see a signal.
Other times it will be off, on, off, on and so you could also see blinking effects
with these molecules at this early time, in 1991.
Another surprise, Thomas Bass, now we've change to perylene in polyethylene, another host and look what happens here.
You scan a molecule, scan it again, scan it again, nothing's changing and then if you go into resonance
and just sit on top of the molecule for a little bit then you see the fluorescence drop down
and if you quickly scan again, you see it's gone.
It's moved somewhere, it's moved somewhere way off the screen to another wave length very far away.
You've changed something in the nearby host.
You've flipped a two level system that's made the molecule's absorption move.
Amazingly, after another moment, the molecule comes exactly back.
You scan it again and you see it again and again and again with no change.
Go into resonance with it, you can drive it away.
So, we were excited about this too because we could switch the molecule back and forth with laser light.
These two effects, blinking and optical control are useful for things that are going to happen later.
So now I want to, just for a moment, ask this question, why should we study single molecules?
Of course you know that we've removed ensemble averaging and we can watch the individuals and see whether they
are behaving differently or not.
I just gave you some great examples of when they do behave differently.
Now for your pedagogical interest, I just want to give you a way of explaining this issue to your friends.
Why study single molecules?
Let's think about baseball for a moment.
If you think back to 2004, there was this team in The United States, the Red Sox, who were the Series champs and here
are the rankings of the various teams and their team batting averages.
So here's the team batting averages here and this is the ensemble measurement of course but if you measure the batting average
player-by-player and then make a distribution like I'm showing here, then you see something interesting beyond the average.
On the left, now you see that there's a distribution but there's also some outliers here.
What are the outliers?
Anybody know? (laughs)
Pitchers, exactly, so here they are. (laughs)
So basically, what we're doing is translating this idea from baseball to the molecular scale.
Are the molecules marching to different drummers or not and maybe that is something else you might use.
Another thing to explain to your friends is well, what is this business of molecular fluorescents?
What is it really?
Well you know it's like their day glow...
ways of having socks or whatever but there's another cool way to demonstrate fluorescents to your friends and I'm going
to show you that in a moment.
But first you have to explain to them that first molecules start in a ground state and after absorbing the proper photon
they go to an excited state of course.
But then, there's some molecular relaxation, some vibrations are emitted etcetera and when the photon is emitted,
it's shifted to a longer wavelength okay?
It has lower energy.
Well, I want to show you a neat way to demonstrate that.
There's a very easy, simple demo that I'd like to describe.
So what you need to do to demonstrate this is get one of these orange highlighters that you can buy.
Make sure you get the cheap one.
Don't get the no smudge variety, the no smudge variety doesn't dissolve as well in water.
The cheap ones dissolve in water much better.
And then you've got a little vial with water in it, that's what I've go here.
All I have to do is open this highlighter and stick it into the water just for a few seconds, twirl it around a little bit,
that's all I had to do.
That's the highlighter done.
Close the vial and we're going to just shake it a little bit, that's all we'll do.
And then, of course the next thing you need is your green laser pointer which you have.
You don't need a fancy one.
You don't need high power.
You don't need anything like that just five milliwatts is plenty.
Now if you're prepared to turn off the lights, I'll show you what you can do with this.
And I'll get this off as well.
Well, it's not completely dark but maybe you'll see it.
I hope you can.
So it's really cool okay? (laughs)
(crowd claps)
Now what's cool about this is that it's a little light saber right?
And it's got that color.
Remember the laser's green and this light coming out of the sample is more like orange and you can demonstrate that even though
when the light goes through this, it emits green when it's going through but look, it's orange when it goes through
but look, the transmitted light is still green.
You haven't used up all of the light from the laser, the transmitted light is green.
And that's a good example of, we only needed a few molecules to get this light coming out and to see it clearly.
So what we're doing in our experiments of course, is reducing the concentration in that sample down to an unbelievably low level.
Down to the single molecule limit.
Also reducing the volume and detecting that light from the individual molecules.
Well it turns out, you can even do that with your own eyes.
In the experiments that are done at room temperature these days now, here let me just remind you how we are
doing them at room temperature.
We're not using FM spectroscopy but we're pumping from the ground state to the excited state and detecting
this fluorescent shift into long wave lengths.
Typically, we look at these little, small molecules which are about a nanometre in size or two.
Aequorea green fluorescent protein and attach that to the molecule of interest and then here is, let's say a cell for example,
where you have these molecules inside but in order to detect single molecules then,
we focus the laser down to the smallest spot possible and that size is limited by diffraction
to lend over two times the numberical aperture.
So to get to the single molecule limit, you have to dilute the molecules.
You have to have them farther apart then the width of this small focal spot.
That's how we detect single molecules at room temperature and work at room temperature of course,
even started back with the work of Keller and many others beginning even in 1990
but the near-field techniques and confocal techniques and wide-field techniques were demonstrated all along the way.
So to show you some real data on a real system just see what it's like.
This is the system composed of a cell where the transmembrane protein, which is called MHCII is in the cell membrane
and it's been labeled by labeling an antigen that has a Fluor-4 on it.
So each one of these spots in this image is coming from the light from individual MHCII complex.
By the way, as I said, you can see this light with your very own eyes.
You can look into our microscopes, get dark adapted, you could see the light from single molecules with your own eyes.
- [William] So that's why it's worth protecting your eyes.
They're very wonderful detectors.
If you now watch this movie as a function of time, that's where the excitement comes.
We're able to do this in living cells and this is room temperature.
You can see that they're diffusing around.
They're moving all over the cell membrane having this dance on the surface of the cell.
They also turn off, that's some of the photo bleaching processes.
There maybe some blinking in this experiment too that we'll talk about in a moment.
That's an example of what can be done with single molecules at room temperature.
We're still in, this happens to be from 2002 but there's one thing that happened in the 1990's that I want to mention.
We decided to look at single copies of these fluorescent proteins that were being developed by Robert Tsien and others
that you've already met and heard about at this wonderful meeting.
I was at UC San Diego at that time, where Roger was and I asked him for some protein and Andy Cubitt gave us something
that was called, at that time, a yellow fluorescent protein which had a few mutations to stabilize
the longer wavelength absorption of GFP and we wanted to see, can we detect them and image them at room temperature
and here's some examples.
Yes, Rob Dickson was able to do that.
This had not been done before at that time and so immediately new surprises appeared.
We saw blinking, that is we saw the molecules emit, emit, emit, frame after frame but then turn off for a long time
and then turn back on again and turn off and turn on and so forth in a stochastic sort of way.
In other words, we were pumping and collecting photons but after some time period, the system went into a dark state,
probably some isomerization of the chromophore from which it could return thermally and start emitting again.
Another surprise is that after a longer radiation, we saw that these molecules went into a long lived dark state.
We thought they might have been photo bleached but Rob used a little bit of shorter wave length
blue light and he could restore those molecules back that had been turned off.
You could then have them emit again for a long time and then if they finished, you could restore them back with 405 nanometers.
So it was both blinking and light induced photo recovery that we observed in these experiments.
This work and others, led to a huge increase in development of switchable fluorescent proteins.
The photo activatable GFP was generated later DRONPA for switching by Miyawaki and others.
The basic idea though is that the sort of dynamics that we had seen at low temperatures was also available at room temperature.
Note by the way, here's this patent that Roger and I got for this particular process (laughs)
at that time but what was in our mind?
We weren't thinking about a super resolution.
We were thinking about optical storage.
We were thinking about using these molecules to store individual bits in the days.
Remember I came from IBM and that was in our minds at the time.
Now let's talk about the super resolution.
Circumventing the optical diffraction limit.
To illustrate this in a simple way for everyone.
Here is a bacterial cell, only a couple of microns long and maybe 500 nanometers across and a particular protein has been labeled
with yellow fluorescent protein in this case and you might think that all you have to do
is buy one of those really, really expensive microscopes, the best microscope you can buy.
Really expensive and here it is.
Here's that expensive microscope but the problem is, you don't see any more detail, you want to see the shapes and the positions
that those molecules might have and you're thwarted, you're thwarted by all these diffraction limit.
Even though the emitters are really small, just a few nanometres in size, they appear to be a few hundred nanometres in size
due to this particular important physical effect that's coming from the wavelength of light
and numerical aperture that you're using.
So, the reason that super resolution is exciting to many people today is that you can go from this kind of an image
to that kind of an image.
Tremendous increase in detail.
Tremendous increase in what can be measured and quantified about what's going on inside the cell.
In order to explain this, even though you heard beautiful explanations earlier in week from both Stefan and Eric.
Let me just describe it in a very general way that I like to use.
I like to say that first you need to detect single molecules and then you need two essential ingredients.
Please note, I'm only going to describe the super resolution based on single molecules.
Stefan already did a wonderful job talking about STED and the other methods that depend upon a patterned light beam
to turn molecules on and off.
First you have to super localize the individual molecules.
A neat way to think about that.
Here's a cinder cone inside a volcanic lake at Crater Lake Oregon and if you get a chance to go there, you should go.
It's really a beautiful lake and here's a cinder cone inside that lake and of course, I have my obligatory scale bar.
120 times 10 to the 9th nanometers.
Now you know very well that if you just walk up to the top of this mountain with your cell phone, you can read out
the GPS coordinates of the peak of the mountain.
That's basically what we're doing.
Here's one of our single molecule images.
Now this is a molecule YFP, Yellow Fluorescent Protein, in a bacterium and I've now turned it
into a 3-D picture just to show you the brightness here on the Z-axis.
You see that that single molecule has a width that is coming from diffraction.
What we do is we spread out the spot on a pixilated detector and we get multiple samples of the shape of that spot.
By having those samples, you can fit the shape and get a very good estimate of the position that's much better then
the diffraction limit, then the width of the mountain.
The other key idea, I like to think of it as active control of the emitting concentration.
This is something the experimenter has to do, has to actively choose a mechanism that controls the emitting concentration.
So why is that at all useful?
Well first of all, here's the structure.
Suppose that we want to observe that structure and you decorate it with those fluorescent labels that I've been talking about.
Now if you just turn on the laser then all of them will emit at the same time and that of course, is the problem.
You see that all of the spots from the individual molecules overlap and that's the low resolution situation.
What we do then is we use an on/off process.
We use some way of having the molecules be in either an admissive state or in a dark state
and use that to control the concentration at a low level.
Then, you only turned a few on and then localize them.
Do that again and do it again and ultimately, you can use a pointillist technique,
connection to art here, a pointillist technique to reconstruct the underlying structure.
This idea I heard about first in 2006 from Eric Betzig calling it PALM.
Later we heard about STORM from Zhuang, F-PALM and then a whole variety of acronyms appeared
showing different mechanisms for doing this.
We started to use, at that time then, our YFP reactivation that I just showed you
and we didn't give it an acronym so, of course, that's been forgotten right?
So here's an acronym to add something back to the pile just for fun, that's mechanism independent, Single-Molecule
Active Control Microscopy or SMACM.
How does this work in a real system?
Here's an image with many single molecules.
There are these spots appearing on the right that are coming from localizing the molecules and when you do this many times,
then you can build up the data required to reconstruct this high resolution image of the underlying structure.
It also works in bacteria but I want to show you in bacteria how yellow fluorescent protein does this for you.
YFP, that I was showing you earlier, blinks beautifully.
Here's some bacterial cells and here's the fluorescents from those cells where there's something labeled in the cells.
It's this that we're really using in our data taking.
This beautiful blinking of the individual fluorescent proteins gives us information about where those molecules are located.
And that approach then allows us to go from these kinds of diffraction limited images
of several different proteins to these kinds of super resolution images of all those proteins.
It's really a wonderful way to see the structure that was not observable before.
These are actually our live cells.
This is a fixed cell.
We have this both in fixed and live situations.
To show you that you can get more information that's a little bit of time dependence from this scheme, I want to mention
the paint idea that came from Robin Hochstrasser.
The way that paint works is that you have a structure that you want to observe and you bring fluorophores in from the outside,
from the solution, fluorophores are zooming around while they're in the solution and you can't detect them easily
but when they bind to something, they sit there and give you all that light and then you localize the ones that have bound.
So we use that idea based on now a fluorescently labeled ligand, fluorescently labeled saxitoxin,
to light up voltage gated sodium channels in a neuronal model cell, a PC12 cell.
That's what this picture's showing you.
This is one of the axonal projections outside of a differentiated PC12 cell and the beauty, of course, is in the movie.
This is a movie where we're essentially averaging on the 500 millisecond time scale.
All localization's we see within the 500 millisecond time and then playing that back as a sort of
a sliding box car and you see all of these structures that grow and disappear over time as the cell is continuously growing.
Time is very short so I'm going to have to skip a few things.
I'll skip the applications of this to Huntington's disease.
Here's some beautiful aggregates that we can observe by super resolution that are aggregates of the Huntington protein.
I'm going to skip another example and end up with a few lessons that I would like to sort of give you that you can
go tell your friends to end up today.
You've all seen this beautiful Nobel medal.
You can see, of course, Alred Noble's picture on this side of the medal.
But I bet you haven't seen the back of the medal.
Here's the back of the Nobel medal.
It's really very, very beautiful.
It's a beautiful, beautiful rendition.
Here on the left is nature.
Nature's holding a cornucopia and on the right is science.
And science is lifting the veil off of nature.
It's just amazing.
So you want to communicate that science is fun.
Help lift this veil of nature in our world.
But there's more that you want to think about and communicate to those younger.
Basically, the undergraduates and those who are getting interested in science.
It's always important to find your passion.
That's incredibly essential because it can be hard work to work through the problems that we do in our very methodical way.
You have to be determined, persistent and methodical but having fun along the way is really important.
It's all based on asking how things work.
There's too many people that take their cell phone for granted, take some of the technology in our world so much for granted.
We really ought to be asking more about how things actually work on the deep fundamental level
all the way down to single molecules.
So that means pushing beyond conventional wisdom and questioning assumptions.
So, of course, we believe that science provides a rational and predictive way to understand our world and that's why we do it.
So I want to thank my past students, postdocs and collaborators and of course, the current Guacamole team.
Here they are being very serious as you can see.
It's right before Halloween.
Thanking our agencies as well.
Here's a little fun, it's our No Ensemble Averaging logo.
Which stands for single molecule spectroscopy.
I forgot to say, why are we called the Guacamole team?
Well, you know, one molecule is one guacamole right?
One over avocado's number of moles.
So with that, thank you very much for your attention.