Thank you, well since the important audience here are the students and the young people, not the old folks in front.
And also because you're such a wide and diverse group of people in all sorts of areas,
I thought I would try to slightly deemphasise the details of the science,
though I want to get into the science and this is not just a talk about, just for personal philosophy.
But I want to sort of focus I think on, what I think are some of the most difficult challenges you will have to face,
which is how do you choose a problem in science, or a general area.
So I will try to go through how I made many decisions in getting to where I am.
And in the hope that this will help all of you
and the next time that you come to Lindau that you’ll be up here, instead of out there.
So when I started really thinking as an undergraduate, what did I really want to concentrate on,
actually I had tried pretty much everything, I had tried being,
I even tried for a little while to be a theoretical physicist and very rapidly realised I did not have the talent.
I was very good at chemistry in high school and found that college courses made me totally disillusioned with chemistry.
I tried biology but didn’t really feel I had much of a talent.
But in the end, this is basically sort of what I came to near the end of my undergraduate career, beginning of graduate school.
Which is that mainstream biological research consists of asking how do natural bio molecules and biological systems work.
Meanwhile the most prestigious form of chemical research that I came across back in the late ‘60’s, early ‘70’s
which is when I was having to make this decision, was this question of total synthesis of natural products.
This was and still is of course a very dominant theme in chemistry.
How do we artificially re-synthesise natural bio molecules.
But often without much concern for what they actually do.
Mainly for whether they are beautiful and whether they are difficult to make
and whether they will show off the latest synthetic technology, organic synthetic technology.
And instead I wanted to take some sort of weird combination of these 2,
I decided can we design and build new molecules and ones that may never have existed in nature.
That will nevertheless perform amusing and useful functions in biology.
So I felt that this was and I still feel that this is rather like architecture or sculpture on the molecular scale,
with one great advantage is that in many of the other areas you are in tense competition with other groups
going after the same goal, the same biological problem or the same area of chemistry, the same target molecule.
But if you're doing sculpture or architecture nobody is going to design exactly the same thing as you will
and so you have a greater chance of showing off without having to, you know directly be,
you can’t quite be beaten by somebody else to the given goal, you can’t be scooped.
And of course then there’s, this is what really gives you understanding I think, is when you,
there’s a famous quote from Richard Feynman, ‘what I cannot create I do not understand’, he certainly had this feeling too.
Traditionally this type of molecular design was assumed you could only do it in a drug company
because only there would you have the huge resources, the big teams necessary to do it.
And now why did I wind up doing this, well partly because engineering runs in my family, you know my father,
my older brothers were both engineering majors, my uncles on various sides of the family.
Even my mother was a nurse and that was the closest a woman in China could come to being a scientist.
And nevertheless I felt that biology had the most interesting questions, that at least an individual could experimentally answer.
I felt that particle physics for example had some wonderful questions
but I was scared of the headlines when you read how many hundreds of people had to be involved in an experiment.
I wanted something that was a little bit more personal.
I have been fascinated since childhood by pretty colours,
that’s what first got me interested in chemistry was not a chemistry set
but something better that had more colours and actually was more dangerous, that did attract me when I was little.
I was the youngest of 3 brothers, I needed to find an ecological niche that was distinct,
so despite the engineering running in the family, none of them wanted to touch molecules,
they were all mechanical or electrical or aircraft engineers.
And this psychological need has an entire book written about it if any of you are interested.
Finally I then realised that there were many, again I worried about the competition,
I realised that most biologists were prevented from competing with me because they knew how to manipulate alphabetic strings,
that is 20 amino acids, 4 bases of DNA one extra for RNA typically and they couldn’t deviate outside that alphabet.
And that’s like if you are a, want to compose a scientific paper and all you know is a word processor
and you can type letters one by one but you can’t draw graphs.
And to me the ability to build unusual molecules is like the ability to use a power point or adobe illustrator
or something compared to Microsoft word or what have you.
So at least back in those early days there wasn’t much competition.
And the first place we had some success, after some false starts admittedly, I didn’t get it right first time.
Was to work on intracellular calcium and first I have to explain to you why that’s important but challenging.
Fluctuations in free calcium turn out to be important in all sorts of signal transduction,
ranging from every motion of my muscles,
every communication basically in the brain between one neuron and another through synaptic signalling.
Even at the beginning of life when the sperm first meets the egg etc, etc, these signals can be very fast,
down to milliseconds and localised below microns.
But chemically there’s the challenge that the concentration of calcium that we’re trying to measure
is on the order of 10 to the minus 8 to 10 to the minus 6 molar and that’s what's controlling.
But you cannot measure that simply by destroying the cell and measuring a total calcium.
Because there’s many orders of magnitude.
More total calcium and worse yet this low calcium is surrounded by a sea, an ocean of high, much higher calcium.
Again orders of magnitude greater so that it’s like if you were trying to measure
the contents of a sealed container of this fluid underneath the ocean and you're not allowed to lift it above the ocean.
And if you stick a needle in to here the salt water will flood in and immediately destroy the contents
of what you are trying to analyse underneath all this water.
And yet there was finally that there’s a vast excess, again several orders of magnitude more of magnesium
which is a divalent ion that is chemically fairly similar to calcium and that we needed the discrimination.
And then I realised also nobody had ever tried to design an indicator specifically,
no human being had designed an indicator for calcium.
And every time people had used an indicator dye it had been something designed for other purposes.
Now this, when you find this out, this can be both elating because you know you have virgin territory or very scary
because you’re desperately afraid of somebody, many people probably tried, failed quietly and not published,
which is the nature of, you know we tend not to publish our failures.
And maybe you will just be doomed to retrace their steps in wasted effort.
So where we actually, what I actually started from and this was as a graduate student,
I looked at the structure, EGTA was the only known molecule that had the necessary affinity and selectivity for calcium.
And biologists used it but it had no optical properties.
It never gave us a report of how much calcium it had bound.
And so I realised I needed to give it some absorbance, some, you know optical property
and the simplest way to give this absorbance is to just tie on 2 benzene rings like that.
And I gave myself about 2 or 3 weeks to try it because actually I had done 3 failed projects in my PhD
and I wasn’t supposed to be doing anymore, I was supposed to be fixing those 3.
But quietly without the knowledge of my supervisor who certainly would have forbidden it,
I started to make this molecule and I knew I was on a short leash, I was running out of time.
And fortunately in about 2 or 3 weeks I was able to make it and it actually worked, it had the desired properties.
So I was hooked.
But it’s still not good enough to measure inside cells because this only has ultraviolet absorbance.
So it took, nevertheless the hook was in my mouth.
And we continued and continued and eventually with the help of even more skilled organic chemists than myself,
when I could eventually hire them, we evolved this molecule called fura-2.
You can see just by continual extension around the core binding site that we did not change.
And this molecule does measure calcium because it can be excited by,
it will fluoresce and when it binds calcium the calcium sits in this cavity here and twists the nitrogen carbon bond.
The lone pairs on the nitrogen are sort like the tip of my nose and the calcium is sitting over here,
in order to bind I have to twist like that.
And that twisting basically disconnects the top of me from the bottom of me with a spectra electronic structure
and therefore we shift the shorter wavelengths.
But then came the problem, I had to get this into a living cell and remember I’m not allowed to puncture the living cell
because that membrane once punctured the calcium rushes in.
And there are people who are good enough to stick glass needles into cells without disrupting them and I was not one of them.
I’m a little too impatient and to clumsy.
And I also knew that there was too much competition in the area, other people were better.
So if I could work on cells that nobody could micro inject then I would have the field to myself.
But how do you accomplish that.
Well here’s the plasma membrane, the cell membrane and we have to get this molecule through
and it won’t get through with 5 negative charges on it, 1, 2, 3, 4 which are absolutely essential for binding the calcium.
And there’s a fifth one down here.
So what do we do, well the chemical solution turns out to be you cover up all the negative charges
with something called an ester group and you have to pick a very special ester group
that the cell itself knows how to get rid of for you.
So we’ve synthesised this molecule and it can walk across the membrane,
just by simple diffusion because it’s somewhat hydrophobic.
And once it hits the inside of the cell fortunately for us the most mammalian cells have the enzymes
to cut off these R groups and convert them back to the material we wanted.
And then we had a solution.
And to give you just one little biological example amongst the 20 or 30,000 that have been published since,
this is one of the first ones and I’m rather found of it because it was one of the first things that worked,
it’s also from the beginning of life for a sea urchin that is.
This is just after adding sperm to a sea urchin egg.
Here was the egg, the sperm have just been put into the dish 15 seconds ago but none of them has yet found the egg,
so the egg is this sphere that is coated here, is a deep blue colour because it’s calcium is very low.
So this is a false colour scale that actually is derived
by measuring constantly the 340 to 380, 340, 380, 340, 380 nanometre ratio back and forth a couple of times a second.
And then at this moment here or at least a second ago, the sperm just hit over here and the calcium is going up in this region,
in the red region, it has risen from about .1 micro molar which is 10 to the minus 7,
up to about 1.2 or 1.3 micro molar, a factor of 13 or so.
And as we go on here over the next few seconds the high calcium envelops the entire egg
and basically tells the maternal, the mother’s DNA that daddy has arrived.
In other words the sperm has come, you now have a full complement of DNA, start the process of development.
And then the calcium comes back down because you can’t stay high in calcium for very long without it becoming toxic.
So that was all well and good and it got me tenure.
But I found that things were still, we wanted new challenges
and also politically it wasn’t good just to work on calcium which has an inorganic ion,
I had to become more bio chemically respectable and so the next target that we picked was cAMP,
the prototypic second messenger for which the concept of second messenger had been developed.
And that’s so important that Nobel prizes had been given to Earl Sutherland, Ed Krebs and Eddie Fischer I believe
who may be somewhere in the audience, he’s here at the meeting.
This is the structure of cAMP but despite all this work, the existing ways to measure cAMP basically required
that you take millions of cells, kill them, grind them up and do a biochemically assay.
And that meant that the dynamics of intercellular cAMP in precise analogy to the calcium were pretty much obscure.
And again there’s a selectivity problem because there is less than micromolar levels of cAMP
in the presence of millimolar levels of ATP, the main fuel of the cell from which cAMP is made.
So it became obvious that there was no hope here of organic chemically synthesising a molecule
with all the selectivity properties big enough to wrap around a molecule of decent size anyway.
So we had to hijack the natural cAMP sensor which is called cAMP dependent protein kinase or nowadays protein kinase A.
And we hoped to label it with fluorophores in a way to detect the cAMP binding.
So once again we had a natural starting point but it didn’t give us a signal, this is the natural starting point,
this enzyme and schematically just shown here is 4 subunits, 2 regulatory, 2 catalytic
and when it binds cAMP the regulatory subunit undergoes a conformational change and dissociates the catalytic subunit.
And I think this is, you know much of what Ed Krebs’s Nobel was about.
So how do we turn this into an indicator.
Well, eventually we figured out that if we put fluorocene on the catalytic subunit,
rhodamine on the regulatory subunit, then in this complex we’d get something called fluorescence resonance energy transfer
which is a quantum mechanical interaction between 2 fluorophores
which was well known in biochemistry for detecting protein, protein proximity.
And once we broke that up with cAMP then we would lose this quantum mechanical phenomenon and get a change of colour.
And indeed this principle worked but the problem was that original protein sensor required
that somebody had to express and purify the regulatory and catalytic subunits of the protein in E-coli
because that’s where we got pure protein, you had to make a lot of this protein.
And that was Susan Taylor’s job.
In fact that was so important that that was one of the main reasons I moved from Berkley to San Diego was to be near Susan.
Also labelling with rhodamine and fluorecene I can blindly just draw how we just added them like that
but it’s actually not quite so simple.
And to figure out how to add them without screwing up the interfaces between the catalytic and regulatory subunit
took a huge amount of work.
And finally then you had to put them back together and micro inject it into cells.
And remember I told you I hate to do micro injection, I’m no good at it.
Now of course at this point I’m tenured, I can hire post docs and tell them you practice for 6 months until you get good at it.
But that’s still not desirable.
So we wanted a general means to fluorescently label proteins using genetics because then,
and do it in the living cell because then we aught to be able to fuse some naturally fluorescent protein,
ideally we wanted 2 colours, the equivalent of fluorescence and rhodamine.
Fuse them to the R and C subunits and then express and we would generate what we wanted in situ.
So I remembered that our big competitor in calcium measurement was the stuff called aequorin
which was the natural jellyfish protein that measured calcium.
So the jelly fish has sort of wanted to measure calcium, remember I said no human being had tried to design one.
And this is Professor Shimomura and he’s somewhere I hope here.
And those of you who choose will hear him later on.
Of course this is a much later picture and he had discovered the green fluorescent protein as a contaminate of aequorin.
And I remember that there was such a thing.
And one thing that I thought I would show you, which is a fairly unique sequence.
It is remarkably hard to find a video of the jelly fish glowing, which is the basis of all of this stuff.
And I asked Shimomura and he couldn’t give me a video.
And, Marty, if I understand you’ve never even seen it.
So I had to go to Alaska last summer and chose to visit this place and ask them to get me some jelly fish
and I hope this, I’m sorry the beaker is sort of fluorescent so that obscures it
but you notice that the jelly fish itself is fluorescent, we are shining ultraviolet light on it
and this ring here is the cells that are going to glow and then we’re getting ready to poke it.
If you turn of the lights, you see that flash, that was it.
So that’s the pleasure of doing an experiment with your own hands.
So of course the next crucial step is that this man, Douglas Prasher in 1992 cloned a gene for GFP
which made all the subsequent work possible.
And then Marty who you will hear in a moment, in a few minutes here,
of course was the first to show that the GFP would actually work outside the jelly fish.
And I’ll let him tell the rest of the story, his part.
So the original jelly fish had some pretty poor spectra, it was made for the jelly fish for its own reasons,
which we still do not really understand.
But its spectrum clearly needed improvement and chasing a bunch of wild ideas we mutagenised it
and eventually found that by changing the amino acid next to the one that makes most of the chromophore
we could make its spectrum much better.
So this was an early example of redesigning the protein to make it more useful for the human beings or the scientists.
And then of course it was helpful to get the crystal structure which was mainly done in the lab of Jim Remington.
This protein proved to be an 11 stranded beta barrel, almost beautiful cylindrical shape and symmetry.
With the chromophore deeply embedded right in the middle of this cylinder.
There’s much interest among young people and all sorts of people these days in chemistry
that is green and environmentally friendly and doesn’t use toxic organic solvents and doesn’t leave waste products.
I would like to point out this is green chemistry in several ways.
Obviously because it’s green literally and also because every fluorescent in protein is actually encoded by DNA,
that’s what makes it so powerful.
And it’s not toxic.
Synthesisable by any aerobic organism.
Here is a mouse that has just made it under our direction because we put the DNA in it.
It takes 230 successive condensations, one cyclosation and 1 or 2 auto-oxidations.
Consumes oxygen gas, works in a few minutes.
A 100% yield in aqueous solution at room to 37 degrees.
And the only by-product is hydrogen peroxide.
So I think that’s pretty good as a goal for green chemistry.
So eventually we managed to get lots and lots of colours, I’ve shown you some of the ones from the jelly fish,
later we got one from coral, there was a lot of fun in the lab or argument as to what we should call them
and eventually after consulting the Crayola website, we came up with these names which are honey dew,
banana orange tomato, tangerine etc which are a little bit more mnemonic to help you remember the colours in which they go with.
And these were evolved by a deliberate process of evolution, which I think is, you know many people,
we’re not the only ones to have done this, but is a form of biological engineering
that I think for example has a lot of potential to help in the energy crisis in the way,
when we need to generate renewable fuels, bio fuels and so on.
Just to give you one pretty, visually pretty example of the use of the fluorescent proteins,
here I’m going to take from the work of my former post doc Atsushi Miyawaki in his own lab now at Riken
who built some very clever molecules that used cell cycle dependent synthesis in breakdown of particular proteins
involved in cell cycle that’s Tim Hunt and Hershko and Ciechanover’s work all wrapped into that.
And they’re all here at the meeting.
So Atsushi arranged for a green protein to be made during mitosis and broken down the rest of the time.
And there’s other proteins that are made in exactly out of phase way, almost exactly.
And he chose to make them red or tag them with a red protein so that they are on when you are not in mitosis
and then they turn off when you’re in.
Now, that's a very clever choice of colour because green therefore means “go” and red means “stop”
and even in Japan we use the same colour code and that makes it easier for people to remember.
And so here’s a week’s worth of these cells and as you see that they are flashing red, green, red, green, red, green
as they go through the cell cycle.
And you may think that this is also sort of trivial because we can here, directly see my morphology
what stage of the cell cycle they’re in.
But many times inside a living organism say we do not know what stage a cell was at because we only get to see it once.
But with the colour code now we can always work out what stage it was at retrospectively.
Ok so that’s enough on fluorescent proteins.
And then more recently I have been switching much more to trying to apply molecular imaging to human beings
and in particular to actual patients.
And this is sort of translational and it’s motivated for a couple of reasons.
First I would like to do something more directly clinical relevant than just providing tools for other people
to go screen drugs for, like the drug companies.
When we develop these techniques I think that’s sort of cool chemistry but from the biological community’s point of view,
this is often disparaged or dismissed as it’s just techniques development, ok.
Whereas if I do this now for patients it’s actually just the same molecular engineering
but now it’s translational research and the funding agencies think that’s all cool and it’s all fashionable.
But more personally my father, my nephew, my PhD supervisor, many other colleagues died from cancer,
my mother had a stroke, this is going to get me, too, eventually, so I would like to do something about it.
Now, the problem is that fluorescent proteins require gene transfer,
to use their main virtue which is that they’re encodable by DNA.
And that’s not really technically feasible to do in sick people.
Gene therapy isn’t advanced well enough and usually it’s not ethically allowable,
we’re not going to make transgenic human beings the way we made a transgenic mouse.
As you saw a little while ago.
It might be very useful to us researchers to have fluorescent human beings but they might not feel quite so pleased by it.
And then furthermore humans are basically too thick and opaque, if you want to scan the entire body with fluorescence,
you can get away with it in a zebra fish and in a worm and so on but we are a bit too thick and fat basically.
And for that we need techniques like magnetic resonance imaging which of course earned Nobel Prizes of its own.
And we need amplification.
So what we’ve done instead is to develop contrast agents that go after proteases in cancer and proteases are important
because for a cancer cell to invade healthy tissue and get into the blood circulation
and out into a distant tissue which is what kills you in the end, it needs the activation of these proteases.
And at a molecular level what we did, again we started with a known mechanism that wasn’t quite suited to our purpose
which was that polycationic peptides, rich in argenines have been long known to be able to stick to the outside of the cell
and eventually go into the cell.
At least into some organelles of the cell by endocytosis,
even a little bit can escape from the endosomes and into the cyctosol and nucleus.
And in some systems, particularly cell-free systems there’s some evidence that they don’t need endosomes
and can go through to plasma membrane.
But this process is pretty non-selective between cells and to make it selective for tumour cells
and particularly to read out those proteases, what we did here is to deliberately cripple the polycation or mask it up
or hide it by giving it an equal sequence of negative charges connected by a hair pin linker.
And in this mode this molecule is internally self neutralised and doesn’t have the stickiness anymore.
But if we come along with a protease and we arrange that the protease should be the one that the cancer cells have,
they cut this apart and they’re the ones which will then take up the prob.
Where if the normal tissue can’t cut it apart and remains, the stuff remains non-sticky,
and it comes and checks out tissue and leaves without sticking, the cancer cells cut it
and then they get loaded up with the payload.
So to show you how this works in vivo , I skipped the cellular work, this is a peptide with the appropriate structure,
9 negative charges, a cleavable sequence, cleavable by the tumour proteases called matrix metalloproteinases 2 and 9.
These are 9 argenines that are the sticky portion, being masked for the time being by the 9 negative charges here.
And a deep red dye Cy5 to mark, so that we can see through some tissue that has blood in it say, where the cancer is.
And in this special case here we cheated, we marked the tumour with GFP
to give you an example also of how GFP can be used in a mouse to mark the cells that you care about.
And so there’s the tumour glowing green and the point is that we can get the same image equivalently in the red dye
by this stuff that we inject and it’s not genetic and it sort of basically finds the same tissue that was tumorous.
And if we, an important control is if we remake the molecule with diamino acids here, then the enzyme doesn’t recognise it
and the molecule is the same size and hydrophobicity but it doesn’t load that tumour because it can’t be cut.
Can we reach things like magnetic resonance imaging,
which are the techniques that we’ll work on intact human beings and the answer is we think we’re getting there.
Here is a mouse with a tumour and this is before we’ve injected it
and the tumour doesn’t really contrast much against the rest of the mouse, this is a section,
you know using the computer MRI scanner to cut across the waist so to speak or up in the shoulder level actually, arm pit level.
Here however is a mouse whose tumour, well we have injected the entire mouse through its tail vein 24 hours ago
with this construct which is a nanoparticle.
Loaded with gadolinium and our activatible cell penetrating peptides, these targeting motifs that are sensitive to the protease.
And yeah the tumour now has lit up and we can see it contrasting with the rest of the tissue.
Here again is the control where we made the same particle with uncleavable amino acids and it doesn’t light up.
But the use of fluorescence is, there is one place fluorescence is still valuable.
Which is in surgery because that’s where we actually cut away the overlying tissue anyway
and the patient needs to have that cut away.
So if I, here’s a piece of a mouse, a segment, the mouse is alive, can you see where the tumour is?
This is what surgeons have been faced with for thousands of years, when they cut out, try to cut out your cancer.
It’s pretty hard to tell where the cancer starts and ends.
So when we start the movie if we switch into fluorescent mode, there’s the fluorescence,
deep red fluorescence that actually shows us where the tumour is, based on this biochemical mechanism I just described.
But to actually cut, it’s much better if we overlay one on the other in the computer in real time such that we fake GFP.
Now there is really no GFP in there, but we’re faking with this deep red dye
because we’re not allowed to put genetic probes in and here for example again we see the tumour
and it’s much, much easier for the surgeon to cut when they can see where all the green stuff is.
And there’s my surgical collaborator Doctor Nguyen, she actually is a real surgeon
who cuts people Monday and Thursday and works in the lab the rest of the time, Tuesday, Wednesday and Friday.
And so you can see how it helps her a great deal.
But the other thing you want to see are the nerves.
Because if you cut the nerves then you will have long term paralysis or loss of sensation.
Other things grow back, the nerves have a hard, hard time if they do at all.
So you really want to see where the nerves are, to avoid cutting them.
And we developed a separate peptide with a different fluorophore that stains the nerve.
Now this is not a complicated biochemical amplification.
Nevertheless we have false coloured that light blue.
Now you can’t see here any more than 2 branches of the sciatic nerve running here.
But in a moment we’ll switch to the fluorescence and what now is revealed
is that there’s a hidden branch of the nerve diving into the tumour tissue that you wouldn’t,
or close to the tumour tissue that you would like to avoid chopping.
And in overlay, again we can see that much more clearly and then we go back to the tumour view
and then that tells us where we want to cut.
So it’s very much like when the gas company is digging under the side walk to put in a new gas line,
you hope they know where the electricity cables are buried.
And with techniques like this, we are beginning to actually improve the survival rate of cancers at least in mice
where with guidance by surgery, in this case we were able
to cure about half the mice where traditional surgery got less than 10%.
I know my time is running out, I just want to mention on the science side that we can also look at atherosclerotic plaques
which are the root causes of strokes and heart attacks, there we have to use a different protease sequence
because we’re looking at the enzyme thrombin rather than MMP matrix metalloproteinase.
Most of you looking at this wouldn’t even know where the carotid artery is
but when you see the fluorescence you can actually light up the plaques and even get a bit of a hint
as to how dangerous the plaques are because it turns out
that the more ready they are to rupture the more enzyme activity they have.
So let me end up with what I hope are lessons for young scientists.
I hope that you find important problems, obviously that would give the maximum pay off for the least pain.
Now that sort of sounds trivial and of course this is judgement, luck,
this is what distinguishes successful people from not so successful people.
One hint is to try to find projects that give you some sensual pleasure because then the pain at least will be less.
You want to do this anyway if it’s fun.
Or in my case a lot of the times it was to put my neurosis to constructive use.
It isn’t just pleasure, I’m afraid, at least I have and maybe you too have some demons driving you
and if you can use your projects to sort of quieten the demons down, that will also minimise the pain involved.
And then you’ll do it vigorously and then if you do it for fun and joy and for your own purposes
then you are best likely to do a good job that will eventually earn the recognition
which should not be your primary goal, outside recognition.
Accept that your batting average will be low but hopefully not zero
and that especially includes accept that your best papers may be rejected from the fashionable journals.
Or sometimes when you’re accepted it’s for the wrong reasons.
Learn to make lemonade from lemons, at least that's what we say in America.
When nature hands you an unexpected result or sometimes a failure, sometimes you can make, that’s a great opportunity.
Persistence often pays off.
But in order to be persistent one thing you haven’t been told is, gratuitous advice,
is that my advice is that if you want to stay alive and especially if you want to keep your brain alive for long enough,
like persistence and to stick it out to become a grey haired old fart like us, exercise.
Prizes are a matter of luck, so avoid being motivated or impressed by them.
And I particularly want to tell the students who always seem to want to have,
Dr. Tsien can I have a photograph with you, can I have your autograph, I feel a bit like a brass idol
in which you think that if you rub me you get luck and I’d much rather discuss science with you
than simply pose for a photograph or sign some piece of paper.
Of course all of this requires finding the right collaborators whom you must exploit but in a kind way for mutual benefit,
those are the only useful collaborations.
And with that I’ll show you the people who did the work
and a picture of green flashes viewed from our lab but drawn but bacteria expressing the different colours.
(applause).