>> GOOD AFTERNOON, EVERYONE. WELCOME TO A VERY SPECIAL LECTURE TODAY BY DR. WILLIAM E MOERNER WHO GOES BY W.E. BECAUSE HIS FATHER WAS NAMED WILLIAM AND HIS GRANDFATHER WAS NAMED WILLIAM AND THEY HAD TO CALL HIM SOMETHING ELSE. SO W.E. IT WAS AND HAS BEEN SINCE. IT'S A SPECIAL TREAT TO HAVE HIM HERE WITH US AT NIH TODAY, EVEN MORE SO WHEN IT'S LESS THAN TWO MONTHS AFTER HE RECEIVED THE NOBEL PRIZE IN STOCKHOLM. AS YOU PROBABLY KNOW, W.E. IS ONE OF THREE SCIENTISTS WHO SHARED THAT PRIZE IN CHEMISTRY FOR THE DEVELOPMENT OF SUPER-RESOLUTION MICROSCOPY. A LITTLE OF THE WORK WAS DONE HERE AT NIH SO WE FEEL CONNECTED HERE IN THIS PARTICULAR CYCLE AND DELIGHTED W.E. CAN BE HERE WITH US TODAY. THIS TECHNOLOGY OPENED A NEW FRONTIER WITH RESOLUTION OF 20 TO 40 NANOMETERS, THERE'S AN UNDERSTANDING GOING BACK TO ERNST EBBY, YOU COULD NOT NORMALLY EXPECT WITH LIGHT MICROSCOPY TO RESULT IN STRUCTURES CLOSER TOGETHER THAN 200 NANOMETERS. THAT'S ALL CHANGED AS A RESULT OF THIS VERY CLEVER USING OF SINGLE MOLECULES. LIKE MANY GREAT SCIENTISTS HIS INTEREST BEGAN WHEN HE WAS A KID AND SPENT TIME TAKING APART OLD TELEVISION SETS, DOING CHEMISTRY EXPERIMENTS IN THE BACK YARD, LOVED FIXING THINGS AND FIGURING HOW THEY WORKED. SO HE TOOK AN INTERESTING PATH, FROM THE PHYSICAL SCIENCES TO BIOLOGY IN HIS TRAINING, HE GOT A BACHELOR'S DEGREE IN PHYSICS, ELECTRICAL ENGINEERING AND MATHEMATICS FROM WASH U, AND THEN MASTERS AND Ph.D. IN PHYSICS FROM CORNELL. HE THEN WENT TO WORK AT IBM'S RESEARCH CENTER IN SILICON VALLEY, AND THERE IN 1989 HE WAS THE FIRST PERSON TO DETECT AND OBSERVE A SINGLE FLUORESCENT MOLECULE WHEN HE AND HIS POSTDOC DEVELOPED A TECHNIQUE THAT COULD DETECT A SINGLE MOLECULE IMPURITY IN A SINGLE CRYSTAL. LATER AT U C SAN DIEGO AND STANFORD I PIONEERED APPLICATION OF THESE SINGLE MOLECULE BIOPHYSICS APPROACHES TO BIOLOGICAL SYSTEMS, IN 1997 HE DISCOVERED A WAY TO TURN THE GLOW OF A VARIANT OF GLEAN FLUORESCENT PROTEIN ON AND OFF AND WILL LAYING THE FOUNDATION FOR SUPER-RESOLUTION TECHNIQUES. THEY HAVE CHANGE THE ONCE BLURRY IMAGES INTO WELL RESOLVED PORTRAITS WITH DETAILS NEVER SEEN BEFORE IN BIOLOGY. YOU MIGHT SAY BACK THEN IT WAS UNUSUAL FOR SOMEBODY WITH DEGREES IN PHYSICS, MATH AND ELECTRICAL ENGINEERING TO APPLY EXPERTISE TO BIOLOGICAL PROBLEMS, BUT THESE DAYS WE SEE THIS HAPPENING MORE AND MORE. THE NOTION OF CONVERGENCE BETWEEN DISCIPLINES BRINGING THE SKILLS OF VARIOUS EXPERTS TOGETHER TO TACKLE PROBLEMS HAS TURNED OUT TO BE ONE OF THE MOST EXCITING DEVELOPMENTS OF THE LAST FEW YEARS AND W.E. WAS AHEAD OF HIS TIME BY ONE OF THOSE WHO MANAGED TO SUCCESSFULLY DO JUST THAT. WE'RE VERY PROUD THAT HE HAS BEEN SUPPORTED OVER THE COURSE OF MANY YEARS BY GRANTS FROM THE NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES AND IS CONSIDERED ONE OF OUR OWN, AND WE'RE DELIGHTED TO SEE, NOT ONLY WHAT HE'S ALREADY DONE, BUT TO HEAR MORE ABOUT WHAT HE'S UP TO TODAY AND I THINK HE WILL GIVE US A MIX OF THOSE PERSPECTIVES. HE'S THE HARRY S. MOSHER PROFESSOR OF CHEMISTRY AND PROFESSOR OF APPLIED PHYSICS AT STANFORD UNIVERSITY, MANY HONORS IN ADDITION TO THE NOBEL PRIZE ARE THE WOLF PRIZE IN CHEMISTRY, ELECTION TO THE NATIONAL ACADEMY OF SCIENCES. I'M DELIGHTED HE MADE TIME IN A CHAOTIC POST-PRIZE SCHEDULE TO VISIT NIH AND TELL US ABOUT HIS JOURNEY. JOIN US IN GIVING A WARM WELCOME TO W.E. MOERNER. >> THANK YOU VERY MUCH. I WANT TO IMMEDIATELY SAY THANK YOU VERY MUCH TO DR. COLLINS FOR THAT WONDERFUL INTRODUCTION. I'M PLEASED AND HONORED TO BE HERE. I WOULDN'T MISS IT FOR THE WORLD. SINCE THIS IS A PLACE WHERE I'VE HAD A LOT OF WONDERFUL INTERACTIONS OVER THE YEARS. SO THIS NOBEL PRIZE IN CHEMISTRY THIS YEAR IS SOMETHING VERY SPECIAL, AND I AM TRULY HONORED TO SHARE THE NOBEL PRIZE WITH STEPHAN AND ERIC, TWO OF MY FRIENDS AND COLLEAGUES IN THE FIELD. NOW, BEFORE I GET STARTED ON THE SCIENCE, I WANTED TO GIVE YOU A LITTLE PHOTO TOUR OF SOME OF THE THINGS THAT HAPPENED AROUND THE NOBEL PRIZE. SO BEFORE THE NOBEL PRIZE CEREMONIES IN SWEDEN, YOU FIRST COME TO WASHINGTON, AND VISIT WITH THE SWEDISH AMBASSADOR AND SO FORTH AND VISIT WITH SOME OTHER REALLY IMPORTANT PEOPLE, AND HERE IS A FEW OF THEM. HERE IS CATHY LEWIS AND BOB STACK. I DON'T HAVE A PICTURE OF JIM DETHERIDGE, BUT WE START BY VISITING THE KEY PEOPLE BEHIND THIS WORK. TURNING TO STOCKHOLM YOU MAY HAVE SEEN THINGS ON TV AND THE WEB BUT IT'S A SPECTACULAR ACTIVITY, COMPLETELY OFF SCALE IN EVERY DIMENSION. THIS IS ME RECEIVING THE PRIZE FROM THE KING OF SWEDEN, HERE IS STEPHAN AND ERIC, THE OTHER LAUREATES, MY WIFE SHARON AND SON DANIEL, AND THIS BEAUTIFUL DIPLOMA THAT IS A ESPECIALLY COMMISSIONED PAINTING AND SO FORTH, EVERYTHING IS AMAZING. THE SPECTACLE IS SO GRAND. HERE IS THE REALLY MONSTER CITY HALL ROOM WHERE 1200 PEOPLE HAVE THE NOBEL BANQUET, AND THERE'S JUST SOME CIRCLES ON HERE THAT MY BROTHER-IN-LAW DREW TO SHOW VARIOUS PEOPLE, BUT IT'S REALLY A SPECTACULAR EVENT. HERE IS ME YOU CAN SEE FROM BEHIND. LET ME SHOW YOU FROM THE FRONT. I'M HERE WITH THE PRINCESS, AND THIS IS ONE OF THESE DIFFICULT MOMENTS. I HAVE TO WALK DOWN THESE STAIRS, OKAY, WHILE NOT LOSING MY BALANCE WITH THE FANCY PATENT LEATHER SHOES AND ON TOP OF THAT SHE'S WANTING ME TO GO FASTER AND FASTER, BUT RIGHT IN FRONT OF ME IS THIS PRINCESS-TO-BE WITH THE BEAUTIFUL LONG TRAIN AND THE LAST THING YOU WANT TO DO IS STEP ON THE TRAIN WHILE YOU'RE COMING DOWN THE STAIRS. [ LAUGHTER ] SO I HOPE YOU GET AN IDEA OF THE MIX OF EXCITEMENT AND AMAZING THINGS THAT HAPPENED AND CHALLENGE AND STRESS. THESE ARE THE OTHER PRINCESSES. SO THE SINGLE MOLECULE STEER AND SUPER-RESOLUTION, I WANT TO SHOW YOU A ROAD MAP AFTER 25 YEARS, WE'RE STILL HAVING FUN WITH SINGLE MOLECULES. I'LL TALK ABOUT THE FOUNDATION AND APPLICATIONS OF THE METHODS FOR THREE DIMENSIONAL LOCALIZATION AND SUPER LOCAL ALIZEATION AND SUPER-RESOLUTION AND I'LL END UP WITH MORE SURPRISES, MORE RECENT SURPRISES FROM SINGLE MOLECULES IN CELLS. LET'S GO TO THE MID-1980s, A BUNCH OF AMAZING STEPS WERE GOING ON AROUND THE WORLD, SINGLE IONS AND TRAPS AND PEOPLE WERE THINKING CAN WE DETECT SINGLE MOLECULES? THIS FAMOUS SCIENTIST SAID IN 1952 WE NEVER EXPERIMENT WITH JUST ONE ELECTRON OR ATOM OR MOLECULE. IT WOULD BE LIKE EXPERIMENTING LIKE RAISING ICTHYSAURA IN A ZOO. THE FIRST STEP WAS BELIEVING IT WAS POSSIBLE AND THAT CAME THROUGH A CIRCUITOUS ROUTE AT IBM. EARLY SPECTROSCOPY, AT LOW TEMPERATURES. IF I COOL IT, OPTICAL ABSORPTION GETS NARROW AND YOU SEE NARROW ABSORPTION LINES AT LOW TEMPERATURES, COMING FROM MILLIONS AND BILLIONS OF MOLECULES. AND I'M SHOWING A COLOR SCALE, FREQUENCY SCALE, THE LIGHT AT THOSE FREQUENCIES IS ABOUT 500 TERAHURTZ IN THAT REGIME. BUT THE LOWEST TRANSITION IS IMPORTANT HERE BECAUSE IN A RIGID MOLECULE THE LOWEST ELECTRONIC TRANSITION CAN BE WHAT'S CALLED A ZERO PHONON TRANSITION, NO VIBRATION, AND NO PHONONS AROUND OR VIBRATIONS IN THE SOLID SO IT SHOULD BE NARROW. YOU SEE AGAIN NOW FOR A SIMILAR MOLECULE THERE ARE MANY BENZENE RINGS FUSED TOGETHER, THAT'S THE SAME BUT YOU SEE THESE LINES THAT HAVE A WIDTH THAT'S RESOLVED, AND YOU MIGHT ASK YOURSELF, WELL, IS THAT ALL THERE IS? IS THAT ALL THERE IS? IS THIS THE NARROWEST THAT YOU COULD GET? THIS LINE HERE IN THE YELLOW AND SO FORTH, THE ORANGE, IS WHAT WE CALL AN INHOMOGENOUSLY BROADENED LINE. IT'S A LINE LIKE THIS, AN ABSORPTION LINE THAT'S COMPOSED OF MANY MANY NARROW FEATURES BECAUSE THE MOLECULES HAVE GOTTEN SO INCREDIBLY NARROW, BUT THEY SPREAD OVER A RANGE OF WAVELENGTHS BECAUSE THEY ARE DIFFERENT AND THEIR LOCAL ENVIRONMENTS ARE NOT EXACTLY THE SAME. THIS IS A CRYSTAL, THERE ARE STRAINS AND STRESSES THAT PUSH THOSE MOLECULES TO SLIGHTLY DIFFERENT CENTER FREQUENCIES, RESONANCE FREQUENCIES. THAT'S THE SET UP, AT LOW TEMPERATURES. WHAT GOOD IS IT? TURNS OUT THERE'S LOTS OF THINGS THAT YOU CAN DO WITH THIS. YOU WANT TO MEASURE THE HOMOGENOUS WIDTHS, AND SOME PEOPLE DID THAT WITH METHODS LIKE PHOTON ECHOES, BUT IN THE '70s SPECTRAL HOLE BURNING WORKS THE FOLLOWING WAY, IF I HAD THE INHOMOGENEOUS BROAD LINE, I BRING A LASER INTO RESONANCE WITH A PIECE OF THE LINE I'M GOING TO PUMP MOLECULES IN RESONANCE WITH WHERE THE LASER IS, EVEN NARROWER YET. IF YOU MAKE A CHANGE IN THOSE MOLECULES, YOU CAN DRIVE THEM TO SOME OTHER FREQUENCY. PHOTOCHEMISTRY, PHOTOPHYSICS, DON'T WORRY ABOUT THE MECHANISM, IT LEAVES BEHIND A DIP THAT IS WHAT WAS CALLED A SPECTRAL HOLE, A HOLE IN THE SPECTRUM, AND IT WAS A MARK, IF YOU LIKE, IN THE SPECTRUM, NOT A TRUE PHYSICAL HOLE. YOU COULD USE THIS FOR POTENTIALLY OPTICAL STORAGE, THAT WAS THE SCENE WHEN I WAS AT IBM AT THAT TIME. IBM'S SCIENTISTS WERE EXPLORING THIS AS A NEW KIND OF OPTICAL STORAGE WHERE YOU COULD TURN TO DIFFERENT WAVELENGTHS, THOUSANDS OF BITS COULD BE RECORDED BY THAT ONE METHOD. WHAT HAPPENED THEN IS THAT -- SORRY. WE HAD AN INTERESTING PROBLEM. THIS IS THE TIME OF GREAT IBM RESEARCH CENTER WHERE YOU COULD STUDY SOMETHING THAT MIGHT BE AN APPLICATION BUT ASK FUNDAMENTAL SCIENCE QUESTIONS ABOUT HOW IT REALLY WORKS AND ITS FUNDAMENTAL LIMITS, THAT'S THE SCENE AT THE TIME WHEN I ARRIVED, AND I WANTED TO ASK THIS QUESTION. WHAT ARE THESE ULTIMATE LIMITS TO THE FREQUENCY DOMAIN OPTICAL STORAGE? WELL, AT THIS POINT, THIS INHOMOGENEOUSLY BROADENED LINE WAS THOUGHT TO BE SMOOTH AND I SHOWED YOU THE SPECTRUM. PEOPLE THOUGHT IT WAS SHAPED LIKE A GALCEON FUNCTION. IS THERE SOME SPECTRAL ROUGHNESS THAT'S COMING FROM THE DISCRETENESS OF THE INDIVIDUAL MOLECULES THAT CONTRIBUTE TO THIS ABSORPTION LINE, THAT WOULD DEFINE THE SMALLEST SPECTRAL HOLE. SPECTRAL ROUGHNESS HAD NOT BEEN DETECTED IN 198. MY POSTDOC TOM CARTER AND I SET OUT TO DETECT IT AT LOW TEMPERATURES AND HERE IT IS. IT'S AMAZING. SO THAT SMOOTH LINE I SHOWED YOU, IF YOU SPREAD IT OUT, OKAY, OVER A VERY WIDE RANGE, I'M JUST LOOKING AT A TINY PIECE NEAR THE CENTER OF THE WHOLE ABSORPTION LINE, YOU SEE THAT THERE'S AN AMAZING SPECTRAL STRUCTURE. AND THIS IS NOT NOISE. THIS IS NOT SOMETHING THAT CHANGES WITH TIME. IF YOU MEASURE IT ONCE AND MEASURE IT AGAIN, YOU SEE THE SAME THING. THIS IS COMING FROM THE FACT THAT THE ABSORPTION IS BUILT UP OF INDIVIDUAL MOLECULES. SO IT TURNS OUT THAT AT ANY ONE WAVELENGTH THERE MIGHT BE N MOLECULES IN RESONANCE BUT THE NUMBER WILL FLUCTUATE FROM WAVELENGTH TO WAVELENGTH BY THE SQUARE ROOT OF THAT NUMBER, N, AND SO IT WAS A SPECTRAL FEATURE THAT SCALED AS THE SQUARE ROOT OF THE MOLECULES, COMING FROM ONE OF THE FUNDAMENTAL THINGS YOU'VE SEEN BEFORE, THROWING BALLS IN BINS, YOU WON'T GET EXACTLY THE SAME NUMBER EVERY TIME, YOU'LL GET SLIGHTLY DIFFERENT NUMBERS, THAT'S WHAT THIS EFFECT IS. NOW IN THE OPTICAL ABSORPTION SPECTRUM. WE CALLED THIS STATISTICAL FINE STRUCTURE FOR OBVIOUS REASONS AND USED FM SPECTROSCOPY TO SEE IT. THE AMAZING ASPECT ABOUT IT, CONTRARY TO MOST OF THE FEATURES THAT YOU MIGHT THINK ABOUT WHERE YOU'VE PUT A CERTAIN N NUMBER OF MOLECULES IN A SAMPLE, N TIMES THE SIGNAL, NOW THIS AMPLITUDE OF THIS IS SCALING THE SQUARE ROOT, DIRECTLY ARISING FROM THE DISCRETENESS OF INDIVIDUAL MOLECULES AND TURNED OUT BY BEING ABLE TO SEE THIS YOU I REALIZED YOU COULD DETECT A SINGLE MOLECULE. IF THIS IS COMING FROM A THOUSAND MOLECULES THAT MEANS ITS AMPLITUDE IS SQUARE ROOT OF A THOUSAND, 32 OR SO, SO MY TECHNIQUE IS ALREADY DETECTED A SIGNAL THAT CORRESPONDS TO 32 MOLECULES WORTH OF SIGNAL. THAT MEANS IT'S ONLY 32 TIMES HARDER TO GET TO THE SINGLE MOLECULE. NOT A THOUSAND TIMES HARDER TO GET TO THE SINGLE MOLECULE LIMIT, A MAJOR STEP. SO VERY, VERY SOON SINCE WE REALIZED IT WAS POSSIBLE, WE HAD TO WORK 32 TIMES HARDER, WE DETECT THE SINGLE-MOLECULES USING THE METHOD I'LL DESCRIBE IN JUST A SECOND. IT USED THIS METHOD CALLED FM SPECTROSCOPY AND I WON'T GO INTO THE DETAILS, BUT FM WAS A QUANTUM LIMBED TECHNIQUE GARY BJORKLAND INVENTED, PERFECTLY SUITED FOR DETECTING THE FINE STRUCTURE, WE HAD TO PUSH THE EXPERIMENT DOWN TO HIGHER LEVELS OF SIGNAL TO NOISE TO DETECT THIS FEATURE FROM A SINGLE MOLECULE. IT LOOKS LIKE A LITTLE W SHAPE. SO IT'S VERY MUCH LIKE FM RADIO AT 506 TERAHERTZ, THAT'S VERY MUCH LIKE HOW THIS EXPERIMENT WORKS. BUT THE IMPACT OF THE EXPERIMENT, EVEN THOUGH YOU MIGHT THINK THIS IS VERY ESOTERIC, LOW TEMPERATURE, FANCY LASERS, IT PROVES SINGLE MOLECULES CAN BE OPTICALLY DETECTED AND PROVIDED THE KEY MODEL SYSTEM THAT GOT EVERYBODY STARTED IN THIS AREA BECAUSE THIS SYSTEM HAS WEAK SPECTRAL HOLE BURNING, YOU WANT THAT TO DO THE EXPERIMENT. A YEAR LATER, MICHELLE IN FRANCE USED THE SAME SYSTEM AND DETECTED THE SINGLE-MOLECULE ABSORPTION DETECTING EMITTED FLUORESCENCE. TURNS OUT THAT IS A SUPERIOR METHOD, BUT IT REQUIRES THERE BE A NEARLY PERFECT SAMPLE BECAUSE NOW ANY SCATTERING FROM THE SAMPLE, ANY STRAIGHT LIGHT WOULD COMPETE WITH THE LIGHT THAT'S EMITTED BY THE MOLECULE. NEVERTHELESS, IF YOU MAKE A CAREFUL SAMPLE AND SO FORTH, THESE ALSO DOTS HERE WERE THE ORIGINAL SORT OF SINGLE-MOLECULES DETECTELED BY THE FLUORESCENCE METHOD. QUICKLY AFTER THAT EVERYONE MOVED TO FLUORESCENCE DETECT OF THE ABSORPTION. I WANT TO GIVE YOU A FLAVOR OF WHAT YOU SEE NOW THAT YOU HAVE THE TECHNIQUES TO LOOK AT MOLECULES AT LOW TEMPERATURES, PAT AMBROSE IN MY LAB NOW SCANNED THAT INHOMOGENEOUS LINE. THIS IS THAT LINE I WAS TALKING ABOUT BEFORE, THE THING THAT LOOKED SMOOTH BUT NOW WE HAVE SO MUCH MORE SENSITIVITY AND LOWER CONCENTRATION YOU CAN SEE ALL OF THESE SPIKES, AND IF YOU SPREAD IT OUT YOU SEE INDIVIDUAL COPIES, IF YOU JUST LOOK AT ONE OF THEM, IT TURNS OUT THAT IT IS ONLY ABOUT 7.6 MEGAHERTZ IN WIDTH. I'VE BEEN THROWING OUT FREQUENCIES FOR A WHILE BUT I SAID THE CENTER OF THIS IS ABOUT 506 TERA HERTZ AND THE WIDTH IS 7.6 MEGAHERTZ. SO THE Q OF THESE FEATURES IS NEARLY 10 TO THE 8th, THE RATIO BETWEEN, YOU KNOW, THE FREQUENCY AND THE WIDTH IS INCREDIBLE. THAT'S WONDERFUL FOR SPECTROSCOPY, BECAUSE A NARROW FEATURE IS VERY SENSITIVE TO LOTS OF PROTEVATION. WE RECORDED THE LIGHT HERE, BUT THIS AXIS' FREQUENCY, OR COLOR, THINK OF IT AS COLOR, YOU MOVE THE LASER SPOT ACROSS THE SAMPLE AND RECORD SIGNAL. DIFFERENT PIECES HAVE MOLECULES, OTHERS DON'T. WE KNEW THE MOLECULES BEING USE TO MEASURE THE SIZE OF THE LASER SPOT, MOLECULE IS A NANOMETER IN SIZE, THE LASER WAS BIGGER, THAT'S WHAT THE DIFFRACTION LIMIT IS BUT WE'LL TALK ABOUT THAT MORE IN A MOMENT. THERE WERE TWO DIMENSIONAL SPECTROSCOPIES THAT FOLLOWED QUICKLY AT LOW TEMPERATURES, TWO DIMENSIONS ON X AND Y, TWO SPATIAL DIMENSIONS. WELL, WHEN YOU GET INTO THE NEW REGIME OF SINGLE-MOLECULES FOR THE FIRST TIME, THERE ARE SURPRISES. YOU DON'T KNOW EXACTLY EVERYTHING THAT'S GOING TO HAPPEN, BECAUSE THE PREVIOUS ENSEMBLE AVERAGING OVER MILLIONS AND BILLIONS OF MOLECULES IS REMOVED. SO WHAT WAS FUN WAS SOME OF THE SURPRISES. I'M GOING TO SHOW YOU NOW SPECTRA, THIS ACTS AS A SPECTRA, AN OLD OSCILLOSCOPE. THE MOLECULE IS SITTING AT ONE FREQUENCY AND JUMPING TO ANOTHER AND ANOTHER FREQUENCY, AND JUMPING TO ANOTHER FREQUENCY. IT WAS TOTALLY AMAZING. WHEN WE SAW THIS, PAT CAME RUNNING IN THE MOLECULES ARE JUMPING AROUND! WAIT A MINUTE, IT'S LOW TEMPERATURE, IT TURNS OUT THERE ARE DEFECTS, AND THE MOLECULE CAN BE MODULATED BY DEFECTED. IF YOU SET A FIXED-FREQUENCY LASER WITHOUT MEASURING THE SPECTRUM, NOW THE MOLECULE WILL JUMP INTO AND OUT OF RESONANCE, YOU'LL SEE THE SIGNAL GOING ON-OFF, ON-OFF, SPECTRAL DIFFUSION. THEORETICAL EXPERTS LIKE JIM SKINNER HELPED FIGURE THIS OUT. ANOTHER AMAZING THING THAT HAPPENED, NOW ANOTHER POSTDOC, FOR THIS MOLECULE, AND FOR THIS SYSTEM HERE IS THREE SCANS OF A MOLECULE, NOT MOVING AROUND, STAYING CONSTANT, NO BIG CHANGE, BUT IF YOU COME INTO RESONANCE WITH IT, PUT THE LASER AND SIT FOR A MOMENT, IT GOES SOMEWHERE ELSE IN FREQUENCY SPACE. WAY FAR AWAY, NOW IT'S GONE. IT SEEMED TO DISAPPEAR. BUT YOU WAIT A MOMENT, IT COMES RIGHT BACK, YOU CAN WATCH IT AGAIN AND COME INTO RESONANCE, DRIVE IT AWAY AND SO FORTH. WE COULD DRIVE THE MOLECULES BACK AND FORTH FROM ONE FREQUENCY TO ANOTHER USING LIGHT SO THAT WAS THE BEGINNINGS OF OPTICAL SWITCHING. SO THESE KINDS OF THINGS THAT WENT ON AT LOW TEMPERATURE WERE SORT OF FORERUNNERS OF LATER WORK BUT IT SHOWS YOU WHAT CAN HAPPEN WHEN YOU GET INTO A NEW REGIME. I WANT TO START TALKING ABOUT ROOM TEMPERATURE NOW, IN THE NEXT FEW SECONDS. AND FIRST JUST SORT OF SUMMARIZE, WHY YOU WOULD EVEN WANT TO DO ALL OF THIS NOW, ONCE YOU START THINKING ABOUT, WELL, SINGLE MOLECULES AND WHO CARES, HERE IS THIS OLD SITUATION, VERY FIRST ONE, SINGLE DOPANS IN A CRYSTAL OR POLYMER. THE CHALLENGE IS TO LOOK AT CELLS, MUCH MORE INTERESTING SYSTEMS THAT HAVE LITTLE MACHINES THAT ARE DOING THEIR BUSINESS, LIKE THIS, ALL THESE PROTEINS AND ENZYMES INVOLVED IN BACTERIAL SYSTEMS AND SO ON. THIS IS THE GRAND CHALLENGE THAT DRIVES ALL OF US TO LEARN MORE AND MORE ABOUT THESE COMPLEX SYSTEMS BY WATCHING INDIVIDUALS. AND OUR MOTIVATION REALLY IS RESTING ON CONCEPTS LIKE REMOVING ENSEMBLE AVERAGING. SO LET ME SAY THAT DIFFERENTLY. WHEN WE'RE MEASURING ONLY INDIVIDUALS, SINCE WE CAN SEE ONE AT A TIME WE CAN EXPLORE WHETHER THERE'S HETROGENEITY, ARE SINGLE COPIES THE SAME OR DIFFERENT? DO THEY BEHAVE DIFFERENTLY BECAUSE THEY ARE IN A DIFFERENT LOCAL ENVIRONMENT? YOU CAN SEE THE INDIVIDUAL STATES RATHER THAN HAVING TO SMEAR OVER ALL OF THEM. THE MOLECULE IS A NANOMETER SIZE REPORTER AND LIGHT SOURCE EMITTING LIGHT, SO THERE'S MANY THINGS YOU CAN DO, FOR EXAMPLE, FRET AND OTHERS, MEASURING DISTANCE AND CONFIRMATIONAL CHANGES ON A SHORT SCALE AND I'M TALK MORE ABOUT SUPER-RESOLUTION IN A MOMENT. THERE ARE COMMERCIAL APPLICATIONS OF THESE MOLECULES, BESIDES SELLING MICROSCOPES FOR SUPER-RESOLUTION. WELL, AT ROOM TEMPERATURE NOW, THERE WAS A PARALLEL SET OF THINGS GOING ON IN THE '90s. I TOLD YOU ABOUT THE '80s AND EARLY '90s OF LOW TEMPERATURES, BUT IN ROOM TEMPERATURE THERE WERE A LOT OF COOL THINGS GOING ON AS WELL. AND DON'T BE AFRAID, BUT I JUST PROVIDE HERE ONE OF THE SLIDES THAT YOU DON'T WANT TO READ BECAUSE IT OBVIOUSLY HAS TOO MANY THINGS ON IT. THERE WERE A LOT OF REALLY WONDERFUL BRILLIANT INVESTIGATORS DOING INTERESTING THINGS, AND MOVING THE FIELD FORWARD, BIT BY BIT. LOOK AT THE COLORED THING. SINGLE BURSTING FROM ONE FLEUROFLORE IN SOLUTION FLOATING THROUGH A FOCUS WAS ONE OF THE THINGS THAT WAS DONE TO SEE A SINGLE MOLECULE AT ROOM TEMPERATURE. HERE IS A NEAR FIELD OPTICAL MICROSCOPY EXPERIMENT IN 1993, IMAGING A SINGLE FLEUROPHORE IN A POLYMER. AND 1995 WAS AN IN VITRO EXPERIMENT. SO WHAT WAS GOING ON HERE, PEOPLE WERE LEARNING BIT BY BIT BY BIT THAT MOLECULES COULD BE DETECTED AT ROOM TEMPERATURE AS WELL, WITH A WIDE VARIETY OF METHODS. AND SO TO JUST MAKE SURE WE'RE ALL ON THE SAME PAGE, I WANT TO JUST REMIND YOU NOW HOW WE DO THESE EXPERIMENTS, AT ROOM TEMPERATURE, WITHOUT THAT FANCY TUNEABLE LASER BUSINESS. WE'RE PUMPING MOLECULES FROM A GROUND STATE TO ELECTRONIC EXCITED STATE WITH A LASER, THERE'S RELAXATION AND EMISSION OF FLUORESCENCE THAT CAN TERMINATE ON LEVELS OF GROUND STATE. SO WE'RE INTERESTED IN THIS RED SHIFT AT FLUORESCENCE, AS THE MOLECULE WAS CYCLED MANY TIMES. MOLECULES CAN GO INTO DARK STATES BUT YOU DON'T SEE THEM, THEY CAN HAVE USES, WE'LL TALK ABOUT THAT LATER. ALL OF THE MOLECULES OF INTEREST ARE THESE SMALL FLUORESCENT LABELS. THESE "THEYS," AND THE GREEN FLUORESCENT PROTEIN AND MANY DERIVATIVES, THE LABEL CAN BE GENETICALLY ATTACHED. IF YOU IMAGINE THE OBJECT OF INTEREST IS CONNECTED TO A LABEL, THEN, FOR EXAMPLE IN A CELL, IN ORDER TO SEE A SINGLE MOLECULE YOU FOCUS YOUR LASER BEAM DOWN TO THE SMALLEST SPOT POSSIBLE, BUT WHAT IS THAT? IT'S LAMBDA OVER TWO TIMES THE NUMERICAL APERTURE OF THE MICROSCOPE, THE DIFFRACTION LIMIT MENTIONED BY DR. COLLINS. YOU CAN'T FOCUS DOWN TO THE MOLECULE DUE TO THE FUNDAMENTAL DIFFRACTION LIMIT. IF THE MOLECULES ARE SPACED APART BY MORE THAN THIS FOCAL SPOT, THEN YOU CAN COLLECT THE LIGHT FROM THAT ONE. AND TURNS OUT TO BE DOABLE, AND PERFECTLY FINE, AND MANY, MANY EXPERIMENTS WERE BASED ON THIS REGIME. I WANT YOU TO NOTICE THE DIFFERENCE BETWEEN THE SIZE OF THE FOCAL SPOT AND MOLECULE THAT'S BEING PUMPED. IN THE MID-'90s, BECAUSE ROOM TEMPERATURE WAS AVAILABLE THEN FOR SEEING SINGLE MOLECULES, I MOVED TO UC-SAN DIEGO AND WAS ABLE TO BEGIN BIOLOGICAL STUDIES AWAY FROM THE THING AND SORT OF NOT PURSUE THE THINGS THAT WERE GOING ON AT IBM AT THAT TIME AND WE GOT INTERESTED IN FLUORESCENT PROTEIN, GREEN FLUORESCENT PROTEIN, FROM ROGER CHEN AND HIS POSTDOC BROUGHT US ONE OF THE LATEST PARTICULAR MUTANTS THEY WERE MAKING BECAUSE THERE WAS SO MUCH INTEREST IN UNDERSTANDING GFP AT THE TIME THEY MADE A YELLOW EMITTING, YELLOW FLUORESCENT PROTEIN, WE LOOKED AT IT IN GEL AND SAW SINGLE MOLECULES, EXCITING TO BEGIN TO SEE SINGLE MOLECULES OF GREEN FLUORESCENT PROTEINS OF DIFFERENT SORTS. BUT IN THAT VERY FIRST EXPERIMENT, ROB DIXON, MY POSTDOC, 1997, DIDN'T SEE JUST THIS, MOLECULES THAT ARE SITTING THERE NOT DOING ANYTHING. IT'S THE NEW REGIME THAT HAD NOT BEEN EXPLORED, SURPRISES START TO APPEAR. ONE SURPRISE IS THE MOLECULES WOULD BLINK. THAT IS, THEY WOULD EMIT, EMIT, EMIT, EMIT FOR MULTIPLE FRAMES AND TURN OFF AND COME BACK ON AGAIN AND TURN OFF, COME BACK ON AGAIN AND SO FORTH, SUGGESTING THAT OUR OPTICAL PUMPING CYCLE GIVING PHOTONS WAS ALSO CONNECTED TO SOME DARK STATE WHERE THE MOLECULE SITS FOR A WHILE BUT CAN THERMALLY COME BACK AND EMIT AGAIN. THAT'S INTERESTING, BUT THAT WASN'T THE ONLY THING WE SAW AM HE WOULD PUMP THE MOLECULE FOR A LONG TIME, IT COULD GO INTO A LONG-LIVED DARK STATE, YOU MIGHT THINK OF PHOTOBLEACHING, THE MOLECULE IS GONE AND NO LONGER ABLE TO EMIT BUT HE LEARNS BY TURNING ON SHORTWAVE LENGTH LIGHT, YOU COULD RESTORE A MOLECULE PUT IN THIS LONG-LIVED DARK STATE AND HAVE IT EMIT FOR A LONG TIME AND MAYBE ALSO BLINK, AFTER IT FINALLY DECIDES TO GIVE UP YOU CAN TURN IT BACK ON, SO THIS EXPERIMENT GAVE US THE SORT OF INTERESTING PROPERTY THAT YOU COULD PHOTORESTORE A MOLECULE THAT HAD BEEN PUT INTO A DARK STATE. SO THIS LED TO SOME OTHER AMAZING DEVELOPMENTS THAT STARTED OCCURRING BECAUSE THERE WERE MANY PEOPLE TRYING TO MAKE BETTER FORMS OF FLUORESCENT PROTEINS AND ONE OF THE MOST IMPORTANT HERE EARLY ON, PHOTOACTIVATABLE, DRONPA, AND OTHERS THAT CAN BE SWITCHED BACK AND FORTH, MAYBE WITH BETTER SWITCHING PROPERTIES, ET CETERA. THAT WAS ONE OF THE AMAZING THINGS THAT HAPPENED WITH SINGLE MOLECULES IN THAT TIME PERIOD AM WE LOOKED AT SINGLE MOLECULES IN INTERESTING CASE WAS COLLABORATORS, HERE LOOKING AT SINGLE MHC2 PROTEINS ON THE SURFACE OR MEMBRANE OF A LIFE CHO CELL, AND THIS PARTICULAR LITTLE MOVIE FROM EARLY ON, I THINK IT'S SO FASCINATING BECAUSE WHAT WE OBSERVED IS THE MOTION OF THE DANCE OF THE INDIVIDUAL TRANSMEMBRANE PROTEINS ON THE SURFACE OF THE CELL AT ROOM TEMPERATURE. YOU SEE THEM ALL MOVING AROUND OF COURSE, AS IS OCCURRING IN OUR CELLS RIGHT NOW, WITH THE PROTEINS IN OUR CELL MEMBRANES. YOU SEE THESE EVENTUALLY TURNING OFF BECAUSE THE FLEUROPHORES DON'T GIVE PROTONS, AND SO ON. WE WERE LOOKING AT BACTERIA, WITH LUCY SHAPIRO, AND I'LL SHOW YOU SOMETHING DIFFERENT OBSERVED IN THAT CASE FOR A PARTICULAR PROTEIN FUSED TO YFP, YELLOW FLUORESCENT PROTEIN. WATCH WHERE I'M HOLDING THE LASER POINTER. THIS IS TIME LAPSE IMAGING, YOU SEE THE MOLECULE MOVED ACROSS THE CELL IN A LINE, TURNS AROUND AND GOES BACK THE OTHER WAY MUCH LIKE THIS LITTLE DRAWING HERE. THIS ISN'T DIFFUSION ANYMORE. THIS IS DIRECTED MOTION. DIFFUSION IS RANDOM. YOU CHANGE DIRECTION ALL THE TIME. YOU DON'T GO IN A LINE FOR A LON TIME. SO HERE WE'RE OBSERVING CIRCUMFERENCEIAL MOTION, DIRECTLY INVOLVED WITH THE CELL WALL FORMATION MACHINERY. SO THE MOTION OF THE MOLECULE IS TELLING US SOMETHING. HERE ARE TRACKS IN THE CELLS, MOSTLY PERPENDICULAR TO THE LONG AXIS, DIFFERENT BEHAVIOR IF YOU WATCH THE SINGLE MOLECULE. THIS IS ROOM TEMPERATURE AGAIN STILL NOT COOLING DOWN AND HERE IS ONE FRAME OF A MOVIE. YOU CAN SEE LITTLE RINGS, MANY MOLECULES ARE IN ORDERED PIECES OF CRYSTAL SO THE Z AXIS -- OR THE -- OF THE MICROSCOPE IS ALIGNED WITH THE DIPOLE, WATCH IF YOU VIEW AS A FUNCTION OF TIME. FIXED MOLECULES AND OTHERS AT ROOM TEMPERATURE THAT ARE MOVING AROUND INSIDE THE SOLID CRYSTAL, AND THEY ARE MORE OVER MOVING IN A BIASED MOTION, ALL UP AND DOWN OR RIGHT AND LEFT, FOR THE MOST PART IF YOU LOOK AT THE AVERAGE BEHAVIOR. THESE MOLECULES ARE EXPLORING THE CRACKS IN THE CRYSTAL. THE MOLECULES ARE TELLING US WHERE THE DEFECTS ARE IN THE CRYSTAL. SO, YOU KNOW, ANOTHER EXAMPLE OF THE KIND OF THINGS YOU CAN SEE JUST BY WATCHING INDIVIDUALS. WELL, LET'S TURN NOW AWAY FROM JUST WATCHING INDIVIDUALS TO THIS WHOLE PROBLEM OF THE DIFFRACTION LIMIT AND SUPER-RESOLUTION. THE PROBLEM IS EASILY SHOWN HERE. WE HAVE A BACTERIAL CELL, OF COURSE THEY ARE VERY TINY AND SPECIFIC PROTEINS HAVE BEEN LABELED NOW INSIDE THAT BACTERIUM WITH YELLOW FLUORESCENT PROTEIN. YOU MIGHT SAY I WANT TO SEE THE STRUCTURE, WHAT'S THE STRUCTURE THE MOLECULES ARE MAKING. YOU MIGHT THINK, OKAY, LET'S BUY THE MOST EXPENSIVE MICROSCOPE SO HERE IT IS BUT UNFORTUNATELY YOU STILL SEE A BLUR. THAT'S WHAT THE DIFFRACTION LIMIT MEANS, THEY ARE A FEW NANOMETERS IN SIZE, THIS SAYS USING OPTICAL LIGHT, LET'S SAY 500 NANOMETERS, MEANS THE SPOT OF THE IMAGE OF A SINGLE MOLECULE DOESN'T LOOK A FEW NANOMETERS BUT LOOKS 200 NANOMETERS IN WIDTH. SO THIS IS THE PROBLEM. THIS IS THE PROBLEM TO BE OVERCOME. SO THE WAY IN WHICH IT GETS OVERCOME, IS TO REALLY CIRCUMVENT THE DIFFRACTION LIMIT, THAT'S WHAT SUPER RESOLUTION MEANS. IT TURNS OUT THERE'S NOT JUST ONE WAY TO DO IT. THERE'S SEVERAL WAYS NOW, AND THERE'S SOME APPROACHES THAT DON'T REQUIRE SINGLE MOLECULE IMAGING. I WANT TO MENTION STED, STRUCTURE ELIMINATION, THERE ARE POWERFUL METHODS BUT SIM DOESN'T IMPROVE BY MORE THAN A FACTOR OF TWO. I'M GOING TO TALK ABOUT APPROACHES, WITH ERIC BEING THE FIRST NAME TO LIST THERE, SO WHAT I WANT TO EXPLAIN IS HOW CAN WE USE THESE SINGLE MOLECULE EMITTERS TO SURPASS THE DIFFRACTION LIMIT. I WANT TO DESCRIBE THE ANSWER IN A SIMPLE WAY AND SORT OF THE MOST GENERAL WAY, SO THAT YOU SEE THE BEAUTY OF THE OVERALL IDEA. SO THE ANSWER REALLY IS, FIRST YOU HAVE TO IMAGE SINGLE MOLECULES AND THEN YOU NEED TO ADD TO THAT TWO KEY IDEAS. SO THE TWO KEY IDEAS ARE THE IMPORTANT THINGS TO DESCRIBE. AND OUR CONTRIBUTION REALLY FROM MY LAB IS REALLY IN THE FOUNDATIONS OF SINGLE MOLECULE IMAGING. LET'S TALK ABOUT THE TWO KEY IDEAS NOW. THE FIRST KEY IDEA I WANT TO CALL IT SUPER LOCALIZATION. WHAT DOES THAT MEAN? THINK ABOUT THIS LITTLE CINDER CONE THAT'S IN CRATER LAKE, OREGON. IF YOU'VE EVER BEEN TO CRATER LAKE, IT'S A BEAUTIFUL PLACE. IN THE CENTER OF THIS VOLCANIC LAKE IS A BEAUTIFUL CINDER CONE, SO THERE'S ONE OF MY PICTURES OF THAT CINDER CONE. HERE IS A SCALE BAR, 120 TIMES 10 TO THE 9th NANOMETERS. SO WE KNOW THE NANOMETER SCALE, RIGHT? EVERYBODY KNOWS THAT IF YOU TAKE YOUR CELL PHONE NOW, AND SIMPLY WALK UP TO THE TOP OF THIS MOUNTAIN, YOU CAN RECORD AND READ OUT THE COORDINATES OF THE POSITION OF THE MOUNTAIN WITH GREAT ACCURACY. MUCH BETTER THAN THE WIDTH OF THE MOUNTAIN. THAT'S REALLY THE BASIC IDEA OF SUPER LOCALIZATION. WE FIND THE POSITION OF A MOLECULE BY LOOKING AT THE SHAPE, FITTING THE SINGLE MOLECULE IMAGE. LET ME EXPLAIN IT. HERE IS ONE OF THESE IMAGES OF SINGLE MOLECULES, OKAY, FROM A TWO DIMENSIONAL CAMERA COLLECTING THE FLUORESCENCE FROM THE MOLECULE. THE SPOT, THE DISC, THE DIAMETER IS COMING FROM THE DIFFRACTION LIMIT, IT HAS TO. LET'S NOW DO WHAT YOU NEED TO DO TO FIND OUT WHERE THEY ARE AND WHAT YOU DO FIRST IS TO SPREAD OUT THEIR PHOTON, SPREAD THE PHOTONS OVER MULTIPLE PIXELS OF THE DETECTOR. MAKE THE IMAGE BIGGER. YOU HAVE TO SAMPLE MULTIPLE POSITIONS ON THE SPOT, BECAUSE HERE IN CROSS-SECTION YOU SEE THE SPOT IS NOT JUST A ROUND DISC BUTS THAT A SHAPE, AND THE SHAPE IS CALLED AN ARIE FUNCTION, CLOSE TO A GALCEON FUNCTION, SINCE YOU KNOW THERE'S A FITTING FUNCTION YOU CAN USE FOR THE SHAPE OF THE SINGLE MOLECULE SPOT, YOU SIMPLY FIT IT WITH THE FUNCTION THAT HAS A WIDTH AS A PARAMETER WHICH HAS TO BE THE DIFFRACTION LIMIT BUT ANOTHER PARAMETER IS THE POSITION, CENTER POSITION OF THE OBJECT. IT TURNS OUT BY DOING THIS FIT, WHAT YOU CAN DO IS DETERMINE THE VALUE OF THE CENTER POSITION TO MUCH, MUCH GET ACCURACY OR PRECISION THAN THE FULL WIDTH OF THE POINT SPREAD FUNCTION. NOW I SAID THAT WORD, POINT SPREAD FUNCTION, IT'S THE IMAGE OF A SINGLE POINT SOURCE. AND SO THESE ESTIMATES OF THE CENTER ARE MUCH, MUCH BETTER THAN THE FULL WIDTH OF THE SPOT, AND WE -- SIGMA IS THE STANDARD DEVIATION OF THE ESTIMATES OF THE CENTER. SIGMA SCALE SAYS THE OTHER LIMITS, DIFFRACTION LIMIT, DIVIDED BY THE SQUARE ROOT OF THE NUMBER OF PHOTONS DETECTED, THE NUMBER OF PHOTONS CONTRIBUTING TO THE IMAGE BECAUSE EACH PHOTON IS LIKE A MEASUREMENT GIVING YOU INFORMATION ABOUT WHERE THE MOLECULE IS AND YOU MAKE MORE AND MORE MEASUREMENTS, PRECISION IMPROVED BY ONE OVER THE SQUARE ROOT OF THE MEASUREMENT. IF I CAN RECORD A HUNDRED PHOTONS, I CAN GO TO 200 DOWN TO 20-NANOMETER LOCALIZATION. SUPER-RESOLUTION IS THE FIRST KEY THING YOU HAVE TO DO -- SUPER-LOCATION IS THE FIRST THING YOU HAVE TO DO. IT'S BEEN AROUND A LONG TIME. I WANT TO POINT OUT VERY EARLY EXAMPLES OF USING SUPER LOCALIZATION IN BIOLOGICAL IMAGE, LOOKING AT THE POSITIONS OF SINGLE LDL PARTICLES, THESE EARLY EXPERIMENTS INVOLVE FINDING THE CENTROID, BUT WE'RE TRYING TO FIND A SINGLE MOLECULE, SO THAT WAS PRETTY MUCH FIRST DONE BY SCHMIDT AND CO-WORKERS IN 1996 AND APPLIED BY A LOT OF PEOPLE. IF THE PHOTONS COME FROM A NANOMETER SIZE PARTICLE, HERE IS WHAT THEY LOOK LIKE. HERE IS ONE OF THE IMAGES AND THERE'S THE LIGHT COMING FROM THE MOLECULE, AND HERE IS THAT DATA, NOW FIT WITH A FUNCTION, AND MY ESTIMATE OF THE CENTER ARE GOING TO HAVE THIS MUCH NARROWER PRECISION, AND SO YOU CAN DO THIS NOW IN REALTIME WITH A FAST MOVIE, TAKING ONE FRAME AFTER ANOTHER AFTER ANOTHER, AND TRACK AND FOLLOW SINGLE MOLECULES WITH NANOMETER SCALE PRECISION IF YOU HAVE ENOUGH PROTONS PER FRAME, A POWERFUL WAY OF GETTING PRECISE INFORMATION. BUT OF COURSE REMEMBER, IT IS NOT RESOLUTION. THIS IS ONLY ONE OBJECT. RESOLUTION MEANS DISTINGUISHING TWO OBJECTS VERY CLOSE TOGETHER. RESOLVING THEM IN SPACE. SO SUPER-RESOLUTION, AS AN IDEA, SORT OF HAS AN INTERESTING HISTORY, I'M SHOWING YOU AGAIN A BUNCH OF KEY PAPERS. DON'T TRY TO READ IT ALL. LET ME POINT OUT A KEY PAPER OCCURRED IN ERIC BETZIG IN 1995. HE WAS THINKING ABOUT IT FOR MANY YEARS. I WASN'T. I WAS THINKING ABOUT SPECTRAL RESOLUTION BUT OTHER PEOPLE WERE THINKING ABOUT RESOLUTIONS, A SPATIAL RESOLUTION. AND IN THIS IMPORTANT PAPER HE POINTED OUT THAT YOU CAN SEPARATE MOLECULES THAT ARE IN THE SAME DIFFRACTION LIMITED SPOT IF YOU HAVE ANOTHER CONTROL VARIABLE THAT LET'S YOU DISTINGUISH THE DIFFERENT MOLECULES, THAT IDEA WAS ACTUALLY PROVED IN 1988 USING THE LOW TEMPERATURE METHODS WE PIONEERED TO GET 40-NANOMETER AND 100-NANOMETER AXIAL AT LOW TEMPERATURE. OTHERS STARTED TO DISTINGUISH BY VARIOUS TRICKS, EITHER PHOTOBLEACHING OR LIFETIMES OR COLORS OR BLINKING AND SO FORTH. THESE PARTICULAR METHODS IN THESE EARLY DAYS REALLY ONLY GAVE YOU A FEW MORE MOLECULES PER SPOT, AND SO THEY WEREN'T THE BIG GENERAL STEP FORWARD. SO THE BIG GENERAL STEP FORWARD OCCURRED IN ABOUT 2006, AND I WANT TO AGAIN DESCRIBE IT IN GENERAL AND TELL YOU ABOUT THE KEY PLAYERS THAT WERE FUNDAMENTAL PIONEERS IN THIS AREA. SO THIS IS KEY IDEA NUMBER TWO. IF I GOT -- I CAN SUPER-LOCALIZE MOLECULES IF THEY ARE SEPARATED FROM OTHER PARTNERS. HERE IS THE PROBLEM. I HAVE A STRUCTURE YOU WANT TO SEE, IF YOU LABEL IT WITH LOTS OF LABELS, HERE YOU CAN SEE THE LABELS, AND IF YOU ALLOW ALL OF THEM TO EMIT, ALL AT THE SAME TIME, THEN YOU GET THESE BLURRY PICTURES BECAUSE THEIR POINT SPREAD FUNCTIONS OVERLAP. THAT'S THE PROBLEM, YOU'RE LETTING THEM EMIT OVER THE SAME TIME AND HAVE NO WAY TO DISTINGUISH THEM. THE KEY IDEA OF WHAT I CALL ACTIVE CONTROL IS TO SIMPLY NOT ALLOW THEM TO EMIT AT THE SAME TIME, AS AN EXPERIMENTER USE SOME METHOD THAT CAN BE -- THERE CAN BE MANY BUT USE A METHOD YOU PICK AND YOU CAN CONTROL, THAT CONTROLS THE CONCENTRATION THAT'S EMITTING. YOU WANT TO MAKE IT LOW SO THAT THE MOLECULES ARE SEPARATED AT ANY GIVEN FRAME OF THE CAMERA. IF THEY ARE SEPARATED BY MORE THAN THE DIFFRACTION LIMIT THEN YOU CAN SUPER-LOCALIZE THEM AND FIT THEM AND FIND THEIR POSITIONS. THEN YOU MAY PHOTOBLEACH THEM FOR EXAMPLE AND DO IT AGAIN, ALLOW OTHERS TO EMIT AND FIND THEIR POSITIONS. DO THIS AGAIN AND AGAIN, AND IN SEQUENTIAL IMAGING YOU BUILD UP MANY POSITIONS, OR SAMPLES, OF THE LOCATIONS ALONG THE OBJECT. IF YOU NOW THEN TAKE THOSE SPOTS THAT YOU'VE LEARNED THE POSITIONS THAT YOU'VE MEASURED, YOU CAN RECONSTRUCT THE UNDERLYING STRUCTURE WITH DETAILS BEYOND THE DIFFRACTION LIMIT. THE IDEA IS TO TEMPORARILY SEPARATE THE DIFFERENT MOLECULES SO THAT YOU CAN IMAGE THEM AND FIND THEIR POSITIONS AND REALLY USE THE POWER OF THE MOLECULES BECAUSE THEY ALREADY ARE NANOMETER-SIZED LIGHT SOURCES INSIDE THE SAMPLE. WELL, THIS IDEA APPEARED ON THE SCENE AND I FIRST HEARD OF IT HERE AT THE NIH IN APRIL OF 2006 AT A CONFERENCE ON CELLULAR IMAGING WHEN ERIC BETZIG REPORTED WHAT HE HAD BEEN DOING WITH HARALD HESS AND JENNIFER AND WITH GEORGE. IT WAS A WATERSHED MOMENT BECAUSE WHEN HE GAVE THAT TALK WHICH YOU CAN NOW GO BACK AND HEAR ON THE WEB BECAUSE IT'S BEEN RECORDED, YOU CAN SEE THAT IT WAS A GREAT STEP FORWARD AT THAT TIME. AND VERY SOON AFTER, ZHUANG CAME UP WITH SOMETHING CALLED STORM, SAM HESS SOMETHING CALLED F-PALM AND SOMETHING CALLED PAINT BY ROBIN THAT I'LL TALK ABOUT LATER, THE STORM, BLINK, SPDM, ALL THESE SCHEMES AND YFP REACTIVATION ARE DIFFERENT WAYS OF ACHIEVING ACTIVE CONTROL BECAUSE SCIENTISTS CAN SAY I CAN THINK OF ANOTHER WAY TO TURN THE MOLECULES ON AND OFF, LET'S SEE IF I CAN MAKE IT WORK AND SO IT WAS A BRILLIANT EXPLOSION OF INTERESTING IDEAS. OF COURSE, THERE'S A BIT OF A MENAGERIE HERE OF ACRONYMS THAT CAN CONFUSE PEOPLE, SAYING, WHAT'S THE DIFFERENT, AND SO FORTH. IN A SENSE THEY ARE ALL ONE AND THE SAME AND THE WAY I TOLD YOU, AND SO WHAT IS NICE IS TO REMEMBER THAT WE DID SOMETHING IN THIS TIME PERIOD, SOME YEARS AFTER THE FIRST EXPERIMENTS, WE DID SOMETHING CALLED YFP REACTIVATION THAT I'LL TALK ABOUT IN A MOMENT BUT WE HAD NO ACRONYM FOR IT. EVERYBODY FORGETS IT, RIGHT? YOU GOT TO HAVE AN ACRONYM. THAT'S WHY PEOPLE WERE DOING IT. [ LAUGHTER ] JUST TO, YOU KNOW, RECTIFY THAT SITUATION HERE IS A NEW ACRONYM, A MECHANISM INDEPENDENT, SINGLE MOLECULE ACTIVE CONTROL MICROSCOPY, OR "SMACK 'EM." THIS HAS SINGLE MOLECULE IN THE TITLE AT LEAST. ALL RIGHT. SO LET'S GO BACK TO THESE DAYS, IF YOU LIKE, AND NOW I WANT TO SHOW YOU ACTUALLY THE WAY THE YFP EXPERIMENTS WANT, SOME EXAMPLES OF REAL IMAGES. SO FIRST I WANT TO TALK ABOUT BACTERIA IN EXPERIMENTS WITH LUCEY SHAPIRO LOOKING AT HER FAVORITE ORGANISM, VERY INTERESTING BECAUSE IT DIVIDES ASYMMETRICALLY, TWO DAUGHTER CELLS HAVE DIFFERENT PHENOTYPES, WE LIKE TO UNDERSTAND HOW PROTEIN LOCALIZATION PATTERNS PRODUCE THOSE DIFFERENT PHENOTYPES IN THIS SMALL ORGANISM. SO YOU WANT TO IMAGE BUT THE PROBLEM IS THE BACTERIA, HERE IS A WHITE LIGHT IMAGE OF BACK YEAH, ARE TOO SMALL. THAT IS, THEY ARE CLOSE TO THE DIFFRACTION LIMIT. YOU WON'T LEARN A LOT IF YOU CAN ONLY DO DIFFRACTION LIMITED MICROSCOPY. HERE IS THE FLUORESCENT IMAGE OF EYFP FUSED TO A SICK PROTEIN AND YOU SEE FOUR SINGLE YFPs WITH PROPER LASER POWERS THEY BLINK, THAT'S FROM 1997, ALL OF THESE SPOTS ARE COMING FROM POSITIONS INSIDE THOSE INDIVIDUAL CELLS. THIS IS THE RAW DATA. THIS IS WHAT WE LOOK AT. YOU FIT EVERY ONE OF THESE FRAMES, FIND THE POSITIONS OF THE MOLECULES AND THEN PUT THEM ALL TOGETHER IN THE FINAL RECONSTRUCTION. SO THAT'S THE BASIC SORT OF IDEA OF THE EXPERIMENTS, AND HERE IS SOME QUICK EXAMPLES OF WHAT YOU CAN SEE IN BACTERIA, USING THIS METHOD, AND USING REACTIVATION AS WELL. SO IN THESE THREE IMAGES, THREE DIFFERENT PROTEINS, INSIDE THE BACTERIA HAVE BEEN LABELED WITH MREB, PAR-A AND H U, WITH SUPER-RESOLUTION YOU SEE THIS. THE FACT THAT YOU CAN GO FROM THIS SITUATION TO THESE KINDS OF SITUATIONS WHERE YOU HAVE NOW RESOLUTION DOWN IN THE ORDER OF 40 NANOMETERS OR BELOW IS MANY FACTORS BEYOND THE DIFFRACTION LIMIT, AND YOU START SEEING STRUCTURES THAT YOU MIGHT NOT HAVE SEEN BEFORE. USING LIGHT MICROSCOPY, IN SOME CASES DOING THIS ON LIVING CELLS, THIS IS A FIXED CELL, HU BINDS TO THE NUCLEOID, SHOWING YOU WHERE THE POSITION IS IN THE CELL. TO SHOW YOU EUKARYOTIC EXAMPLE, MANY PEOPLE WANT TO LOOK AT MICRO-TUBULES IN A CELL, HERE IS ONE FRAME FROM A WHITEFIELD IMAGE, IN THIS CASE ANTIBODY LABELS, ON MICRO-TUBULES, AND ON THIS SIDE IF YOU LOOK CLOSELY YOU SEE ALL THE SPOTS WHICH ARE THE LOCALIZATION OF THE MOLECULES FROM THAT FRAME. NEXT FRAME YOU SEE DIFFERENT MOLECULES ON. NEXT FRAME DIFFERENT MOLECULES ON, ET CETERA. AND AS YOU THEN -- OOPS, I DIDN'T WANT TO DO THAT. FROM THE MOVIE OF MANY SUCH LOCALIZATIONS, YOU BUILD UP THESE FINAL RECONSTRUCTIONS WHICH HAVE SUCH WONDERFUL ADDITIONAL RESOLUTION. THIS IS BASICALLY USING THE ALEXIS 647 AND REDUCTANT IDEAS THE ZHUANG LAB PIONEERED. THAT'S NOT THE ONLY WAY TO ACHIEVE ACTIVE CONTROL SO I WANT TO ILLUSTRATE OTHER WAYS OF GETTING ACTIVE CONTROL OF THE MOLECULE. THIS IS A PAINT IDEA FROM ROBIN HOCHSTRASSER. IN THIS EXPERIMENT, WE'RE GOING TO HAVE WHAT I CALL TARGET-SPECIFIC PAINT. WELL, HOW DOES IT WORK? WITH JUSTIN DUBOIS AT STANFORD, HE CAN MAKE SAXOTOXIN, A NEUROTOXIN, YOU CAN MAKE ILLAND ATTACH A FLEUROPHORE, ADDED TO THE LIGAND, IT WILL BIND TO SODIUM CHANNELS ON THE SURFACE OF CELLS, SO OUR EXPERIMENT INVOLVES HAVING A LIVE CELL GROWING AND YOU ADD THIS MOLECULE TO THE SOLUTION, SO THE LABELED SAXITOXIN, AND THEY ARE MOVING FAST AND YOU DON'T GET A SINGLE SPOT, YOU GET A DIFFUSE LOW-LEVEL BACKGROUND. WHEN THEY BIND TO THE CHANNELS YOU SEE BRIGHT FLASHING OF FLUORESCENCE. THE MOVIE SHOWS MANY LOCALIZATIONS AS TIME GOES ON, COMING FROM SAMPLING OF THE POSITIONS OF THE BINDING SITES, IN THIS CASE CHANNELS ON THE CELL AND WE USED THAT SCHEME -- BY THE WAY YOU CAN SEE HOW TO CONTROL CONCENTRATION, YOU HAVE DIFFERENT CONCENTRATIONS OF MOLECULES IN SOLUTION AND SAY HOW MANY MOLECULES ARE APPEARING AT WHAT DENSITY AT ANY GIVEN TIME, EXPERIMENTER CONTROL. WE APPLIED THIS TO SODIUM CHANNELS ON PC-12 CELLS DIFFERENTIATED INTO NEURONAL LIKE BEHAVIOR, AND HERE IS AN AX ON. WE IS PRODUCE A TIME-DEPENDENT MOVIE OF THE BEHAVIOR. THESE ARE AVERAGED ON THE 500 MILLISECOND TIME SCALE, SO THIS IS NOT INTENDED TO BE EXTREMELY HIGH TIME RESOLUTION OR INTERMEDIATE, 500 MILLISECOND TIME INTERVALS. YOU CAN SEE AS TIME GOES ON NEURITIC GROWTH THAT DECAY AND DISAPPEAR, THIS IS ALL OF COURSE STRUCTURES BEYOND OPTICAL DIFFRACTION LIMIT. SO THAT'S TO SHOW YOU ANOTHER WAY OF DOING IT. NOW, I'D LIKE TO MOVE FORWARD TO THE CASES THAT INVOLVE LOOKING AT OTHER SORTS OF INTERESTING STRUCTURES INSIDE CELLS, SO I'LL SKIP ONE SLIDE HERE, BECAUSE OF TIME. AND TELL YOU ABOUT HUNTINGTIN AGGREGATES IN CELLS WHERE YOU MIGHT WANT TO SEE WITH HIGH RESOLUTION WHAT THE AGGREGATE STRUCTURES LOOK LIKE INSIDE CELLS. SO WE KNOW THAT HUNTINGTIN PROTEIN CAUSES HUNTINGTON'S DISEASE, THIS IS THE WORK OF STEPHAN SAHL IN MY LAB. THERE'S A HUGE AGGREGATE THAT PRODUCES A GREAT DEAL OF FLUORESCENCE, SO WE FIRST PHOTOBLEACHED THAT INCLUSION BODY AND LOOKED INSIDE THE CELL AFTER BLEACHING THE INCLUSION BODY, YOU SEE THE TINY STRUCTURES, SCATTERED AROUND, THAT ARE AGGREGATES OF SOME PARTICULAR SHAPE THAT'S HARD TO SEE IN THE DIFFRACTION LIMITED IMAGES. BUT WITH SUPER-RESOLUTION IMAGES BEING PRODUCED HERE BY FUSIONS, INVERTED CONTRAST TOO, YOU SEE THE SLENDER FIBRIL-LIKE STRUCTURES, ALSO PRESENT IN THE AXONAL PROCESSES, WHEN YOU LOOK AT THE DIFFRACTION LIMIT YOU SEE A BLUR BUT WITH SUPER-RESOLUTION YOU SEE GREATER DETAIL. SO WE THINK THERE'S SOME APPLICATIONS OF THESE IDEAS TO A DIFFERENT KIND OF NEURONAL AND DEGENERATIVE PROCESSES. NOW, WHAT ABOUT THREE DIMENSIONS, LOOKING AT SINGLE MOLECULES NOT JUST IN X AND Y BUT ALSO IN Z? AND WE'RE PARTICULARLY INTERESTED IN MY LAB IN -- NOT JUST PRECISION BUT ALSO ACCURACY OF THE SINGLE MOLECULE LOCALIZATION. I WANT TO TELL YOU ONE OF THE MANY WAYS THAT PEOPLE ARE USING TO GET THREE DIMENSIONS BUT WITH OUR PARTICULAR FLAVOR. TURNS OUT THAT YOU CAN UNDERSTAND THIS BY REALIZING THE CONVENTIONAL MICROSCOPE USES WHAT I'LL CALL A CLEAR PUPIL. HERE IS A SAMPLE, HERE IS LIGHT COMING, COLLECTED BY AN OBJECTIVE, IT GOES THROUGH THE MICROSCOPE AND THEN TUBE LENS FORMS THE FINAL IMAGE. INTERMEDIATE IS THE PUPIL PLANE, THIS IS TYPICALLY EMPTY, PRODUCING A POINT SPREAD FUNCTION, IMAGE OF A SINGLE MOLECULE. AS YOU MOVE THE MOLECULE UP AND DOWN IN Z, ALONG THE AXIS NOW, YOU SEE THIS PARTICULAR IMAGE IS ONLY BRIGHT AND TIGHT WHEN IT'S IN FOCUS BUT QUICKLY IT DEFOCUSES AND DIDN'T GIVE YOU NEARLY AS MUCH INFORMATION ABOUT WHERE THE MOLECULE IS. WE'VE CHANGED THAT MICROSCOPE BY NOT HAVING A CLEAR PUPIL TO GET AWAY FROM THESE PROBLEMS, WE PUT IN THE PUPIL PLANE, WHAT'S CALLED A PHASE PATTERN OR PHASE PLATE, PLACED AT THIS LOCATION, NOW A SINGLE MOLECULE HAS AN IMAGE THAT HAS TWO SPOTS. AND IF I MOVE THE SPOTS, MOVE THE MOLECULE UP AND DOWN IN THE Z AXIS, THESE SPOTS REVOLVE AROUND ONE ANOTHER. SO YOU SEE THE IDEA HERE, IT'S THIS ANGLE BETWEEN THE TWO SPOTS THAT WILL ENCODE AND TELL ME THAT THE Z IS FOR THE POSITION OF THE MOLECULE. THAT'S HOW THIS WORKS. IT'S A MODIFIED POINT SPREAD FUNCTION THAT GIVES YOU INFORMATION, NOT ONLY ABOUT X AND Y, THE MID-POINT BETWEEN THE TWO, BUT ALSO Z. AND THESE THINGS ARE NOT HARD TO IMPLEMENT. YOU CAN EASILY IMPLEMENT THIS PARTICULAR DOUBLE HELIX, WE CALL IT, POINT SPREAD FUNCTION, A DOUBLE HELIX IF YOU THINK ABOUT IT IN THREE DIMENSIONS IT'S LIKE A DOUBLE HELIX, ADD IT TO A CONVENTIONAL MICROSCOPE, JUST ADD OPTICS, 4F OPTICAL PROCESSING, NOTHING BUT A BOX WITH TWO LENSES IN IT, THE PHASE MASK IS PLACED IN BETWEEN. I'LL SKIP THE MATH. LENSES HAVE THE BEAUTIFUL PROPERTY THAT THEY COMPLETE A TRANSFORM OF AN IMAGE, THIS IS WHERE THE TRANSFORM WILL BE. THE DOUBLE HELIX MICROSCOPE IS INTERESTING. NOW HERE ARE SOME REAL DATA. THE SINGLE MOLECULES LOOK LIKE PAIRS AND HERE IS THE NEXT IMAGE. THE ANGLES ARE SLIGHTLY DIFFERENT, AND IF WE NOW TAKE THAT DATA AND ENCODE THE Z INTO COLOR YOU GET A THREE DIMENSIONAL IMAGE OF WHAT'S GOING ON INSIDE THE CELL. NOW, I WANT TO EMPHASIZE THAT DOUBLE HELIX WAS IN FOCUS OVER TWO MICRONS, OVER A LARGE RANGE, MUCH LARGER THAN THE CONVENTIONAL MICROSCOPE. SO YOU DON'T HAVE TO DO ANY SCANNING HERE. ALL OF THIS INFORMATION COMES OUT ALL AT THE SAME TIME. X, Y AND Z. SO IT'S AN EXCITING GROWTH AREA THAT WE'RE INVOLVED IN, OTHER PEOPLE ARE INVOLVED IN AND SO ON. YOU CAN DO THIS, THERE'S JUST SOME COMPARISONS, BUT YOU CAN DO THIS WITH DIFFERENT COLORS, DIFFERENT PATHS OUTSIDE THE MICROSCOPE WITH DIFFERENT PHASE MASKS, NOW YOU SEE DIFFERENT PROTEINS BEING IMAGED. IN THIS CASE POP-Z IS SHOWN HERE, AND CRE-S IS SHOWN HERE, THREE DIMENSIONAL INFORMATION. LET ME SAY ALSO THAT BESIDES HAVING A LOT OF EXCITING THINGS GOING ON THESE DAYS, TO LOOK AT SINGLE MOLECULES, AND TO COME UP WITH INTERESTING NEW MICROSCOPIC WAYS OF ENCODING THE LIGHT FROM THE MOLECULES WITH NEW POINT SPREAD FUNCTIONS, THERE'S STILL A NEED FOR BETTER MOLECULES FOR ALL OF THESE EXPERIMENTS. MOLECULES THAT WOULD BE PHOTOCONTROLLABLE, THAT WOULD GIVE YOU LOTS OF PHOTONS, SMALLER THAN GFP AND SO ON. ONE QUICK EXAMPLE, MARISA IN MY LAB IS WORKING WITH SYNTHETIC CHEMISTS WITH SUPPORT FROM A COMMON FUND PROGRAM TO WORK ON PHOTOSWITCHABLE RHODAMINE SPIROLACTAMS. YOU CANTIC MA -- YOU CAN MAKE IT A FLUORESCENT MOLECULE, SHIFTING TO BLUE RATHER THAN ULTRAVIOLET. NORMALLY THEY SWITCH IN ULTRAVIOLET BUT BLUE IS SAFER FOR THE CELLS. FUNCTIONALITY BINDS WITH SURFACE LYSINES, HERE ARE THE SURFACES, LIT UP IN SUPER-RESOLUTION USING THIS MOLECULE, AND YOU SEE THAT THE STALKS WHICH ARE FAR BELOW THE DIFFRACTION LIMIT ARE EASILY OBSERVABLE, YOU CAN EVEN DO THIS IN 3D USING THE DOUBLE HELIX METHODS AND SEE THAT THE SAMPLING ON THE CELL VERY HIGH WITH THIS PARTICULAR METHOD. THERE ARE ALSO NOT ONLY NEW MOLECULES THAT NEED TO BE DEVELOPED WHICH I WANT TO ENCOURAGE, BUT ALSO NEW OPTICAL IDEAS. MY STUDENTS, I REALLY CAN'T STOP THEM, THEY COME UP WITH ALL KINDS OF COOL THINGS, SO HERE IS SOME NEW PHASE MASKS. I TALKED ABOUT THE DOUBLE HELIX. HERE IS ONE CALLED THE BISECTED MASK THAT MAKES IMAGES LIKE THIS AND ALSO GIVES YOU X, Y AND Z, THAT'S FROM ADAM. FROM YOAV A YIN-YANG OR SADDLE POINT. I'VE BEEN TALKING FOR ABOUT 54 MINUTES, I THINK I'M GETTING NEAR THE END OF MY TIME. I WANTED TO TELL YOU ONE MORE COOL STORY ABOUT THE PRIMARY CILIUM, NOT A UP -- NOT A SUPER-RESOLUTION STORY. THE PRIMARY CILIUM, ONE QUICK STATEMENT, IF WE OBSERVE THE MOTION OF SMOOTHANT, AN IMPORTANT SIGNALING PROTEIN IN CELLS THAT ARE RECOGNIZING EITHER THE HEDGE HOG PATHWAY OR OTHER DEVELOPMENTAL SIGNALS, IF YOU OBSERVE SINGLE SMOOTHEN, THEY MOVE ALL OVER THE CILIUM AND BIND TO SITES, YOU SEE IT MOVE AND IT STOPS, MOVES, STOPS, MOVES, STOPS. AND SO WE CAN SEE THERE ARE BINDING SITES OF UNKNOWN CHARACTERS BEING EXPLORED. THAT'S THE BASIC PART OF THE WHOLE STORY, THIS EVEN OCCURS WHEN THE PATHWAY IS NOT EVEN ACTIVATED. AND PATHWAY ACTIVATION CHANGES THE BINDING AFFINITY OF THESE MOLECULES AT THE BASE. SO THERE'S THERE'S A WHOLE STORY ABOUT INTERESTING SINGLE MOLECULE STUFF BUT I KNOW I HAVE A TIME LIMIT SO I'M GOING TO SKIP IT AND GIVE YOU FINAL COMMENTS ABOUT THE WHOLE AREA, THE IMPACT OF SINGLE MOLECULE SPECTROSCOPY, IT SPANS MANY FIELDS, A WONDERFUL INTERDISCIPLINARY. IT'S AN ACTIVITY OF THE SCIENTIFIC COMMUNITY THAT'S BEEN EXCITING TO BE INVOLVED IN AND I THINK THERE'S STILL A GREAT DEAL MORE TO DO. AND SO I NOW WANT TO START SORT OF THANKING PEOPLE FOR ALL OF THIS WORK THAT'S BEEN GOING ON. WELL, HERE IS THE MOERNER LAB ALUMNI. YOU CAN SEE IT ON THE NOBEL LECTURE SITE. MY CURRENT GROUP OF COURSE IS THE ONE THAT'S GOING TO TAKE THE BRUNT OF MY TOUGH TRAVEL THIS YEAR, HERE IS THE GUACAMOLE TEAM. YOU CAN LEAD THESE AT THE NOBEL LECTURE SITES, THE INSTITUTIONS AND FAMILIES I THANK AS WELL, EARLY WORK SUPPORTED BY THE ONR AT IBM, NSF AND NIH AND DOE, AND OF COURSE THANK YOU VERY MUCH FOR LISTENING TODAY. [APPLAUSE] >> THANKS, W.E., FOR A WONDERFUL ROMP THROUGH EXCITING TECHNOLOGY AND ITS APPLICATIONS. WE HAVE TIME FOR QUESTIONS, ALTHOUGH THE HOUR IS UP, THERE'S I'M SURE LOTS OF PEOPLE WHO WANT TO CONTINUE THE CONVERSATION SO THOSE OF YOU WHO ARE ABLE TO STAY, PLEASE DO SO. THERE ARE MICROPHONES IN THE AISLES, AND BECAUSE WE'RE WEBCASTING IT WOULD BE GREAT IF YOU HAVE A QUESTION IF YOU WOULD GO TO THE MICROPHONE SO THAT PEOPLE LISTENING ON THE WEB CAN HEAR. IT LOOKS LIKE WE'RE GETTING UNDERWAY, YES. >> FABULOUS PRESENTATION. AND JUST WONDERING IF THIS BEAUTIFUL TECHNIQUE OF MICROSCOPY HAS BEEN USED IN MISFOLDED PROTEINS IN ALZHEIMER'S OR PARKINSON'S DISEASE? >> YES. OH, WELL, DEFINITELY. YOU'RE ASKING HAS IT BEEN USED. YES, A NUMBER OF CASES PEOPLE HAVE BEEN USING DIFFERENT SINGLE MOLECULE METHODS TO LOOK AT PROTEINS THAT ARE INVOLVED IN MANY NEURODEGENERATIVE DISEASES, SOMETIMES FRET LIKE, IT'S AN EXCITING AREA. >> THANK YOU. >> W.E., TELL ME WHERE YOU ARE IN TERMS OF HAVING THE ABILITY TO LOOK SIMULTANEOUSLY AT MULTIPLE STRUCTURES, THOSE OF US WHO DREAM ABOUT REALLY UNDERSTANDING HOW BIOLOGY WORKS WOULD LOVE IT IF YOU COULD LABEL TEN THINGS AT ONCE AND WATCH THEM DANCE WITH EACH OTHER, HOW CLOSE ARE YOU TO HAVING THAT KIND OF MULTIPLE DETECTION SYSTEM THAT YOU CAN DO SIMULTANEOUSLY IN LIVING CELLS? >> YEAH, WELL, THAT'S OF COURSE A GREAT CHALLENGE. WE'RE NOT QUITE THERE YET. THERE'S MULTIPLE LABELS AVAILABLE, HOWEVER. THERE'S A NUMBER OF CASES WITH TWO COLORS, THREE COLORS AND FOUR COLORS, THAT SORT OF THING. AT SOME POINT THERE MAY BE SOME ISSUES ABOUT DIVIDING FREQUENCY SPACE, SO BECAUSE AT ROOM TEMPERATURE THE LENS ARE BROAD, NOT NARROW. HOWEVER, YOU CAN STILL BELIEVE IN THE CLEVERNESS OF SCIENTISTS, I WANT TO SAY. I DON'T WANT TO SAY IT CAN'T BE DONE. I DON'T WANT TO SAY THIS WILL NOT WORK OR WHATEVER. BECAUSE YOU CAN ALSO DISTINGUISH MOLECULES BY LIFETIME, YOU CAN HAVE MOLECULES WITH DIFFERENT EXCITED STATE MOLECULES BUT SIMILAR OTHER PROPERTIES AND THERE WAS ONE OF THOSE THINGS THAT WAS ACTUALLY EXPLORED FOR EARLY SUPER-RESOLUTION, AND YOU COULD ADD THAT TO THE MULTIPLE COLORS, OR YOU CAN ALSO MEASURE ORIENTATIONS OF THE MOLECULES AND USE THAT, ORIENTATIONAL FLEXIBILITY WHICH WE CAN SENSE BY LOOKING AT SINGLE MOLECULE IMAGE, HOW OF THE MOLECULE IS WIGGLING COULD TELL US DIFFERENCES IN THE MOLECULE. BACK TO THE IDEA ERIC POINTED OUT, WHAT VARIABLES CAN WE COME UP WITH TO DISTINGUISH THOSE MOLECULES. >> WELL, PERHAPS PEOPLE ARE SLIGHTLY INTIMIDATED BY THE SIZE OF THE ROOM. I WOULD ENCOURAGE THOSE WHO WANT TO CONTINUE THE CONVERSATION TO COME DOWN FRONT AFTERWARDS, BUT PLEASE LET'S GIVE ANOTHER ROUND OF APPLAUSE TO OUR NOBEL LAUREATE, W.E. MOERNER. [APPLAUSE] >> THANK YOU.