IT IS MY GREAT PLEASURE TO INTRODUCE HARI SCHROFF, WHO IS TODAY'S NIH DIRECTOR'S LECTURER. I ENJOYED READING HARI'S CV, I'LL SHOW YOU WHY IN A MOMENT. HE WAS AN UNDERGRADUATE AT THE UNIVERSITY OF WASHINGTON, WHERE HE GOT A BACHELOR'S DEGREE IN BIOENGINEERING. HIS Ph.D. WAS IN BIOPHYSICS FROM THE UNIVERSITY OF CALIFORNIA BERKELEY, POSTDOCKED IT ERIC AT JANELIA FARM, CURRENT POSITIONS INCLUDE CHIEF OF HIGH RESOLUTION IN OPTICAL, AND ADVANCED IMAGING AND MICRO SCOPEY, AN EFFORT TO BRING TOGETHER NEW INSTRUMENTATION PHYSICISTS AT THE NIH HAVE BEEN PRODUCING. I WANT TO READ A FEW PROJECTS, UNDERGRADUATE WORKED TOWARDS MEASURING LIFT PRESSURE GENERATED BY A HONEYBEE IN HOVERING FLIGHT, AS Ph.D. RESEARCH WAS ON DEVELOPING SINGLE MOLECULE SENSORS OF MECHANICAL FORCE, BASED ON A FLUORESCENCE READOUT AND BUILD A MICROSCOPE WITH MAGNETIC TWEEZER IN ORDER TO FACILITATE THAT. BUILT THE FIRST FUNCTIONAL F POM WITH ERIC BEDSEN AND JANELIA FARMS AND TECHNIQUES FOR HIGH RESOLUTION OF LIVING CREATURE, WORKING WITH C. ELEGANS. WE DON'T HAVE A TITLE SLIDE, IT HAS SOMETHING DO WITH DECONVOLUTING C. ELEGANS. I'M TWISTING IS. >> THIS IS THE TITLE. >> HIGH SPEED IMAGING BEYOND THE DIFFRACTION LIMIT. >> THANK TO THE PEOPLE COMING AND A SHOUT OUT TO THE AUDIO/VISUAL PEOPLE HELPING ME MY SLIDES. YEAH, IT'S A PLEASURE TO TELL YOU ABOUT THE WORK IN MY LAB. I'M A MICROSCOPIST, I GET UP THINKING ABOUT HOW TO MAKE OPTICAL MICROSCOPES FASTER, BETTER, SHARPER, MORE GENTLE. THE MOTIVATION FOR MY WORK AT THE NIH 6 OR 7 YEARS, BIOLOGY IS ACROSS MULTIPLE ORDERS. IF YOU WANT TO CATCH ACTION YOU HAVE TO BUILD MICROSCOPES TO IMAGE ACROSS MANY LIGHT SCALES AND TEMPORAL SCALES. THINK ABOUT FOCAL ADHESION AND FLOW OF MOLECULES INSIDE A FOCAL ADHESION ON THE ORDERS OF NANOMETERS PER SECOND. FILAMENT POLYMERIZATION, ORDERS ON MAGNITUDE FASTER. SORT OF THE FASTER END OF THE SPECTRUM IN A CELL, MOVEMENT OF MOLECULAR MOTORS OR SIGNAL TRANSDUCTION LIKE CALCIUM WAVES, THE OVERARCHING THEME THAT I'VE BEEN THINKING ABOUT IS HOW TO IMPROVE IMAGING TECHNIQUES FOR IMAGING AT THE FAST END OF THE SPECTRUM. SO EVERYTHING I TELL YOU TODAY IS ANCHORED IN TWO TECHNOLOGY IS FURTHER DEVELOPED AT THE NIH, ONE IS STRUCTURED ILLUMINATION MICROSCOPY, SIM, YOU SHINE SHARPLY PATTERNED LIGHT ON THE SPECIMEN, TYPICALLY RECORD A SERIES OF IMAGES AND PROCESS IMAGES IN ORDER TO RECOVER SHARPER SUPER-RESOLUTION IMAGES. WE DIDN'T INVENT BUT IMPROVED IT, INSTEAD OF A SINGLE CELL HUNDREDS OF MICRONS THICK AND IMPROVING THE SPEED, HUNDRED OF RESOLUTION PER SECOND. UNPUBLISHED WORK APPLIES TO INTERNAL REFLECTION FLUORESCENCE MICROSCOPY AND DEPTH PENETRATION WITH ADAPTIVE OPTICS. THE SECOND CLASS OF TECHNIQUES ARE LIGHT SHEATH MICROSCOPY TECHNIQUES IF YOU WANT TO IMAGE OVER MANY HOURS OR DAYS WITH MINIMAL DOSE. FANTASTIC REALLY IMPORTANT TO KEEP YOUR SAMPLE HEALTHY OR PREVENT PHOTOBLEACHING. OUR CONTRIBUTION HAS BEEN IMPROVING THE SPATIAL RESOLUTION TO SORT OF SUBCELLULAR SPATIAL RESOLUTION WHILE IMPROVING SPEED AND COLLECTION EFFICIENCY. A PARTICULAR APPLICATION NEAR AND DEAR TO MY HEART IS ATLAS BUILDING, STEPS TO BUILDING IN C C. ELEGANS. THIS IS A GORGEOUS MOVIE TAKEN NOT FROM MY LAB BUT FROM THE INVENTOR OF THE TECHNIQUE WHO PASSED AWAY FROM BRAIN CANCER A FEW YEARS AGO. MITOCHONDRIA ARE CRAWLING AROUND A HeLa CELL. IT'S GOOD ENOUGH TO SEE SUBSTRUCTURE IN THE MITOCHONDRIA. THE MOVIE WAS RECORDED AT A FAST ENOUGH SPEED THAT YOU CAN RESOLVE MITOCHONDRIA MOVING AROUND WITHOUT TOO MUCH MOTION BLUR, AND WE'RE ALSO LOOKING AT VOLUMES OVER TIME. THIS IS A FOUR DIMENSIONAL IMAGING SERIES AT SUPER-RESOLUTION. THE OTHER REMARKABLE THING, OPTICAL SECTIONING, LOOK AT THE MITOCHONDRIA, THEY APPEAR CRISP ON A DARK BACKGROUND. IF YOU LOOK AT 3D SAMPLE, THE OUT OF FOCUS FLIGHT IS ONE OF THE FIRST PROBLEMS TO DEAL WITH. WE DON'T HAVE THAT PROBLEM IN THIS MOVIE. TURNS OUT THE REASON FOR THAT IS THAT THE MATH ASSOCIATED WITH THIS MOVIE WITH GENERATING THE MOVIE REJECTS OUT OF FOCUS LIGHT WELL FOR SIM SAMPLES. FOR THE CELL HERE, WE SEE VERY LITTLE OUT OF FOCUS LIGHT. WHEN STUDYING THIS TECHNIQUE AT JANELIA FARM, ALMOST ALL APPLICATIONS WERE LIMITED TO SINGLE CELLS, THE REASON FOR THAT IS THAT MATH ALONE CAN'T GET RID OF THE NOISE, SO IF YOUR SAMPLE IS DENSELY LABELED YOU HAVE TO DO MORE THAN COMPUTATION IN ORDER TO GET RID OF NOISE ASSOCIATED WITH OUT-OF-FOCUS LIGHT. ON THE OTHER HAND MOST OF US HEARD OF CONFOCAL MICROSCOPES, YOU REJECT OUT-OF-FOCUS LIGHT WITH A PIN HOLE. TAKE A LASER PEOPLE, FORM A DIFFRACTION LIMITED SPOT, ENABLES SPEECH OF A CONFOCAL IS A PIN HOLE. PIN HOLE PREVENTS OUT OF FOCUS LIGHT FROM REACHING YOUR DETECTOR. IF YOU GET RID OF THE OUT-OF-FOCUS THERE'S NO NOISE WITH THAT OUT-OF-FOCUS, THAT'S WHAT I MEAN WHEN I SAY REJECT OUT-OF-FOCUS LIGHT PHYSICALLY. WHETHER OR NOT YOU COULD BUILD A CONFOCAL MICROSCOPE WHILE RETAINING THE BENEFIT OF THE PIN HOLE. LIKE MOST IDEAS, I WASN'T THE FIRST TO THINK OF THIS. GO BACK 30 YEARS, COLIN SHEPHERD PUBLISHED A THEORETICAL PAPER IN AN OUT OF PRINT JOURNAL CALLED "OPTIC" WHERE HE DESCRIBED THIS IN THEORY. IF YOU LOOK AT THE OFF-AC AXIS RAY, RECORD WITH A CAMERA, ALL THE INFORMATION IS THERE TO INCREASE RESOLUTION OF YOUR MICROSCOPE BY A FACTOR OF TWO. THE MATH IS ALL IN THIS PAPER, LARGELY FORGOTTEN BECAUSE COLIN SHEPHERD NEVER BUILD A MICROSCOPE LIKE THIS. THE PAPER I ACTUALLY READ FIRST CAME OUT DECADES LATER IN PRL. IN THE PHYSICS JOURNAL A GROUP BUILT A MICROSCOPE CALLED THE IMAGE SCANNER MICROSCOPE, ISM. THE HARDWARE IS A CONFOCAL MICROSCOPE WITH A CAMERA, WORKING OUT HOW TO OPERATE SYSTEM TO DOUBLE RESOLUTION OF CONFOCAL MICROSCOPE. HE SCANNED POINT BY POINT THROUGH THE SAMPLE, RECORDED ONE IMAGE FOR EVERY LOCATION OF ILLUMINATION SPOT AND DID THE MATH. I WAS STRUCK BY THE ELEGANCE. ONE PROBLEM IS THE FLOW SPEED. TAKE ONE IMAGE FOR EVERY POSITION, IT TAKES 20 OR 30 MINUTES TO COVER A CELL. THAT'S AN UNACCEPTABLE TRAIT. NOBODY WANTS TO WAIT HALF AN HOUR FOR A DOUBLING. IF YOU WANT TO GO FASTER, PARALLELIZE. INSTEAD OF USING ONE SPOT, MAKE 10,000 SPOTS. WE BUILT A MICROSCOPE, MULTI-FOCAL SIM, MSIM, THE RAW DATA IS ON THE RIGHT SIDE, A SERIES OF DIFFRACTION SPOT, AN IMAGE FOR A THOUSAND SPOTS, THROW AWAY THE LIGHT, THE PINHOLDING, SCALE AND SHRINK THE SPOTS WHERE THE ENHANCEMENT HAPPENS. AS IMAGE FORMATION OCCURS, COLLECT AND ADD THEM IN SOFTWARE, DONE RELATIVELY FAST, A FEW FRAMES PER SECOND TO GET SUPER-RESOLUTION IMAGES AT A COUPLE FRAMES PER SECOND. THE BENEFIT IS THE PINHOLING. THIS MEANS YOU CAN RECORD IMAGES, FOR EXAMPLE IN A ZEBRAFISH EMBRYO, THIS IS THE ROTATING MOVIE, AN EXAMPLE OF MICROTUBULES, PROVIDED BY NICHD, AND THE IMPORTANT PART IS YOU CAN GO A FACTOR OF 10 DEEPER THAN CONVENTIONAL SIM. THE KIND OF DOSE WE APPLY IS SIMILAR TO A SPINNING DISC, YOU CAN DO MULTIPLE VOLUMES, TAKE FOUR DIMENSIONAL DATA LIKE THE OTHER MOVIE, A GROUP OF CELLS FROM THE POSTERIOR LATERAL LINE PRIMORDIUM. IF I PLAY THE MOVIE AGAIN, YOU CAN SEE RELATIVELY LITTLE BLEACHING. THE MICROSCOPE CAN BE MADE TO TAKE VOLUMETRIC TIME LAPSE SAMPLES. ONE LIMITATION IS THAT YOU NEED MANY RAW IMAGES FOR A SINGLE SUPER-RESOLUTION IMAGE. IN THIS MOVIE I NEED 100 RAW IMAGES FOR A SINGLE SUPER-RESOLUTION IMAGE, SLOWING ME DOWN. I'M SLOWED DOWN BY A FACTOR OF 100 COMPARED TO DIFFRACTION LIMITED MICROSCOPE. THE PRACTICAL ADVANTAGE IS MORE THAN THE COLLECTION OF THE IMAGES. YOU HAVE TO STORE AND PROCESS IMAGES. THIS ADDS ADDITIONAL TIME. CAN YOU BUILD A MICROSCOPE THAT DOES ALL OF THIS IN THE OPTICS? MY POSTDOC AT THE TIME, ANDY YORK, SAW EVERY STEP HAD AN OPTICAL ANALOG. WHAT DO YOU NEED TO DO? YOU NEED TO DELIVER STRUCTURED ILLUMINATION WITH A MICRORAY SIMILAR TO WHAT IS FOUND IN YOUR SPINNING DISC MICROSCOPE, IT TAKES A BEAM OF LIGHT AND MAKES A SERIES OF DIFFRACTION. YOU NEED SHIFT OR SCALE THE LIGHT WHERE THE SUPER RESOLUTION IS COMING FROM, THAT'S OUR INNOVATION, DONE WITH ANOTHER MICROLENS ARRAY IN THE RIGHT CONFIGURATION. FINALLY ADD THE FLUORESCENT EMISSION AS YOU SCAN EXCITATION SPOTS WITH A TWO-SIDED GALVANOMETRIC MIRROR, YOU CAN BUILD THE INSTANT SIM, OCCURRING AT THE SPEED OF LIGHT, SUPER-RESOLUTION IS REALLY LIMITED ONLY BY HOW BRIGHT YOUR SAMPLE IS AND HOW FAST THE CAMERA CAN INTEGRATE. THIS CAN BE AT TENS OF HERTZ OR HUNDREDS OF HERTZ, HUNDREDS OF FRAMES PER SECOND. THE ADVANTAGE OF THIS MICROSCOPE, IN ANY CASE THE POINT IS YOU CAN RECORD MULTIPLE PROTEIN DISTRIBUTIONS AT A FRACTION OF VOLUME PER SECOND AT SPATIAL RESOLUTION AT 150 NANOMETERS, AN ORDER OF MAGNITUDE FASTER THAN CONVENTIONAL SIM, IN A FRACTION OF TIME IT WOULD OTHERWISE TAKE. YOU'RE LIMITED BY HOW FAST THE CAMERA CAN TAKE IMAGES AND HOW BRIGHT THE SAMPLE IS. LIKE THE MULTI-FOCAL SIM WE CAN DO THIS ON THE COVER SLIP AND TENS OF MICRONS AWAY FROM THE COVER SLIP. WE CAN SEE BLOOD CELLS WHIZZING BY THE VAIN IN A LIVE ZEBRAFISH EMBRYO. TEMPORAL RESOLUTION IS BETTER THAN VIDEO. WE CAN CATCH THIS AS IT GOES BY THE VEIN. YOU CAN SEE MICROTUBULE BUNDLES, IF BRIGHTLY LABEL THE AT HUNDREDS OF FRAMES HER SECOND. ENDOPLASTIC RETICULUM, CAPTURING GROWTH AND REMODELING OF INDIVIDUAL E.R. TUBULES. THIS TECHNOLOGY IS COMMERCIALLY AVAILABLE FROM VISIT-TECH, IN BUILDING 10, GET IN TOUCH WITH ME AND I CAN PUT YOU IN TOUCH WITH THE PERSON THAT OPERATES THAT SCOPE. I WANT TO TALK ABOUT WORK FROM MY LAB, APPLYING TO MODALITIES. ONE OF THE ENABLING TOOLS IN CELL BIOLOGY IS TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY. IF YOU'RE A CELL BIOLOGIST AND WANT TO LOOK AT BIOLOGY AT THE COVER SLIP SURFACE, IF YOU BRING IN THE LIGHT AT A SHARP ANGLE, THE ANGLE IS STEEP ENOUGH, TOTALLY INTERNALLY REFLECTING, YOU SEE FLUORESCENCE FROM MOLECULES WITHIN A FEW HUNDRED NANOMETERS OF COVERS IN THE EVANESCENT WAVE. IF YOU LOOK AT FOR EXAMPLE THIS ACTIN DISTRIBUTION YOU SEE ALL OF THE OUT OF FOCUS HAZE COMING FROM ELSEWHERE IN THE SAMPLE IN TOTAL INTERNAL REFLECTION YOU ONLY SEE REFLECTION IN A FEW HUNDRED NANOMETERS. WE'VE ADAPTED TO GET HIGH CONTRAST, HIGH SPEED AND HIGH RESOLUTION IMAGES OF PROTEIN DISTRIBUTION CLOSE TO THE SURFACE. THIS IS JUST AN UNPUBLISHED MOVIE OF THE PROTEIN DISTRIBUTION RAS, A SMALL GTPase. CONTRAST IS GOOD ENOUGH TO SEE INDIVIDUAL MICRODOMAINS OF RAS IN THE INTERIOR. THIS IS NOT RECORDED BEFORE. OUR COLLEAGUES AT NCI ARE INTERESTED BY RAS IS IMPLICATED IN SOMETHING LIKE 30% OF CANCER. SO WE'RE INVESTIGATING WHAT DIFFERENT ISOFORMS OF RAS LOOK LIKE WITH THIS MICROSCOPE. THE SECOND EXAMPLE WAS TAKEN BY OUR GRADUATE STUDENT IVAN, AC ACTIVE IN A JURKAT T CELL, TRYING TO SETTLE ON AN ANTIBODY COATED COVER SLIP, YOU ONLY SEE IT IN A CLOSE PROXIMITY TO THE COVER SLIP. I LIKE THIS MOVIE BECAUSE IT ILLUSTRATES WE'RE IN TIRF. THE MOVIES WERE RECORDED FAST ENOUGH TO CAPTURE BIOLOGY. WE CAN GO FAST. A FINAL EXAMPLE IN THIS MICROSCOPE, WE'RE LOOKING AT A MOLECULE CALLED RAB 11 TAGGED WITH GFP AT 37 DEGREES, THIS IS FAST, MICRONS PER SECOND AT THE BASE OF THE CELL. WE'RE RECORDING AT A HUNDRED FRAMES PER SECOND, MORE THAN ENOUGH TO TRACK THE INDIVIDUAL SORT OF MICROMOTOR-DRIVEN TRANSPORT IN THE CELL. IF A SAMPLE CAN BE BRIGHT ENOUGH WE CAN IMAGE TEN FASTER THAN CONVENTIONAL SIM. IF YOU'RE BLURRED IN CONVENTIONAL WE MIGHT HAVE SOMETHING TO OFFER YOU. NO MICROSCOPE TECHNIQUE IS PERFECT. ONE LIMITATION. INSTANT SIM IS IT'S A LINEAR MICROSCOPE, ONE PHOTON ILLUMINATION. IF YOU LOOK AT SCATTERING SAMPLE LIKE C. ELEGANS EMBRYO, IT'S DIFFICULT TO MAINTAIN RESOLUTION MORE THAN HALFWAY THROUGH THE SAMPLE THICKNESS. WE'RE LOOKING AT GFP LABEL HISTONES, THEY ARE SHARPLY RESOLVED BUT FURTHER IN WE WERE NOT GETTING SUPER-RESOLUTION, MUCH LESS DIFFRACTION. USE LONGER WAVES TO BEAT SCATTERING, WE BUILT A TWO-PHOTON INSTANT SIM. IF WE LOOK, NOW WE GET MUCH CLEARER IMAGES ALL THE WAY THROUGH THE VOLUME OF THE ANIMAL. PRICE WE PAY OVER HERE IS DROP-OFF IN SPEED SO IMAGES WITH TWO-PHOTON EXCITATION ONE IMAGE PER SECOND, ONES ON THE OTHER MOVIE MILLISECONDS, WE LOSE SPEED WILL YOU SLOW ENOUGH TO INCREASE CLARITY IN TWO-PHOTON INSTANT SIM. WE'VE BEEN ABLE TO PUSH THIS TO 100 MICRONS FROM THE COVER SLIP. SEMI TRANSPARENT ORGANISM LIKE ZEBRAFISH WE CAN LOOK AT MICROTUBULES AT SUPER-RESOLUTION, HERE BUNDLES DEFINE RATIO ORGANIZATION OF DEVELOPING LENS IN ZEBRAFISH EMBRYO. THIS WORKS WELL IN SEMI TRANSPARENT SAMPLE. NO MICROSCOPE IS EVER GOOD ENOUGH. ONE PUBLISHED STORY I WANT TO TELL BUT IS TRYING TO VARIATIONS IN REFRACTIVE INFECTION IN THE ATMOSPHERE SMEAR OUT THE PHOTONS -- [ NO AUDIO ] THE ACTIN IS IMMUNOLABELED IN A COLLAGEN GEL, WITH A DIFFERENT ENOUGH REFRACTIVE INDEX THAN WATER, IF YOU GO 150 MICRONS INTO THE GEL, THE REFRACTIVE INDEX VARIATION IS ENOUGH TO SMEAR THE IMAGE. . LOOK AT THE TWO-PHOTON IMAGE IT'S NOT A BAD IMAGE BUT AFTER DECONVOLUTION WE RECOVER WITH THE TECHNIQUE. UNFORTUNATELY THIS MOVIE WON'T PLAY BUT I WAS GOING TO SHOW YOU A PROJECTION SERIES. THE POINT IS EVEN IN THE Z DIMENSION YOU SEE THIS ABERRATION MANIFESTING. IN Z IN THE AXIAL DIRECTION IT'S OFTEN MUCH WORSE. SO IF YOU LOOK AT A MORE CHALLENGING SAMPLE LIKE DROSOPHILA BRAIN TISSUE THIS IS WORSE. LOOKING AT ACTIN IN FIXED DROSOPHILA, YOU CAN LATERALLY AND AXIALLY THE IMAGE IS MESSY. WE CAN SHARPEN THAT, AFTER DECONVOLUTION GET RESOLUTION POSSIBLE BY MICROSCOPE ENABLING IN CERTAIN BIOLOGICAL CONTEXT AND WORKS IN LIVE SPECIMENS, EXAMPLES ARE FIXED. LOOK AT THE ZEBRAFISH EYEBALL AND COMPARE ADAPTIVE OPTICS PLUS DECONVOLUTION YOU RECOVER SIGNAL TO NOISE, PHOTONS ARE MORE CORRECTLY ASSIGNED TO LOCATION. THE MICROSCOPE IS GENTLE ENOUGH WE'VE DONE SOME LIVE IMAGING AS WELL. IF WE LOOK AT C. ELEGANS HISTONES OVER AN HOUR IN THE TWO PHOTON MICROSCOPE WE COLLECT 60 VOLUME WITHOUT PERTURBING DEVELOPMENT. BEFORE GET INTO LIGHT SHEET MICROSCOPY I WANT TO PLACE THIS IN CONTEXT. THERE'S NO OMNISCIENT MICROSCOPE. IF YOU WANT RESOLUTION LESS THAN 100 NANOMETERS YOU'RE LIMITED TO TECHNIQUES THAT ONE THE NOBEL PRIZE LIKE LOCALIZATION MICROSCOPY INSTEAD, LIMITED BECAUSE YOU PAY A HEFTY PRIZE WHEN YOU USE TECHNIQUES IN TEMPORAL RESOLUTION AND DOSE. IT'S A PIPE DREAM TO TAKE MULTIPLE VOLUMES OVER TIME IN A LOCALIZATION MICROSCOPE, THIS WILL NOT CHANGE UNLESS DYES GET BETTER. IF YOU CAN TOLERATE 100 AND 200 YOU SHOULD USE STRUCTURED ELIMINATION, NO TRADEOFF IN SPEED, HUNDREDS OF MICRONS FROM THE COVER SLIP AND YOU CAN TAKE MULTIPLE VOLUMES OVER TIME. MAYBE YOUR PROBLEM DEMANDS OPTICAL SECTIONING OVER MANY TIME POINTS. THIS IS WHERE LIGHT SHEET MICROSCOPES COME INTO THEIR OWN BECAUSE THEY ARE BY FAR THE MOST GENTLE OPTICAL MICROSCOPES AROUND TODAY. I WANT TO TELL YOU ABOUT OUR IMPLEMENTATION OF LIGHT HEAT MICROSCOPY. BEFORE I DO THAT, THE BIOLOGY THAT MOTIVATED US, ONE OF THE GOALS OF THE LAB IS TO TRY AND UNDERSTAND NEURAL DEVELOPMENT AND I'D LIKE TO UNDERSTAND SUBCELLULAR RESOLUTION IN TOTO IN A HUMAN BRAIN. IT'S UNLIKELY IN OUR LIFETIMES WE'LL BE ABLE TO IMAGE A HUMAN BRAIN IN VIVO. WE'RE FORCED TO USE MODEL ORGANISMS. OUR CHOICE IS THE WORM C. ELEGANS FOR MANY REASONS. IT HAS BEHAVIORS, A SIMPLE TRACTABLE NERVOUS SYSTEM, 5,000 CHEMICAL SYNAPSES BUT IT'S TRANSPARENT AND YOU CAN PUT GNP IN THE ANIMAL, IT'S STEREOTYPED. ONE ADVANTAGE OF THIS ORGANISM OVER PERHAPS OTHERS IS A WEALTH OF SYSTEMS BIOLOGY ALREADY KNOWN. WE HAVE A LINEAGE. WE KNOW WHAT THE CELLS ARE, WHERE THEY CAME FROM, WHAT THEY ARE GOING TO BECOME. WE KNOW THE FAMILY TREE OF ALL THE CELLS. WE KNOW THE CONNECTOMES, PATTERNS, WHERE MANY SYNAPSES ARE. WE KNOW THE DNA SEQUENCE, OF COURSE. MORE RECENTLY WE HAVE MAPS OF PROTEIN EXPRESSION SO YOU CAN ASK WHEN IS THE GENE EXPRESS AND HOW DOES THAT PROFILE CHANGE OVER TIME. EVEN MORE RECENTLY WE HAVE BRAIN ACTIVITY MAPS FROM COLLECTIONS OF NEURONS FREELY BEHAVING ADULTS AS THEY EXECUTE SOME TASKS. WE KNOW ALL OF THIS, WHICH IS A GREAT HELP. BUT DON'T KNOW HOW THE BRAIN DEVELOPS AT SUBCELLULAR RESOLUTION. THAT'S THE LONG-TERM GOAL, MEDIUM-TERM GOAL. WE WANT TO KNOW WHERE ALL 222 EMBRYONIC NEURONS ARE, EVERY POINT IN DEVELOPMENT AS GOOD A SPATIAL RESOLUTION IN A LIVE ANIMAL. C. ELEGANS IS STEREOTYPED, ONE CAN ATTACK WITH SPARSE SUBSETS OF GFP AND BUILD A COMPOSITE WHOLE. THIS MIGHT SEEM LIKE A BASIC STEP, KNOWING THE ANATOMY, MAPPING IN SPACE AND TIME, I WOULD ARGUE IT'S AN IMPORTANT FIRST STEP. IT'S HARD TO MAP ON GENE EXPRESSION AND FUNCTIONAL IMAGING DATA. THIS IS REALLY SORT OF A QUAD COLLABORATION BETWEEN MY LAB AT THE NIH, DANIELLE RAMOS AT YALE, SLOAN-KETTERING AND UNIVERSITY OF CONNECTICUT. INCREASINGLY I'M BECOMING MORE INTERESTED IN BIOLOGY, DRIVEN BY THE MICROSCOPY CHALLENGE. THINK ABOUT THE SIZE OF A C. ELEGANS EMBRYO, THE SIZE OF A CULTURED CELL, 30 MICRONS THICK SO WE DON'T HAVE A BIG DATA PROBLEM. THE PROBLEM IS A RESOLUTION PROBLEM. THINK ABOUT RESOLUTION YOU WOULD NEED TO SEGMENT A NEURITE, IT'S DIFFRACTION LIMITED, A THOUSAND TIMES SMALLER THAN THE ANIMAL. THE CHALLENGE IS TAKE A VOLUME, CARVE IT INTO VOXELS SMALLER OVER MANY HOURS WITHOUT KILLING THE ANIMAL. CONVENTIONAL TECHNIQUES FAIL US. IF THE EMBRYO WERE A THIN PANCAKE YOU WOULD USE EPIFLUORESCENCE MICRO SCOPE, VERY FAST, RIGHT? YOU GET THE ENTIRE IMAGING PLANE AT ONCE, YOU ONLY HAVE TO SCAN IN ONE DIMENSION TO BUILD VOLUME. THERE'S NO OPTICAL SECTIONS SO YOU HAVE LOTS OF OUT OF FOCUS LIGHT AND BLUR. IMAGE IN FOCUS WILL BE CONTAMINATED BY NEURONS GOING OUT OF FOCUS. MORE IMPORTANT, THE WHITE FIELD MICROSCOPE IS LITERALLY A KILLER, VOLUMETRIC BLEACHING DESTROYS EMBRYO BEFORE YOU'RE DONE IMAGING. CONFOCAL, YOU CAN GET HIGH QUALITY INFORMATION IN THE FOCAL PLANE, THE PRICE IS YOU HAVE TO SCAN AND SO THE CON FOCAL MICROSCOPE IS TOO SLOW FOR THIS PROJECT. YOU HAVE THE PROBLEM OF VOLUMETRIC BLEACHING AND DAMAGE. THE CONCEPT IS MORE THAN 100 YEARS OLD, SHAPE ILLUMINATION INTO A THIN PLANE AND ONLY ILLUMINATE FOCAL POINT, LIGHT SHEET MICROSCOPY BECAUSE YOU'RE SHAPING ILLUMINATION TO A LIGHT SHEET WITH THE VIRTUE OF BEING FAST BECAUSE YOU ONLY HAVE TO SCAN IN ONE DIMENSION LIKE THE WHITE FIELD MICROSCOPE TO BUILD UP. IT HAS THE ADVANTAGE OF OPTICAL SECTIONING LIKE A CONFOCAL BECAUSE NOW IN PRINCIPLE ONLY WHAT YOU ARE IMAGING IS GLOWING. BUT EVEN MORE IMPORTANT THAN THESE TWO IS THE FACT THE DAMAGE IS GREATLY REDUCED BECAUSE YOU'RE DOSING THE FOCAL POINT. THIS IDEA HAS BEEN AROUND FOR A WHILE. WE HAVE INVENTED OUR OWN IMPLEMENTATION, INVERTED MICROSCOPE BASE SELECTIVE PLANE ILLUMINATION MICROSCOPE. WE COLLABORATE A COMPANY CALLED ASI, A GREAT COMPANY, I RECOMMEND WORKING WITH THEM IF YOU HAVE A CUSTOM PROJECT LIKE FAST TURN AROUND. YOU REPLACE TRANSMISSION COLUMN OF YOUR CONVENTIONAL MICROSCOPE WITH TWO OBJECTIVES THAT ENABLE LIGHT SHEET CAPABILITY. ONE OBJECTIVE BRINGS IN THE LIGHT SHEET. THE OTHER OBJECTIVE COLLECTS FLUORESCENCE FROM THE LIGHT SHEET AND YOU SCAN THIS IN TIME AND SYNCHRONY, YOUR SAMPLE CAN REST ON THE COVER SLIP, YOU TAKE THE BURDEN FROM THE BIOLOGIST, PREPARE CONVENTIONAL AND BRING LIGHT SHEET CAPABILITY TO THE SAMPLE. YOU CAN USE THE LOWER OBJECTIVE, LOWER ILLUMINATION DETECTION PATH TO SCREEN THE SAMPLE FAST AT HIGH SPEED. SO HOPEFULLY THIS MOVIE PLACE. AWESOME. JUST TO SHOW YOU EARLY MOVIES FROM THIS EARLY iSPIM, YOU CAN RECORD TENS OF THOUSANDS OF VOLUMES WITHOUT ANY APPARENT BELIEVING OR DAMAGE AT A SPEED CONSISTENT WITH FOLLOWING THE MOVIES REPRESENTS 13 1/2 HOURS, EVERY ONE ACQUIRED IN TWO SECOND, 20,000 VOLUME, THE RATE OF PHOTOBLEACHS LESS THAN THE RATE OF PROTEIN SYNTHESIS, YOU CAN RECORD IN PRINCIPAL AND TOTO OF THE ENTIRE ANIMAL THE TAKEHOME POINT IS THE EMBRYOS HATCH ON TIME AT THIS LEVEL OF LIGHT DESPITE MASSIVE AMOUNT OF DATA. NO MICROSCOPE IS PURPOSE. ONE CRITICISM OF THIS, THE AXIAL RESOLUTION WAS NOT AS GOOD AS THE LATERAL RESOLUTION. I HID THAT FROM YOU IN THESE MOVIES BECAUSE I WAS SHOWING YOU MAXIMUM INTENSITY BUT IF YOU TAKE THE DATA AND RESLICE OR LOOK FROM THE SLIDE, IF YOU LOOK AT DATA OUT OF THE MICROSCOPE YOU CAN SEE NUCLEI, RESOLVE THEM. TAKE THE SAME DATA SET AND PLAY IT FROM THE SIDE, NOW THE POOR AXIAL RESOLUTION INHERENT TO THE MICROSCOPE SMEARS OUT NUCLEI, THEY LOOK LIKE FOOTBALLS. THIS ISN'T A PROBLEM ENDEMIC TO MINE, ANY SINGLE OBJECTIVE MICROSCOPE COLLECTS FEWER ANGLES ALONG THE OPTICAL AXIS THAN TRANSVERSE, WE NEED TO SEGMENT NUCLEI. THERE ARE MANY WAYS OF IMPROVING THE Z RESOLUTION OF MICROSCOPE. WE TOOK A SIMPLE CONCEPT, JUST TO LOOK FROM TWO DIRECTIONS, IF I TAKE THIS CARTOON OF THE LIGHT SHEET GEOMETRY AND I JUST ROTATE AND LOOK FROM THE SIDE NOW I'VE SWITCHED DIRECTIONS EVER GOOD AND BAD RESOLUTION BY SWITCHING VIEWING DIRECTION, WHAT WAS FORMERLY THE DIRECTION OF POOR AXIAL RESOLUTION IS NOW THE DIRECTION OF MUCH BETTER LATERAL RESOLUTION IF I BRING THE LIGHT SHEET IN PERPENDICULAR. THE IDEA HERE IS SIMPLE. WE COLLECT TWO VIEWS OF THE SPECIMEN, REGISTER, FUSE THEM AND APPLY DECONVOLUTION TO EXTRACT THE BEST POSSIBLE RESOLUTION FROM EACH VIEW. IN ISPIM WE HAVE TO CAPTURE THIS FLUORESCENCE FROM THE SIDE. ONCE WE CAPTURE TWO IMAGING VOLUMES WE REGISTER ON TOP OF ONE OTHER AND DO PROCESSING, RECOVERING SPATIAL RESOLUTION IN Z TWICE AS GOOD AS CONFOCAL, ISOTROPIC IN THREE DIMENSION, USING FAST CAMERAS AND LIGHT SHEET IS FAST. HERE IS A COMPARATIVE DATASET SHOWING HISTONES DEVELOPING IN THE DUAL VIEW SYSTEM, diSPIM COMPARED TO SINGLE VIEW. YOU CAN APPRECIATE Z, PICK UP A FACTOR OF FIVE-FOLD AXIAL RESOLUTION OTHERWISE JUST MISSED. WHERE WE ARE DOSING THE EMBRYO TWICE AS MUCH BUT STILL GENTLE ENOUGH TO RECORD FOR HUNDREDS OF THOUSANDS AND HAVE THE ANIMALS HATCH, C. ELEGANS AT 13 1/2 TO 14 1/2 HOURS, WE DON'T SEEM TO BE PAYING TOO MUCH OF A PRICE FOR THE SECOND VIEW. IN THE END WHAT WE'RE AFTER IS THE NEURONS. HERE ARE JUST A GROUP OF I THINK FIVE NEURONS LABELED WITH GFP. I WANT TO COMPARE RESOLUTION IMPROVEMENT PARTICULARLY ALONG THE AXIAL DIRECTION, HOPEFULLY YOU CAN APPRECIATE THERE'S A LOT OF DETAIL HERE ON THE EDGE OF THE CELLS JUST WASHED OUT BY DIFFRACTION. IS THE GOAL IS TO SEGMENT WE HAVE AN EASIER TIME COLLECTING THE SECOND VIEW. THE diSPIM IS ROBUST, 60 LABS ACROSS THE WORLD BOUGHT THIS SYSTEM AND DEPLOYED IT, UNPUBLISHED DATA WITH JUSTIN TORASCO, IMAGING SMALL ORGANELLES, THESE FOR HALF AN HOUR, AXIAL CUT TO EMPHASIZE AXIAL RESOLUTION, IMAGE OVER MANY TIME POINTS WITHOUT OBVIOUS RESOLUTION. FRIENDS AT HARVARD WITH A diSPIM, COLLABORATING TO RECORD CALCIUM ACTIVITY IN DROSOPHILA NERVOUS SYSTEM. THE SAME UNDERLYING MICROSCOPE CAN BE USED AT MULTIPLE SCALES IN SPACE -- SPACE AND TIME, TRACKING SUBSETS OF NEURONS, IDENTIFYING NEURONS BY USING HISTONES WHICH LABEL ALL OF THE CELLS AND THEN SEGMENT THEM AND INTEGRATE INTO A MODEL. THIS IS ONE EXAMPLE SHOWING NEURONS FORMING THE NERVE RING, THE BRAIN OF THE ANIMAL. THE TWO-COLOR EXPERIMENT, AT THE SAME TIME RECORDING POSITIONS OF OTHER CELLS. AND EXPLAINING IN MOVIE FORMAT, IT WOULD TAKE MULTIPLE DATASETS. IF YOU'RE INTERESTED, VISIT THIS WEBSITE AND DOWNLOAD THE PROGRAM. YOU SHOULD BE ABLE TO DOWNLOAD ON THE DESKTOP. THE POINT IS WE BUILT AN APP THAT TAKES MULTIPLE DATASETS AND LETS YOU INTEGRATE THIS INFORMATION IN SPACE AND TIME SO THE NUCLEI YOU'RE SEEING HERE WERE COLLECTED AT NIH ON OUR diSPIM, NEURONS FROM YALE AND SLOAN-KETTERING, WE CAN GO IN AND BE ON SEARCH AND ROTATE, FOUR DIMENSIONAL, AND ANNOTATE. YOU CAN SEE VARIOUS OUTGROWTHS OCCURRING. THIS IS AN EDUCATIONAL DATA-DRIVEN DISCOVERY, JUST ONE PARTICULAR STORY AS WE'VE SHOWN, SHOWING NEURONS AS THEY FORM THE NERVE RING. YOU CAN APPLY THIS TO ANY GROUP OF CELLS INSIDE THIS EMBRYO. IF YOU'RE INTERESTED IN PHARYNGEAL CELLS, YOU CAN SEE SOME OF THESE CELLS ARE APPARENTLY BEING THREADED THROUGH THE NASCENT NERVE RING, IT MIGHT BE DIFFICULT TO GET WITH A DATASET BUT BY HAVING A SYSTEMS LEVEL VIEW OF THE BIOLOGY, YOU CAN SEE HOW THIS HAPPENS ACROSS TIME AND ACROSS THE ENTIRE ANIMAL. THAT'S THE DREAM, THAT'S WHERE WE'RE GOING. THERE'S A PROBLEM WHICH IS THE ANIMAL TWITCHES. UNFORTUNATELY MY MOVIE WON'T PLAY HERE BUT YOU SAW IN THE PREVIOUS MOVIES AFTER THE MUSCLES FORM THE ANIMAL WRITHES IN THE EGG SHELL. EVEN IF YOU HAVE ENOUGH SPATIAL TEMPORAL RESOLUTION YOU HAVE AN ANNOYING FRAME OF REFERENCE PROBLEM THAT KEEPS MOVING. IT'S ANNOYING WITH ONE, CRIPPLING WITH MULTIPLE ANIMALS. LOOK AT THE SAME GENES EXPRESSED IN MULTIPLE ANIMALS EVEN THOUGH THE OVERALL DEVELOPMENT IS STEREOTYPED EACH ANIMAL IS TWITCHING INDEPENDENTLY, YOU HAVE TO ALIGN IN SPACE AND TIME, BUILDING GOOD MICROSCOPES ISN'T GOOD ENOUGH. WE INVERSED YEARS, UNTWISTING THE EMBRYO. THE IDEA HERE IS WE EXPRESS GENETICALLY ENCODED MARKERS THAT MARK THE LEFT AND RIGHT SIDES OF THE ANIMAL. WE ALSO -- THAT GIVES ENOUGH INFORMATION TO MODEL THE ANIMAL IN THE TWISTED STATE. WE CAN RESAMPLE THE ANIMAL, UNTWISTED AS IT WERE, AND WE HAVE A COMMON FRAME OF REFERENCE, LINEAR FRAME OF REFERENCE TO PLACE VARIOUS DEVELOPMENTAL EVENTS. THE MOVIE WILL NOT PLAY BUT I HAVE A BACKUP, IF THIS WORKS, FROM THE WEB PAGE, FROM THE PAPER. TO WALK YOU THROUGH HOW THIS ACTUALLY WORKS, HOPEFULY THIS MOVIE PLAYS. YES, THERE IT GOES. THE IDEA IS YOU TAKE THESE THEME CELLS, SEGMENT THEM. THEY ARE EASY TO SEGMENT. BILATERALLY SYMMETRIC, YOU CAN PAIR FROM LEFT AND RIGHT SIDES BECAUSE THEY RUN DOWN THE LENGTH, YOU CAN CONNECT ALONG THE LEFT AND RIGHT SIDES, BUILDING THE LADDER SO THE BACKBONE. ONCE WE HAVE THE TWO DIMENSIONAL LATTICE WE REFIT SPINES ALONG THE LEFT AND RIGHT AND MIDLINE, LOOKS LIKE A BACKBONE. REFINE BY SWEEPING ELLIPTICAL CROSS-SECTION FROM HEAD TO TAIL, 3D-IFYING, NOW IT'S A WIRE GRID MODEL. WE TAKE THE MODEL AND EXPAND OUTWARD TO MAKE SURE WE DON'T MISS ANY DATA, WHICH YOU'LL SEE IN A SECOND HERE. NOW WE HAVE A MODEL OF THE TWISTED ANIMAL WE CAN RE RESAMPLE, WE CAN SWEEP TANGENT TO THE MIDLINE. I'M SHOWING TWO VIEWS, TOP RIGHT AND LOWER PART OF THIS STRAIGHTENED ANIMAL. HOPEFULLY YOU CAN APPRECIATE IT'S MUCH EASIER WHERE THE HEAD AND TAIL IS. IMAGINE DOING THIS NOW ANNOTATING WHERE VARIOUS CELLS ARE AND FOLLOWING THOSE ANNOTATIONS ACROSS TIME. YOU CAN BUILD A MODEL IN THIS POST-SWITCHING STAGE HOW THE ANIMAL DEVELOPS. SO MY POSTDOC RYAN WHO LED THIS PROJECT SPENT A YEAR DOING THAT FOR SEVEN ANIMALS, COMBINING DATA RYAN BUILT A SIMPLE BUT POWERFUL BALL AND STICK MODEL, POSITION OF 25 CELLS, ONE PAIR OF NEURITES AFTER THE MUSCLES FORMED. REPRESENTED AT SPHERES, NEURITES ARE THE STICKS AS THE EMBRYO DEVELOPS. IT'S POWERFUL IN THE SENSE YOU CAN NOW START TO DETERMINE WHERE CELLS ARE IN RELATIONSHIP TO ONE ANOTHER, AND YOU CAN ALSO SEE THIS PAIR OF NEURITES CONTINUES TO GROW WELL AFTER THE CELLS ASSUMED FINAL POSITION. THERE'S SOME PROGRAM TELLING THEM TO DO THAT WHICH WE CAN NOW START TO HUNT DOWN. WHERE WE'RE GOING WITH THIS IS TRYING TO FILL IN BLACK SPACE. THIS IS ABOUT 5% OF THE ENTIRE ANIMAL. WE HAVE A LOT OF WORK AHEAD OF US BUT WE'RE STARTING TO DO THAT FIRST WITH ALL OF THE DIFFERENT CELLS. WE STARTED TO IMAGE HISTONES TARGETED TO VARIOUS NEURONS, AND THEN UNTWIST THEM AND TRACK THESE. THIS WILL TAKE SOME TIME BUT ALL THE TOOLS, THE MACHINERY IS THERE TO COMPLETE THE MODEL IN THE BALL AND STICK MODEL. MORE COMPLICATED BUT INTERESTING TO TRY TO GET THE CELL SHAPES INTO THE MODEL. THIS IS A MORE DIFFICULT SEGMENTATION PROBLEM BUT I THINK ONE DAY WE'LL BE ABLE TO GET THAT AS WELL. NOW, EVERYTHING I'VE BEEN TALKING ABOUT SO FAR IS A STRUCTURAL MODEL OF NEURODEVELOPMENT BUT NEURONS AND CELLS ARE COMMUNICATING WITH ONE ANOTHER. WE STARTED TO GAIN A WINDOW INTO THE COMMUNICATION LOOKING AT CALCIUM FLUX IN THE WORM EMBRYO AS IT CELLS. SO THAT'S REALLY TOO BAD. THIS MOVIE SHOWS CALCIUM WAVES OF THE ANIMAL AS IT'S DEVELOPING IN THE TWO-FOLD STAGE. BECAUSE THE DATA ARE FOUR DIMENSIONAL WE CAN TAKE THE DATASETS AND SEGMENT THEM AND PLOT THE ANGLE OF THE ANIMAL FROM SIDE TO SIDE AND WE CAN ACTUALLY CHIMERGRAPH AND SHOW HOW THE INTENSITY CHANGES AS THE ANIMAL IS BEHAVING. WHEN THE ANIMAL MOVES OR ROTATES YOU SEE CALCIUM TRANSIENCE IN THE DORSAL BUNDLES, WHEN IT MOVIES THE OTHER WAY YOU SAY ANTI-CORRELATED TRANSIENCE, WITHOUT INPUT FROM THE NERVOUS SYSTEM. ONE OF THE INTERESTING QUESTIONS NOW IS HOW DOES THIS CHANGE IN THE MUSCLES, HOW DOES THE NEURON START TO INNERVATE THE MUSCLES? THAT WOULD BE AN INTERESTING DIRECTION TO GO IN. ANOTHER PROJECT THAT'S HOT IS SO CALLED BRAIN ACTIVITY MAPPING. USING GCaMP, TARGETING, YOU CAN INVESTIGATE CALCIUM TRANSIENCE, THIS MOVIE WAS GENERATED, THE FIRST CALCIUM RECORDINGS IN THE EMBRYO. I SPENT TIME MANUALLY TRACKING EACH OF THESE CELLS WHICH SUCKS AS MUCH AS YOU MIGHT THINK TO GENERATE THE MOVIE OVER THERE, AN A ASSEMBLY OVER TIME. I THINK THE DAY WILL COME WE'LL BE ABLE TO SEE WHAT LARGE GROUPS OF NEURONS ARE DOING AND HOW THEY ARE COMMUNICATING WITH ONE ANOTHER IN THIS GROWING EMBRYO. ONE OF THE COMPLEMENTARY PROJECTS IN THE LAB WE STARTED IN ADDITION TO THE CALCIUM IMAGING IS BEHAVIORAL IMAGING. WE CAN USE THE SAME MARKERS TO DEVINE CONFIRMATIONAL SHAPE OF THE ANIMAL AS THE ANIMAL IS GROWING IN THE EGG SHELL. EVAN SPENT TIME FIGURING OUT HOW TO TRACK EACH OF THE CELLS TO GENERATE SKELETAL MODEL YOU SEE OVER THERE. WE'RE PRETTY INTERESTED IN THIS BECAUSE WE WOULD LIKE TO KNOW HOW DID THE SHAPE OF ANIMAL CHANGE AT HIGH TEMPORAL RESOLUTION, BUT YOU HAVE A LOT OF DATA. EVAN'S ALGORITHMS ARE 95 TO 99% GOOD ENOUGH THAT STILL MEANS THOUSANDS OF ERRORS A HUMAN HAS TO MAN REALLY CORRECT SO WE'RE COLLABORATING WITH MACHINE VISION EXPERTS IN AND OUT OF THE NIH TO ADDRESS THIS PROBLEM TO MAKE THIS SORT OF A MORE HIGH THROUGHPUT PROJECT. HOPEFULLY AS WE CORRELATE THIS BEHAVIORAL CHANGE WITH CALCIUM IMAGING INSIDE OF THE EMBRYO. NOW, THE IMAGES IS NEVER GOOD ENOUGH, EVER, RIGHT? WE'RE ALWAYS ON THE LOOKOUT TO FIND WAYS OF IMPROVING IMAGE QUALITY, ESPECIALLY IF WE CAN DO THAT WITHOUT LOSING ANY INFORMATION, SO IF WE CAN ENHANCE SPATIA RESOLUTION OR IMPROVE SPEED WHILE COLLECTING MORE LIGHT THAT'S A STRAIGHT WIN. TO SHARE UNPUBLISHED WORK TO CLOSE HERE, ABOUT HOW WE'RE DOING THAT, THIS IS THE LIGHT GEOMETRY, A SAMPLE ON A CLASS COVER, BRING IN LIGHT SHEET, RECORD FROM THE OTHER OBJECTIVE, BY MOVING, YOU CAN BUILD UP A VOLUME. THAT'S GOOD. BUT ONE PROBLEM WITH THIS GEOMETRY, YOU CAN ONLY COLLECT AS MUCH FLUORESCENCE AS OBJECTIVE ALLOWS AT ANY POINT IN TIME. YOU'RE WASTING MOST OF THE FLUORESCENCE EMITTED FROM THE SAMPLE. SO (INDISCERNIBLE) HAD A CLEVER IDEA, INSTEAD OF GROWING ON A GLASS COVER SLIP GROW ON A MIRROR. GET A GLASS COVER SLIP AND COAT WITH A THIN AMOUNT OF ALUMINUM THE ACTION IS TO REFLECT THE LIGHT BACK, VIRTUALLY COPYING THE LIGHT ON THE UNDERSIDE OF THE COVER SLIP. WHAT THIS MEANS IS YOU JUST INCREASED THE COLLECTIVE IMAGING SYSTEM, BOTH CAMERAS RECORDING AT THE SAME TIME. YOU GET FOUR SIMULTANEOUS VIEWS IN THE SAME TIME IT WOULD NORMALLY TAKE YOU TO GET TWO VIEWS, AND YOU'VE DOUBLED YOUR COLLECTION EFFICIENCY AND INCREASED TEMPORAL RESOLUTION. THE PRICE YOU PAY FOR QUAD VIEW IS EPIFLUORESCENCE CONTAMINATION, SEEING THE LIGHT SHEATH AND SEEING THIS CONE OF EPIFLUORESCENCE THAT PERVADES THE SAMPLE SO YOU HAVE TO MODEL THAT, WE'VE BEEN ABLE TO REMOVE IT, KEY TO THE SUCCESS OF THIS IDEA. SO IF YOU LOOK AT THE RAW DATA COMING FROM THE MICROSCOPE IT LOOKS LIKE GARBAGE, RIGHT? YOU SEE NOT ONLY THIS ISOTROPIC RESOLUTION, YOU SEE EPIFLUORESCENCE CONTAMINATION, IF YOU REMOVE THAT YOU DON'T DO A GOOD JOB. YOU CAN STILL SEE IT HERE AND DON'T RECOVER THE FULL RESOLUTION IMPROVEMENT. BY MODELING THE ENTIRE IMAGING PROCESS ABLE TO GET ISOTROPIC RESOLUTION AND REMOVE EPIFLUORESCENCE, GOING FASTER IN THE WORM EMBRYO. IF YOU'RE INTERESTED IN TRANSIENCE AT THE END OF EMBRYO GENESIS WHEN THE WORM IS MOVING FAST YOU HAVE TO GO AT MULTIPLE VOLUMES PER SECOND. WE CAN NOW DO SINGLE SHOT ISOTROPIC IMAGING, WE GET BOTH VIEWS AT ONCE, CATCHING CALCIUM TRANSIENCE LATE IN EMBRYOGENESIS. ONE ADVANTAGE IS THE SPEED AND INCREASED LIGHT EFFICIENCY, YOU CAN USE THIS TRICK TO DO CERTAIN THINGS AT HIGHER NUMERICAL APER -- APERTURE. YOU HAVE A FAT OBJECTIVE TO COLLECTS MORE LIGHT, IF THIS CONFIGURATION HERE NOW, IF YOU YOU COLLECT 1 1/2 TIMES MORE BECAUSE THE OBJECTIVE IS BETTER. IF YOU DO CONVENTIONAL diSPIM, YOU GET MORE CONFOCAL RESOLUTION IN XY, TWICE IN Z. YOU WANT TWO 1.1 NA VIEWS, THERE ISN'T SPACE IN THIS CONFIGURATION TO SHOVE TWO LENSES INTO PROXIMITY. USING REFLECTIVE TRICK YOU GET THAT FOR FREE. IF YOU COME FROM THE LOW OBJECTIVE, NOW YOU CAN COLLECT TWO HIGH NA VIEWS, IMPROVING COLLECTION EFFICIENCY, IT'S EARLY DAYS BUT THE SAME IDEA MIGHT APPLY TO SUPER-RESOLUTION MICROSCOPY, COLLECT MORE INFORMATION IF YOU PUT YOUR SAMPLE ON A MIRROR. WE CAN NOW OBSERVE SUBCELLULAR DYNAMICS, GRADUATE STUDENT IVAN ASKING WHAT MICROTUBULES AND NUCLEAR SHAPE IS DOING AND CAN OBSERVE OVER THE WHOLE VOLUME CHANGES IN SHAPE, AS THE NUCLEUS DEFORMS THERE'S A CORRELATED MOVEMENT IN MICROTUBULES, YOU CAN SEE EASILY IN THE MICROSCOPE. LONGER TIME SCALES, OUR POSTDOC IS INTERESTED IN METABOLISM AND TOOK MITOCHONDRIA AND POISONED THEM SHUTTING DOWN OXPHOS, IT'S EASY TO TRACK SHAPE CHANGES IN RESPONSE TO THIS DRUG USING THIS MICROSCOPE. FOR SUBCELLULAR RESOLUTION WE DO SUB300 NANOMETERS IN XY AND Z USING THIS MICROSCOPE. SO LET ME END ON A CONCEPTUAL NOTE. THE QUESTION ARISES WHAT GOOD ARE MICROSCOPES IN MY LAB? I COMMERCIALIZED, iSPIM AND diSPIM, YOU DID BUY THEM, MAYBE YOU DON'T HAVE THE MONEY OR SPACE TO ADAPT THEM. WITH KEIR, JUSTIN AND DAN, WE THOUGHT IT WOULD BE A GOOD IDEA TO HOUSE HOME-BUILT INSTRUMENTS IN FACILITY TO BE USED BY ALL RESEARCHERS, TO RECYCLE DOLLARS. I'M NOT THE ONLY ONE DEVELOPING INSTRUMENTS. THERE'S iSPIM AND diSPIM, JUSTIN HAS THE MICRO DOPE GOOD -- MICROSCOPE WITH THE HIGHEST RESOLUTION, DAN TRACKS MOLECULES, THE THOUGHT WAS CAN WE CONSOLIDATE INSTRUMENTS LIKE THESE INTO ONE PLACE WHERE ANYBODY COULD COME IN AND USE THEM. ANCILLARY BENEFITS AS WELL. POSTDOCS CAN USE INSTRUMENTS AND LEARN OPTICS. COLLABORATIVE OPPORTUNITIES, SOMETIMES IDEAS FLOW BY HAVING TWO PEOPLE IN THE SAME ROOM TOGETHER. POTENTIALLY THERE ARE EXTRAMURAL POSSIBILITIES HERE TOO. MANY BENEFITS TO HAVING THIS TRANS-NIH FACILITY, SO WE APPROACHED DR. GOTTESMAN AND THE CZARS OF THE INTRAMURAL PROGRAMS, KIND ENOUGH TO GIVE US SPACE AND STAFF. SO WE HAVE NOW SPACE ADJACENT TO MY LAB FOR SIX HIGH-END HOME-BUILT OPTICAL INSTRUMENTS, WE HAVE FIVE MICROSCOPES THAT ARE READY TO BE PUT INTO THIS FACILITY TO STAND MULTIPLE SCALES IN SPACE AND TIME. AND MOST IMPORTANTLY HIRED TWO AWESOME STAFF SCIENTISTS TO DIRECT THE FACILITY, AND THESE GUY, JIJI AND HARSH KNOW THE INS AND OUTS AND CAN HELP DEAL WITH DATA AFTER THE DATA HAS BEEN ACQUIRED, A MAJOR PROBLEM AT HIGH RESOLUTION IN SPACE AND TIME. THESE GUYS ARE AN ASSET AND THE AIM IS OPEN FOR BUSINESS EVEN THOUGH THE FACILITY ISN'T BUILT, YOU SHOULD USE THEM AS A RESOURCE. AS A PLUG FOR THAT EVEN THOUGH THE SPACE ISN'T COMPLETE YET, HARSH AND JIJI HAVE BEEN BUSY. THIS IS AN INCOMPLETE LIST OF USERS INSIDE THE NIH AND THOSE THAT CRASH FROM THE OUTSIDE, FROM OPTIC CLOSURE IN ZEBRAFISH TO CHOLERA, WITH THE diSPIM. IT'S EASIER TO USE, WE CAN TRAIN YOU TO USE IT YOURSELF. WE'VE IMAGED MOUSE EGGS TO NEURONS. IF YOU'RE INTERESTED IN TRACKING, TRANSCRIPTION FACTORS INSIDE THE NUCLEUS, WE CAN HELP YOU THERE AS WELL, WITH ACQUIRING DATA AND ANALYSIS. SO I THINK THAT'S ALL I HAVE TIME FOR. LET ME JUST -- THE MOST IMPORTANT SLIDE THANKING EVERYBODY INVOLVED IN THIS. I'D LIKE TO THANK RICHARD, HANK AND ROD PETTIGREW FOR HIRING ME. THIS IS A VERY TARGET-RICH ENVIRONMENT FOR ME AS A TOOL DEVELOPER. I'M THANKFUL FOR THAT. ON THE EXTRAMURAL COMMUNITY, TOO MANY PEOPLE TO THANK BUT MOSTLY THE WORM COLLABORATORS, ZRIRONG, DAN AND BILL, AND THE UNTWISTING PROJECT, PATRICK AND CORY WITH INVERSE PROBLEMS HELPING US THINK THROUGH REFLECTIVE IMAGING, THE FORMATION PROCESS. IN MY LAB I OWE A DEBT TO ANDY YORK, AND MENTORING PETER WINTER, WEI ZHENG. AND THEN IN MY LAB (INDISCERNIBLE) DROVE THE DEVELOPMENT, RYAN AND EVAN ARE TWO TALENTED BIOLOGISTS DRIVING NEURODEVELOPMENT. ALMOST EVERY MOVIE TAKEN BY PANOS, A CELL BIOLOGIST, AND JOHN AND IVAN WITH GRADUATE STUDENTS AND A TEAM OF POSTBACS. WITH THAT I'D LIKE TO STOP AND THANK YOU FOR YOUR TIME. [APPLAUSE] >> HOPEFULLY THAT DIDN'T LEAVE YOU TOTALLY SPEECHLESS. PLEASE IF YOU HAVE ANY QUESTIONS COME FORWARD TO THE MICROPHONES, SINCE WE HAVE PEOPLE ALL OVER THE NIH WATCHING. >> THIS IS GREAT. IT'S VERY IMPRESSIVE. I WANTED TO KNOW IF YOU HAVE ANY THOUGHTS ABOUT OTHER APPLICATIONS FOR SOME OF THE TECHNIQUES YOU DEVELOPED TO REDUCE THE PROBLEMS OF JITTER AND TWITCH. >> IN PRINCIPAL, THE PIPELINE FOR THE WORM, FOR ANY ORGANISM WITH VERMIFORM GEOMETRY, WE'D BE INTERESTED. WE DON'T HAVE THE BANDWIDTH TO SCALE TO MOUSE OUR ZEBRAFISH, BUT TRYING TO LINEARIZE OR MERGE DATA FROM DIFFERENT ANIMALS MAKES SENSE, RIGHT? >> AND MACHINE LEARNING. >> AND MACHINE LEARNING. GOOGLE CAN IDENTIFY CAT FACES, WE CAN DO MORE TO ADAPT ALGORITHMS FOR DATA ANALYSIS. THE FUTURE OF OUR FIELD CAN'T BE LIKE HUMANS SITTING THERE AND TRACKING EACH NEURON. EVENTUALLY COMPUTERS WILL BEAT US. THERE'S A LOT OF OPPORTUNITY THERE FOR INNOVATION, RIGHT? WE HAVE A TEAM OF PEOPLE IN CIT STARTING TO DO THIS. AND ARE WILLING TO GET INVOLVED, RIGHT? IN MORE -- >> (INAUDIBLE) >> THANK YOU, HARI, FOR ANOTHER SPECTACULAR TALK HERE. MY QUESTION IS, EFFORT YOU PUT IN DEVELOPING THE NEW TECHNOLOGY, WHAT YOU THINK IS LIMITING YOU TO GO EVEN FURTHER? IS IT CHIMERS? IS IT COMPUTING? MICROMECHANICAL DEVICES? WHERE DO YOU SEE GROWTH TO HELP YOU IMAGE FASTER, DEEPER, BETTER? >> THE REAL ANSWER IS IDEAS, ALWAYS. IDEAS ARE ALWAYS THE LIMITING FACTOR IN DEVELOPING NEW TECHNOLOGY BUT I THINK WE'VE SEEN AMAZING THINGS IN CAMERA TECHNOLOGY. EVEN FASTER CAMERAS ARE COMING. SO AS FAR AS PUSHING THE SPEED LIMIT, WE'RE NOT QUITE AT THE LEVEL OF SORT OF THE SHOT NOISE LIMIT BUT WE'RE VERY CLOSE TO THAT. SO I THINK THERE'S MORE THAT CAN BE DONE THERE. THE OTHER MAJOR LIMITING FACTOR, EVERYTHING I'VE DONE, IT DEPENDS ON THE DYES, WE'RE AT THE MUST OF PROTEIN AND SYNTHETIC DYE TECHNOLOGY. MORE TOOL DEVELOPMENT THERE WOULD BE GREAT FOR EVERYBODY, NOT JUST TOOL DEVELOPERS BUT FOR EVERYBODY. >> WELL, WE'D LIKE TO THANK YOU, HARI, FOR THE TOUR DE FORCE. [APPLAUSE]