I'M JOHN NGAI WELCOME TO THE FIRST OF FIVE WORKSHOP SERIES WITH THE BRAIN INITIATIVE AND THE U.S. DEPARTMENT OF ENERGY OFFICE OF SCIENCE. THIS WORKSHOP SERIES CONVENES RESEARCHERS TO DISCUSS THE STATE OF THE ART OPPORTUNITIES AND CHALLENGES OF THE SCIENCE NEEDED TO GENERATE COMPREHENSIVE ATLASES OF BRAIN CONNECTIVITY OR WIRING DIAGRAMS OF MAMMALIAN BRAINS. EACH WORKSHOP WILL EXPLORE A FACET OF THIS GOAL. THE BIOLOGICAL SIGNIFICANCE OF THE UNDERTAKING METHODS OF SAMPLE PREPARATION, EXPERIMENTAL MODALITIES AND HOW TO BE USED BY THE SCIENTIFIC COMMUNITY. THE UNDERSTANDING THE MAMMALIAN BRAIN WOULD BE IMMENSE. THE RESOURCES WOULD ENHANCE THE CAPABILITY OF RESEARCHERS TO FORMULATE AND TEST MODELS HOW ACTIVITY IN BRAIN CIRCUITS DRIVES BEHAVIOR AND SUCH KNOWLEDGE WILL ACCELERATE THE NEXT CIRCUIT BASED THERAPEUTICS. ONE OF THE MAINS GOALS OF THE NIH. AND BECAUSE DATA HAS NOT BEEN COLLECTED AT THIS SCALE THE FULL UNDERTAKINGS REMAIN UNCERTAIN MUCH THE WAY THE TRANSFORMATIVE EFFECT AND FOLLOW ON TECHNOLOGIES WOULD COULD NOT HAVE BEEN IMAGINED FROM THE START OF THE BRAIN INITIATIVE. MORE THAN 30 YEARS AFTER THE PUBLICATION, THE C ELEGANS CONTINUES TO BE WIDELY CITED RESOURCE AND YET WE CONTINUE TO LEARN MORE ABOUT THE VARIABILITY IN THE WORM AND DEVELOPMENT PROCESSES FROM ONGOING WORK. SIMILARLY THE RECENT COMPLETION OF THE FRUIT FLY BRAIN CONNECTOME PROJECT LOOKED AT THE BEHAVIORS AND RODEN RODENT BRAINS ARE DIFFERENT AND HUMAN BRAINS MAGNITUDES LARGER THAN RODENT BRAINS. WE CAN THEREFORE EXPECT THE CONSTRUCTION OF HIGH RESOLUTION ANATOMICAL MAPS WILL RESULT IN DATA GENERATION TO PRESS THE COMPUTATIONAL CAPABILITIES AND DRIVE INNOVATIONS IN DATA SCIENCE INCLUDING ARTIFICIAL INTELLIGENCE AND MACHINE LEARNING. ACHIEVING THE AUDACIOUS GOAL OF THE CONNECTIVITY OF THE MAMMALIAN BRAIN IN OUR LIFE TIME WILL REQUIRE COLLABORATION AMONGST SCIENTISTS AND ENGINEERS TO INVENT, REFINE AND SCALE UP ANDDEMOCRATIZE TO MEET THE CHALLENGE. I LOOK FORWARD TO EXPLORING WITH THE DEPARTMENT OF SCIENCE AND OTHER FEDERAL AGENCIES AND PARTNERS IN THE COMING MONTHS. IN CLOSING I WANT TO THANK THE CO-CHAIRS OF THE SERIES AND THE CO-LEADS OF EACH INDIVIDUAL WORKSHOP FOR THEIR INSIGHTS AND FOR BRINGING AN OUTSTANDING GROUP OF SCIENTIST TO THINK DEEPLY ABOUT THE PROBLEM AT HAND. I WANT TO THANK CHRISTEN AND PETER. ASKING PEOPLE TO ORGANIZE A WORKSHOP IS A BIG ASK. ORGANIZING FIVE WORKSHOPS IS A DEEP ASK AND APPRECIATE THE TIME AND COMMITMENT AND THINK THEY'VE ORGANIZED AMAZING SET OF WORK SHOPS TOGETHER WITH THE WORKSHOP CO-LEADS. I ALSO WANT TO THANK MY COLLEAGUES AT NIH WITHOUT WHOM THIS MAY NEVER HAVE HAPPENED AND DEPARTMENT OF DOE AND WHETHER YOU'RE PARTICIPATING THROUGH THE WEBINAR OR NIH WEBCAST, I THANK YOU FOR YOUR SUPPORT AND HOPE YOU GET A LOT OF IT AND MAKE GREAT CONTRIBUTIONS TO THE THINKING IN THIS EXCITING FIELD. THANK YOU VERY MUCH. I'LL HAND IT OVER TO DR. KUNG. >> GOOD MORNING AND WELCOME. I'M HARRIET KUNG DEPUTY DIRECTOR FOR THE SCIENCE PROGRAMS AT THE U.S. DEPARTMENT OF ENERGY OFFICE OF SCIENCE. TOO A GREAT PLEASURE TO BE HERE AND ON BEHALF OF MY IN THE THEORIES OF SCIENCE, I WANT TO JOIN DR. NGAI FOR JING JOINING US IN THIS WORKSHOP. WE'RE EXCITED TO WORK WITH OUR COLLEAGUES AT NIH TO REALIZE THE EXTRAORDINARY BENEFITS FOR HUMAN HEALTH AND WELL BEING THAT MAY BE POSSIBLE BY DEVELOPING A COMPREHENSIVE UNDERSTANDING OF BRAIN CONNECTIVITY LOOKING BACK OVER THE PAST DECADES THERE'S BEEN A LONG AND ASSISTFUL HISTORY OF WORKING TOGETHER TO ADVANCE OUR SCIENCE GOALS FROM DEVELOPING CANCER THERAPY TO THE HUMAN GENOME PROJECT TO THE USE OF DOE LIGHT SOURCES BY THE LIFE SCIENCES COMMUNITIES AND TO OUR WORK WITH NEXT GENERATION TOOLS AND A.I. AND MACHINE LEARNING. THE PAST COLLABORATIONS HAVE BEEN VERY FRUITFUL AND I HAVE GREAT HOPES AND EXPECTATIONS MORE EXCITING ADVANCES WILL COME. AS A BELIEVE INTRODUCTION TO THE DOE OFFICE OF SCIENCE WE'RE THE LARGEST SUPPORTER OF BASIC RESEARCH IN THE SCIENCES. THE RESEARCH WE SUPPORT AT THE LABORATORIES AND UNIVERSITIES ACROSS U.S. ARE CONSTANTLY PUSH THE BOUNDARIES OF SCIENCE AND HELPING TO BRING THE WORLD LEADING SCIENTIFIC TOOLS AND FACILITIES TO RESEARCHERS BOTH IN THE UNITED STATES AND ALSO ABROAD. OF COURSE AS WE KNOW MAPPING COMPLEX BRAINS AND DEVELOPING A FUNDAMENTAL UNDERSTANDING OF BRAIN ACCIST WILL REQUIRE AN ARRAY OF ADVANCES ASSON ALLUDED TO IN THE HIGH THROUGHPUT AND HIGH END COMPUTING AND DATA ANALYSIS AND DATA MANAGEMENT. ALL THESE WILL REQUIRE THAT WE CAN PERFORM THIS WORK AT SCALE IN FACT THE SCIENCE AT SCALE APPROACH IS SOMETHING WE'RE FAMILIAR WITH. IT'S A CORE COMPETENCY WE CULTIVATED AND UNIQUE CONTRIBUTION WE CAN BRING TO THE TABLE. THIS PAST NOVEMBER WE TOOK AN IMPORTANT FIRST STEP BY HOSTING A ROUND TABLE THAT BROUGHT TOGETHER EXPERTS IN NEUROSCIENCE, BIO IMAGING AND DATA SCIENCE FROM U.S. UNIVERSITIES AND DOE LABORATORIES. TOGETHER THESE RESEARCHERS ARTICULATED THE NEEDS OF THE NEUROSCIENCE COMMUNITY AND CONSIDERED THE LIMITATIONS IN CURRENT TECHNOLOGY AND PROPOSED OPPORTUNITIES TO ADVANCE THE CAPABILITIES THE FINDINGS ARE A VALUABLE CONTRIBUTION TO THE DISCUSSIONS WE'LL HAVE DURING THIS WORKSHOP SERIES. ALSO I'D LIKE TO STRESS WHILE THE CHALLENGE OF THIS OUR ENGAGEMENT BRINGS SIGNIFICANT OPPORTUNITIES FOR DOE TO ADVANCE OUR OWN MISSION IN SCIENCE, ENERGY, SECURITY AND OUR ENVIRONMENT AND THE SCIENTIFIC BENEFITS OF MAPPING COMPLEX BRAINS WILL EXTEND WELL BEYOND HUMAN HEALTH TO OPPORTUNITIES IN REALIZING KEY EVENTS IN ARTIFICIAL INTELLIGENCE AND FUTURE COMPETING IN IMAGING AND DATA ANALYSIS AMONG OTHERS CRITICAL TO THE DOE'S MISSIONS. THE CO-CHAIRS OF THE WORKSHOP SERIES AND OF EACH INDIVIDUAL WORKSHOP HAVE BROUGHT TOGETHER AN INCREDIBLY DIVERSE TEAM OF EXPERTS TO THINK DEEPLY AND CHALLENGE IN THE BRAIN CONNECTIVITY AND PROACTIVELY BROUGHT OUR COLLEAGUES PERFECT THE DOE COMMUNITY INCLUDING OUR LABORATORIES INTO A DISCUSSION AT THIS EARLIER STAGE. I WANT TO THANK THE CO-CHAIRS FOR ORGANIZING THE WORKSHOP SERIES AND THANK THE PARTICIPANTS FOR YOUR CONTRIBUTION TO THE IMPORTANT ACTIVITY. WE LOOK FORWARD WITH GREAT ANTICIPATION TO THE NEW INSIGHT AND PROSPECTIVES -- PERSPECTIVES AND REALIZE THIS TRANSFORMATIVE OPPORTUNITY FOR SCIENCE AND FOR HUMANITY. THANK YOU. >> WELCOME EVERYONE, TO THE BRAIN WORKSHOP 2, SAMPLE PREPARATION IN MAMMALIAN WHOLE-BRAIN CONNECTOMICS. I'M HONGKUI ZENG AND I'M WITH DAVI BOCK. WE WANT TO ESTABLISH WORK FLOW MAPS IN SAMPLE OPERATION FOR FIRST WHOLE MOUSE BRAIN ELECTROMICROSCOPY CONNECTOMES OF MOUSE AND SECOND, COMPLIMENTARY MICROSCOPY METHODS IN SAME OR INDIVIDUAL ANIMALS IN CONJUNCTION WITH THE EM CONNECTOMES AND THIRD, WHOLE-BRAIN LIGHT MICROSCOPY CONNECTOMES AND PROJECTOMS IN THE BRAIN. AND WHAT CAN WE LEARN? WE SHOULD BE ABLE TO LEARN A LOT ABOUT BRAIN ORGANIZATION AT CELL TYPE LEVEL AND AT THE NETWORK LEVEL WITH CELL TYPES WE CAN UNDERSTAND A LOT ABOUT CELL TYPE DIVERSITY FROM THEIR MORPHOLOGICAL AND BRAIN WIDE CONNECTION PATTERNS. BEYOND THE CELL TYPES AND WE THINK WE CAN LEARN STRUCTURE FUNG RELATIONSHIPS -- FUNCTION RELATIONSHIPS BY CONNECTOMES TO UNDERSTAND THE UNDERLYING CONNECTIVITY AND HOW THEY RELATE TO THE FUNCTIONAL PROPERTIES OF THE CELLS AND WE OBTAIN FROM THE CONNECTOMES WITH CELL TYPES THAT CAN BE TARGETED USING TOOLS AND FURTHER MONITOR AND MANIPULATED IN LIVING ANIMALS. BECAUSE WE HAVE LEARNED A LOT FROM THE PREVIOUS WORKSHOP ABOUT THE IMPORTANCE OF STRUCTURE, FUNCTIONAL RELATIONSHIPS HERE WE WANT TO ELABORATE A LITTLE BIT MORE ON THE FIRST POINT. THE RULES OF BRAIN ORGANIZATION. WEN IT COMES TO CELL TYPE DIVERSITY WE WANTED TO KNOW IF CELL TYPES BELONGING TO THE SAME AND CELLS BELONGING TO DIFFERENT TYPES HAVE DIFFERENT CONNECTION PROPERTIES AND INDIVIDUAL CELLS WITHIN A TYPE HAVE THE SAME CONNECTION PROPERTIES. SO BASICALLY THE IDEA IS WHATEVER OUR DEFINITION OF CELL TYPES IS CONGRUENT WITH THEIR CONNECTION PROPERTIES OR IF THERE IS FURTHER DIVERSITY AND HOW CAN WE COME UP WITH THE BEST DEFINITION OF CELL TYPES WE WANT TO ADDRESS THIS NOT ONLY FOR A FEW CELL TYPES AND THERE MAY BE THOUSANDS OF CELL TYPES THAT ARE SPREAD THROUGHOUT THE ENTIRE BRAIN AND IT IS ACTUALLY IMPORTANT TO ADDRESS THIS KIND OF QUESTIONS FOR ALL THE THOUSANDS OF CELL TYPES ACROSS DIFFERENT NEURAL SYSTEMS THAT ARE INVOLVED IN DIFFERENT KINDS OF BEHAVIORS. ACROSS THE DIFFERENT BRAIN SYSTEMS. ONE OF THE MOST EFFICIENT WAYS TO DO THAT IS TO LOOK AT A COMPLETE CONNECTOME. AND WE MAY FIND DIFFERENT CELL TYPES FOLLOW DIFFERENT RULES IN TERMS OF DIVERSITY AND IMPORTANTLY WE CAN INVESTIGATE AT THE ULTRA STRUCTURE LEVEL THE RELATIONSHIP BETWEEN CELL TYPES AND SYNAPSES. BEYOND CELL TYPES OF THE NETWORK LEVEL WE CAN -- IT IS IMPORTANT TO INVESTIGATE FOR A PARTICULAR REGION THE DIVERGENT AND CONVERGENT CONNECTIONS. AND FURTHERMORE AMONG MULTIPLE REGIONS AND AT A DIFFERENT LEVEL OF CONNECTIONS OF THE NETWORK TO LEARN ABOUT A HIGHER ORDER OF NETWORKING TOOLS TO GIVE YOU EXAMPLES REGARDING THE HIGHER ORDER NETWORK AND ONE SINGLE EXAMPLE IS THE RECIPROCAL CONNECTIONS. I USED THE SOMATO SENSORY CORTEX AS AN EXAMPLE. AND THREE NEURONS FROM THE CORTEX AND PROJECTIONS AND PRIMARY MOTOR CORTEX AND SECONDARY OR SUPPLEMENTAL SOMATO SENSORY CORTEX AND WE KNOW NOW THAT THOSE TWO DIFFERENT PROJECTION PATHWAYS ARE LIKELY MEDIATED BY DIFFERENT NEURONS IN PRIMARY SOMATO SENSORY CORTEX. AT THE SAME TIME, PRIMARY MOTOR CORTEX AND SECONDARY SOMATO SENSORY CORTEX SENDS FEEDBACK CONNECTIONS TO PRIMARY SOMATO SENSORY CORTEX AND THE QUESTION HERE IS WHAT EXAMPLE IS THOSE TWO DIFFERENT KINDS OF FEEDBACK CONNECTIONS PROJECTIONS, DO THEY TERMINATE ON ALL KINDS OF CELLS WITHIN PRIMARY SENSORY CORTEX OR WHETHER EACH PATHWAY TERMINATES ONLY ON THE SPECIFIC SUBSET OFF THE CELLS THAT PROJECT TO THOSE HIGHER ORDER AREAS. IN OTHER WORDS, WHAT ARE THE RECIPROCAL NETWORKS ARE REGION SPECIFIC OR BOTH REGIONAL AND CELL SPECIFIC. REFINED SUBNETWORKS AT THIS KIND OF INTERAREA CONNECTION LEVEL. THAT EXAMPLE IS THE FURTHER ELABORATION OF THAT IS MULTI STEP CONNECTIONS OF A PARTICULAR REGION THAT RECEIVE INPUTS FROM A WIDE RANGE OF UP STREAM REGIONS AND CELL TYPES AND ALSO SENDS OUT OUTPUT CONNECTIONS TO A NUMBER OF DOWN STREAM REGIONS AND POSSIBLY CELL TYPES THE CONVERGENT AND DIVERGENT CONNECTIONS AT THE SAME TIME AND ARE THERE MULTI STEP CONNECTIONS? NAMELY DO THE INPUT CONNECTIONS TARGET THE REGION IN A PROMISCUOUS WAY AND IF THE OUTPUT IS NON-DISCRIMINATIVE IN A BROADCAST MANNER OR IF THERE ARE NETWORK AS INDICATED BY THE YELLOW ARROWS AND GREEN ARROWS AND GREEN CELLS HERE. THAT ABROW PROCESSING. -- ALLOW PROCESSING CENTERS. THERE'S FUNCTIONAL CONNECTISTS -- CONNECTIVITIES IN THE CELLS WE'LL BE INVESTIGATING AND THIS KIND OF NETWORK PROCESS CAN BE SHAPED AS YOU CAN IMAGINE BY PLASTICITY AND LEARNING. TO RE-UNDERSTAND AND -- TO IDENTIFY AND UNDERSTAND THE PATTERNS AND RULES OF THE NETWORKS AND THE POSSIBLE EXISTENCE OF SUBNETWORKS WE THINK IT'S NECESSARY TO TRACE SINGLE AXONS FROM THE STARTING CELLS THEMSELVES ALL THE WAY TO THE TERMINAL SYNAPSE. AND WE NEED TO TRACE MULTIPLE CELLS FROM THEIR SOURCE REGIONS INTO THE CONVERGING TARGETS AND TRACE THE MULT INPUT CELLS SIMULTANEOUSLY IN ORDER TO UNDERSTAND THEIR RELATIONSHIP. THIS ARGUES FOR WHOLE GRADE LEVEL DENSE RECONSTRUCTION. >> TO SUMMARIZE THE IDEA OF LOCAL AND LONG RANGE SUBNETWORKS HONGKUI JUST INTRODUCED WE HAVE CORTICAL LAINAE AND INPUT CELL TYPES AND TARGET CELL TYPES SHOWN IN SHAPE AND BORDER PATTERNS. THE CURRENT STATE OF THE ART IS WE ARE JUST BEGINNING AND REALLY MAKING PROGRESS ON DESCRIBING LOCAL NETWORKS AS A FUNCTION OF THESE TYPES. LARGER SCALE VOLUMES ARE ACROSS LARGER DISTANCES. WHAT WE'RE TALKING ABOUT IS REALLY BEING ABLE TO DESCRIBE NETWORKS ACROSS BRAIN REGIONS AND THAT WOULD BE A FANTASTIC ACCOMPLISHMENT IF WE COULD ACHIEVE IT. AND THE CRITERIA IS USEFUL AND IT'S DESCRIBED AS A COMPLETE ACCURATE AND PERMANENT DATA SET AS BEING THE GOAL FOR ANY LARGE SCALE ENDEAVOR SUCH AS THIS. BUT THERE'S A BIG QUESTION ABOUT WHAT COMPLETE MEANS. IF WE DID A WHOLE MOUSE BRAIN WE CAN PROBABLY GET A DIAGRAM WIN SYNAPTIC RESOLUTION TO BE AN INCREDIBLE ACCOMPLISHMENT BUT THERE'S A SPINAL CORD AND WHOLE BODY AND ALL THIS MATTERS AND ARGUABLY THAT WOULD BE COMPLETE AND SHOULD WE GET THIS AND AS FAR AS NEUROTRANSMITTER AND GAP JUNCTIONS AND SO ON AND SO FORTH. WE MAY NOT GET EVERYTHING WE WANT IN A SINGLE WHOLE MOUSE DATA SET BUT SHOULD OUR SAMPLE PREPARATION PIPELINE GEAR TOWARD COMPLIMENTING IT WITH ADDITIONAL DATA SETS AND I WANT TO GIVE EXAMPLES OF INTEGRATION ACROSS DATA SETS WITHIN THE SAME SAMPLE. THERE'S MANY WAYS IT DO THIS AND SHOW ONE TO FRAME IT. YOU CAN TAKE IN VIVO IMAGING HERE CALCIUM IMAGING USING A GENETICALLY CODED INDICATOR LABELLING THE VASCULATURE AND TAKE A SECTION AND DO SINGLE PHOTON IMAGING OF THE SAME BRAIN. HERE LOCAL AND LONG-RANGE TARGETS ARE LABELLED IN RED AND BLUE. YOU CAN SCAN THAT SECTION USING MICRODATA SET. AND MICROSCOPY IS SOMEWHAT COARSE AND LOW RESOLUTION AND YOU CAN IMAGE IT FAST AND SEGMENT IT. IF YOU MADE A GOOD ENOUGH SAMPLE IT MAY PAY TO GET A HIGHER RESOLUTION SO THAT YOU CAN SEE THE DETAILS OF SYNAPSES AND OTHER STRUCTURE TO LOOK AT SYNAPTIC WEIGHT. YOU MAY WISH TO INTEGRATE DATA SETS USING DIFFERENT PROTOCOLS. IN WORK FROM A LAB RECENT DATA SHOWED CRYOFIXATION HAS A DIFFERENT STRUCTURE WHICH CHANGES THE BIO PHYSICAL MODELS. IN SUMMARY, THIS PORTION DATA QUALITY IS A FUNCTION OF ENTIRE PIPELINE. PROFUSION, FIXATION, STAINING, EMBEDDING, SECTIONING, IMAGING, ALIGNMENT AND SEGMENTATION AND THE DESIGN OF THE OVERALL PIPELINE AND SAMPLE PREPARATION NEED TO SHOW HOW YOU'LL DO CROSS-MODAL LABELLING. THE WORKSHOP IS DIVIDED INTO SECTION ONE TO DESCRIBE AND DISCUSS CONTINUOUS WHOLE BRAIN CONNECTOMICS AND A KEY OVERRIDING QUESTION BEHIND ALL OF THESE IS WHETHER WE CAN DO IT AT ALL. IN SESSION IT WE'LL DISCUSS WHOLE BRAIN IMAGING IN MOUSE AND LARGER SPECIES AND AGAIN A SET OF QUESTIONS SHOWN HERE DISSEMINATED TO EACH OF THE SPEAKERS AND THEN HAVE AN INTERACTION PANEL WHERE SPEAKERS AND DISCUSSANTS WILL BE ABLE TOHILL KEY TAKEAWAYS. THE OVERALL TOPIC IS TO CREATE WHOLE MOUSE CONNECTOMES AND WHOLE BRAIN CONNECTOMES. THE FEASIBILITY FOR IMAGING PLATFORM SELECTION AND SAMPLE SELECTION CRITERIA AND ATTEMPTING TO SCALE TO MULTIPLE BRAINS. SO IN THE DISCUSSION, WE WILL TRY TO SUMMARIZE THE CURRENT STATE OF THE ART, IDENTIFY KEY ISSUES AND ARTICULATE THE NEEDS AND ADVANTAGES OF COMPLIMENTARY DATA TYPES BEYOND ELECTROMICROSCOPY AND DEFINE LIMITATIONS AND OPPORTUNITIES IN MOVING TO LARGER BRAINS INCLUDING NON-HUMAN PRIMATES. >> GOOD MORNING. I WILL NOW HIGHLIGHT A FEW LOGISTICAL DETAILS TO MAXIMIZE PARTICIPATION IN TODAY'S WORKSHOP. MANY ARE ATTENDEES IN THE ZOOM WEBINAR AND MANY THROUGH THE VIDEOCAST. BOTH CAN ACTIVELY CONTRIBUTE TO THE WORKSHOP. LET'S BEGIN WITH TIPS. AS A ZOOM ATTENDEE, PLEASE USE THE Q&A BOX TO SUBMIT QUESTIONS FOR EXAMPLE FOR ANY OF THE QUESTION AND ANSWER SESSIONS OR DURING THE DISCUSSION PANEL. YOU MAY ALSO USE THE CHAT BOX TO SEND MESSAGES OR COMMENTS. YOU CAN CHAT WITH SPECIFIC PANELISTS OR ALL PARTICIPANTS IN THE ZOOM. USING THE CHAT YOU CAN INDICATE YOU HAVE A QUESTION OR COMMENT AND WOULD LIKE TO BE UNMUTED TO SPEAK. IF TIME PERMITS AND AT THE DISCRETION OF THE MODERATORS, YOU'LL RECEIVE A PROMPT TO UNMUTE AND SPEAK. WHEN UNMUTED, YOUR PROFILE PICTURE AND NAME WILL BE DISPLAYED AND ALL SPOKEN COMMENTS RECORDED AS PART OF THE VIDEOCAST. HERE'S A FEW TIPS FOR THOSE VIEWING THE HIP -- NIH VIDEOCAST THE QUESTIONS AND ANSWERS ARE LIVE. VIDEOCAST VIEWERS CAN SUBMIT QUESTIONS AND COMMENTS VIA THE SEND LIVE FEEDBACK BUTTON ON THE VIDEOCAST PAGE. WE'LL ADDRESS AS MANY SUBMISSIONS AS POSSIBLE DURING THE PANEL DISCUSSION. FOR QUESTIONS AND COMMENTS WE'RE UNABLE TO GET TO DURING THE WORKSHOP BE AWARE WE'RE SEEKING INPUT VIA AN ONLINE PLATFORM CALLED IDEA SCALE IT'S A GREAT WAY TO SHARE YOUR VIEW ON THE MAPPING OF MAMMALIAN BRAIN CIRCUITRY AND FOR IDEAS FOR ADVANCING THE CHALLENGES THAT WILL BE ADDRESSED TO CREATE COMPREHENSIVE MAPS BRAIN CONNECTIVITY. SEE THE JOINING THE DISCUSSION PAGE. ONCE YOU REGISTER FOR THE CAMPAIGN, YOU'LL BE ABLE TO LOG BACK INTO THE CAMPAIGN ANY TIME VIA THE CAMPAIGN LOG IN BITTON ON THE OVERVIEW PAGE. -- BUTTON. HERE'S A FEW NOTES HOW IT WORKS. AFTER REGISTERING FOR THE IDEA SCALE PLATFORM HOSTING THE CAMPAIGN, YOU CAN START CONTRIBUTING IN THREE EASY STEPS. FIRST GO TO ACTIVE CAMPAIGNS SHOWN AT THE BOTTOM. SELECT BRAIN CONNECTIVITY WORKSHOP SERIES. SECOND, CHECK OUT RECENT OR TRENDING IDEAS. FINALLY, TO SUBMIT AN IDEA CLICK SUBMIT NEW IDEA BUTTON AND YOU'LL BE GUIDED FROM THERE BUT REMEMBER THE JOIN THE DISCUSSION PAGE CONTAINS DISCUSSIONS ABOUT THE PLATFORM TO FURTHER ASSIST YOU. ONE REMINDER TO ALL, TO AVOID CURTAILING SCIENTIFIC DISCOURSE PREMATURELY KEEP IN MIND ALL WORKSHOP DISCUSSIONS SHOULD FOCUS ON THE SCIENCE AND TECHNOLOGY AT HAND RATHER THAN BUDGETS, OPPORTUNITY COSTS AND SIMILAR ISSUES. FINALLY, BEFORE WE HEAR ABOUT OUR FIRST SET OF SPEAKERS I WANT TO THANK THOSE WHO MADE THE HAVEN'T POSSIBLE INCLUDE THE DOE PLANNING GROUP, WORKSHOP CO-LEADS, OUR CONTRACTOR AND VIDEOCAST STAFF AND ALL THOSE YOU'LL BE HEARING FROM TODAY, SPEAKERS AND DISCUSSION PANELISTS AND OTHER PARTICIPANTS WELLS A FEW PEOPLE YOU MAY NOT HEAR FROM WORKING TIRELESSLY BEHIND THE SCENES, DR. AMANDA PRICE MY CO-COORDINATOR AND OUR TWO CO-CHAIRS, DR. HARRIS AND PETER LONG. NEXT WE'LL HEAR FROM DR. DAVI BOCK. >> HELLO. I'LL LIKE TO ECHO THE PREVIOUS SPEAKERS AND THANK EVERYBODY FOR PARTICIPATING. I THINK WE HAVE A NICE LINE UP FOR THE WORKSHOP I'D LIKE TO INTRODUCE THE SPEAKERS FOR SESSION ONE FOCUSSING ON WHOLE MOUSE BRAIN ELECTRON MICROSCOPY AND WE HAVE XIAOTANG BUT AND NUNO RAS -- MACARICO AND ERIC BUSHONG AND THEY'RE CLOSE TO THE BENCH AND DOING HANDS ON WORK ROUTINELY AND I THINK THAT WE'LL SEE THAT REFLECTED IN THEIR TALKS. SO WITHOUT FURTHER ADO, LET'S ROLL RIGHT IN. >> SO ZA I'LL BE TALKING ABOUT -- TODAY I'LL TALK ABOUT ME WHOLE MOUSE CONNECTOME AND THERE'S A GOLD STANDARD AND CAN BE USED FOR THE WHOLE BRAIN CONNECTOME PROJECT AND THERE'S STAINING FOR THE FULL CONNECTOME. FIRST, MEMBRANES MUST RETAIN HIGHER CONTRAST AND AND HUMAN TITERS BELONG TO DIFFERENT CELLS. SO RELIABLY IDENTIFYING MEMBRANES IN THE FIRST STEP AND HIGH MAIM BRAIN CONTRAST WOULD FACILITATE THE MEMBRANE IDENTIFICATION AND THE STAINING MUST BE INTENSE SO IMAGING CAN OCCUR REASONABLY FAST. THE MORE METALS WE PUT IN THE TISSUE THE STRONGER SIGNALS CAN BE ACHIEVED AND FASTER THE IMAGING WILL OCCUR. IT WILL PROBABLY TAKE SEVERAL DECADE SO ANY IMPROVEMENT WOULD BE POSITIVELY IMPACTED IN THE IMAGING EFFORT. AND THIS METHOD MUST BE SCALEABLE IN LARGE VOLUMES. THIS ONE IS THE MOST CHALLENGING PART AND I WILL DIVE INTO MORE DETAILS TODAY. WE CREATE A CONTRAST IN THE EM AND IT ATTRACTS IN THE IMAGES. IN THE TWO ROUND APPROACH WE CAN PRESENT NEW BINDING SITES FOR ADDITIONAL OSMICATION AND THIS WOULD REDUCE THE SITES INTO THE STA STATE. THIS MOLECULE IS IN FATTY ACIDS. THIS TOO RUNS IN CONTRAST AND USED FOR THIS PREPARATION AND THERE ARE PROTOCOLS. ONE IS THE MOST WIDELY USED AND COMPARING WITH OTHER TOOLS THIS OFFER THE CONTRAST AND THE STAINING MEETS THE FIRST TWO REQUIREMENTS I MENTIONED HOWEVER, IT DOES NOT PROVIDE STAINING FOR LARGE BRAIN BLOCKS. TO INCREASE THE STAINING IN 2015 THE LAB REPORTS A PROTOCOL I LIKE TO REFER TO IT TO ORTO AND USED OSMIUM AND IN THE PROTOCOL THE TISSUE WAS TRANSFERRED DIRECT DIRECTLY. THIS IS CRUCIAL FOR INCREASING THE STAINING PENETRATION. THE CONTRAST CAN BE OBTAINED THROUGH THE TISSUE BLOCK. THE MAIN DRAW BACK COMES PERFECT THE USE OF PCH. IT'S A REACTION AND CAUSES WHICH RESULTS IN A LARGE TISSUE FLUX. AND OF THE THE MOLECULES WE IDENTIFIED PYROGALLOL AND THIS CAUSED SOME ISSUES AND THE PROTOCOLS COULD MEET SOME BUT FOT ALL THREE REQUIREMENTS. SCALI SCALING AND A NEW PROTOCOL IS REQUIRED FOR THE WHOLE MOUSE CONNECTOME AND I DECIDED TO DIVIDE IT INTO TWO STEPS. AND HERE'S FOUR TOOLS I WANT TO EMPHASIZE TODAY THE EXTRA MICROSCOPE. HERE THE TISSUE STAINING IS LIKE A BLACK BOX TO US. WE HAVE NO KNOWLEDGE OF WHAT HAPPENS AND GET THE FINAL BLOCK AND CUT IT AND IMAGE IT AND THERE'S ADVANTAGES BECAUSE THE PROCEDURE TAKES WEEKS TO FINISH. SO TO CHANGE THE SITUATION I INCORPORATED MICROCT IN MY EXPERIMENT TO MONITOR THE REACTION. I PUT MY SAMPLE HERE AND SCAN THE X-RAYS TO OBSERVE THE PROCESS AND DETERMINE WHAT HAPPENS IN THE REACTION. BESIDES THE STAINING DURATION IT CAN HAVE PROFOUND INFORMATION ABOUT THE REACTION AND THIS INFORMATION IS CRUCIAL FOR US TO REALLY UNDERSTAND THIS CENTURY OLD SHAPE CAN OPTIMIZE THE STAIN. THE NEW PROTOCOL THAT I'VE BEEN DEVELOPING I LIKE TO CALL IT [INDISCERNIBLE] AND THE FIRST STEP IS OSMIFICTION. AND HERE'S VIDEO OF THE FIRST. YOU CAN SEE THE DIFFUSION OF OSMIUM IN THE BRAIN AND IT'S EASY TO KNOW FROM THE VIDEO THE OSMITATION TAKES ABOUT PO -- 30 HOURS TO COMPLETE AND THE SECOND STEP IS THE BUFFER. HERE'S A VIDEO OF THE REDUCTION WHAT MIGHT BE COUNTERINTUITIVE IS THE BRAIN IS GETTING LIGHTER FROM THE EXTERIOR TO THE INTERIOR AND IT'S SLOWER THAN OSMIUM BINDING I SHOWED IN THE LAST VIDEO. THIS REVEALS OSMIUM REDUCTION WHICH I DON'T THINK HAS BEEN THOROUGHLY UNDERSTOOD IN THE PAST. AND WHERE OSMIUM IS MIXED WITH OSMICATION AND THIS IS WITH UNSATURATED FATTY ACIDS SO THE REDUCED THOUGH IT'S EASY TO GET TO OSMIUM DIOXIDE IS NOT WATER SOLUBLE. IT LEADS TO THIS LAYER AROUND THE MICRON AND PREVENTS FURTHER STAINING. HOWEVER, IF WE SEPARATE OSMICATION OSMIUM SPECIFICITY IS NOT AS HIGH AS IN THE REDUCED FORM SO IT BINDS TO BOTH LIPIDS AND OTHER MOLECULES IN THE CYTOSOME. AND THIS IS WATER SOLUBLE AND CAN BE WASHED OUT OF THE TISSUE WHY WE SEE THE BRAIN GETTING LIGHTER IN THE STEP. AND THE OSMIUM CAN BECOME LESS SOLUBLE. AND THIS CAN BE MORE EFFICIENT. OKAY, THE NEXT IS LIGAND BINDING. WE HAVE TWO CHOICES. AND YOU CAN SEE FROM THE CROSS SECTIONS WHICH USES PCH, ONE AVOIDING THE MICROBUBBLE FORMATION AND PREVENTS CRACKING AND REDUCES [INDISCERNIBLE] CAUSED BY TCH. IN MY PROTOCOL I SIT WITH PYROGALLOL AND THE TOGETHER AND THE GENETICS IS SIMILAR TO THE FIRST OSMICATION AND TAKES ABOUT 30 MINUTES TO COMPLETE THIS REACTION. AND I'D LIKE TO TALK ABOUT THE OUTCOMES FOR THE SMALL TISSUES AND IN LARGE TISSUES LIKE THE WHOLE BRAIN TAKING A BUFFER AS AN EXAMPLE I KEPT USING THE BUFFER IN EVERY STEP EXCEPT PYROGALLAL BECAUSE IT'S AN ORGANIC ASIT BUT BEFORE AND AFTER PYROGALOL I KEPT USING THE BUFFER. YOU CAN SEE THE CHANGE HERE DURING THIS TRANSITION THE LAST THING I'D LIKE TO EMPHASIZE IS THE IMPORTANCE MUCH MONITORING EVERY DETAIL IF YOU WANT TO GET A PERFECTLY STAINED BRAIN. HERE'S A PRELIMINARY RESULT. ONE OF THE CHANGES I WAS EXPECTED TO ADDRESS IN TODAY'S TALK IS HOW TO ASSESS THE PREPARATION PRIOR TO IMAGING AND X-RAY TOMOGRAPHY WOULD BE USEFUL. I'D LIKE TO SEE THE UNIFORM CONTRAST THROUGHOUT THE BRAIN AND IN TRACKS FOR A GOOD PREPARATION. HERE'S SOME IMAGES OF THE MOUSE BRAIN I STAINED THIS IS TOM TOMOGRAPHY OF THE STAINED BRAIN AND FROM THE INTERIOR AND THE TWO HIGH RES IMAGE ON THE RIGHT WERE TAKEN AND YOU SEE THE MEMBRANE CONTRAST THAT IS COMPARABLE TO THE PROTOCOL. SO IN SUMMARY I INTRODUCED THE WHOLE BRAIN STAINING PROTOCOL AND COMBINES THE AND THE RESULT IS PROMISING AND SECOND IT'S POWERFUL FOR ADJUSTING THE REACTION AND INTERPRET MECHANISMS. AND I LOOK AT CHANGES LIKE HYDRATION AND THE ADULT BRAIN TAKES 46 HOURS TO COMPLETE AND THE WHOLE PROCESS WILL TAKE SIX WEEKS AND SLOW BUT WE'LL GET THERE. IN TERMS OF ACKNOWLEDGEMENT I WANT TO THANK DR. WHITMAN FOR HIS SUPPORT AND ADVICE THROUGH THE DISCUSSION AND OTHERS FOR PROCESSING AND I WANT TO THANK ADRIAN WARNER AT PRINGS TON UNIVERSITY THE FIRST ONE PROPOSED FOR WATCHING STAIN AND THANK YOU FOR YOUR ATTENTION. I'M HAPPY TO TAKE QUESTIONS IN THE Q&A SESSION. >> WELCOME, EVERYONE. THANK YOU TO THE OPPORTUNITY OF PRESENTING ON THE WORKSHOP. HI MY NAME IS NUNO MACARICO AND IN TERMS OF THE QUESTIONS THE ORGANIZERS ASKED TO ADDRESS IS THE CONNECTOMICS PIPELINE DEVELOPED DURING THE PROGRAM WHICH HAS BEEN A WONDERFUL COLLABORATION BETWEEN THE TEAM IN THE COLLEGE OF MEDICINE WHICH SENT THE MICE TO US AT THE INSTITUTE PREPARED THE SAMPLE AND CUT IT AND IMAGED IT AND STICHD THE IM -- STITCHED THE IMAGES AND WE HAD BEAUTIFUL RECONSTRUCTIONS AND THIS IS A LAYER FIVE IN THE PRIMARY CORTEX OF THE MOUSE AND THE DENDRITES ARE IN BLUE AND THE AXON IN PURPLE AND WE CAN ZOOM IN AT ANY PART OF THE AXON AND GO TO A SIGN UP AND LOOK AT THE SYNAPTIC CELLS WHICH WILL BE AN EXCITATORY NEURON IN THE SECONDARY AREA. AND THIS DATA SET HAS ABOUT 100,000 NEURONS AND THOUGH THIS IS PROBABLY THE MOST COMPLICATED CONSTRUCTION OF THE LAYER FIVE NEURON IT'S A PORTION AND YOU CAN SEE IT'S RECONSTRUCTED AND IN A WAY PART OF SCALING FROM THE GOLD STANDARD IN THE MIRE -- MICRO RONS IS THE WHERE WE MOVE FROM THE PHASE 1 DATA SET THE GOLD STANDARD AT THE TIME AND NOW TO THE PHASE IT DATA SET AND SCALING TO THE PULL MOUSE BRAIN. DURING MY PRESENTATION I'LL FIRST TALK ABOUT SAMPLE PREPARATION NOT AS A RELATED STEP BUT IN THE CONTEXT OF THE FULL PIPELINE AND OUR EXPERIENCE IN DOING THAT AND THEN I'M GOING TO DISCUSS THE CONTINUITY OF RECONSTRUCTIONS WITHOUT CONTINUITY OF IMAGES. I'LL STOP BY SHOWING THE PIPELINE TO POINT OUT THAT SAMPLE PREPARATION IS CRUCIAL FOR EVERYTHING THAT HAPPENS DOWN STREAM AND SUCCESS IS ONLY MEASURED AND EVALUATED WHEN WE GET TO THE LAST ROW WHEN WE GET TO DO DATA ANALYSIS. SO AGAIN IT'S CRUCIAL HISTOLOGY IS NOT AN INSULATED STEP BUT DONE IN THE CONTEXT OF WHAT HAPPENS AND CONSIDERED A MEASURE OF SUCCESS AND IN THIS SLIDE SHARING OUR VIEW OF IMPORTANT STEPS THAT WE TOOK AND FROM OUR EXPERIENCE TOO PREPARATION AND IN THE CONTEXT OF THE PIPELINE AND THE FIRST IS IMPORTANT TO DEFINE SUCCESS AND HAS BEEN DISCUSSED EXTENSIVELY IN THE FIRST WORKSHOP IN OUR CASE WAS FINDING ALL THE NEURON AND DURING THAT WE DID A CERTAIN TIME. AND THIS IS A BIG OF A CONSTRAIN OR THE MAPPING OF THE SYNAPSES ITSELF. AND THIS BRINGS FOCUS AND UNDERSTAND THE TRADEOFFS AND TO SAY THE DATA SET IS NOT USEFUL FOR OTHER THENGS IT'S JUST THE OPTIMIZATION IS FOCUSSED AND THE TESTAMENT TO THAT IS AT LEAST HALF THE PAPERS I'VE BEEN SUBMITTING ARE IN THE PROCESS OF BEING WRITTEN ARE NOT ON CONNECTIVITY BUT CELL TYPES AS DIVERSE AS GLIAL CELLS OR ORGANELLE CELLS. ONCE WE OPTIMIZE FOR THIS SUCCESS YOU OPTIMIZE IT IN THE CONTEXT OF THE OLD PIPELINE AND THIS IS SOMETIMES QUITE A DIFFICULT DECISION BUT IMPORTANT. FOR EXAMPLE, EVEN THOUGH WE COULD USE STAINING TO INCREASE THE CONTEXT OF OUR SAMPLES WE DECIDED NOT TO DO IT BECAUSE GIVEN THE TIME WE HAVE THAT WOULD INCREASE THE RISK OF THE PROJECT. AND THIS HELPS MAKE THE REST OF THE PROCESS MUCH MORE PREDICTABLE AND YET INCREASES THE RISK AND ONCE YOU HAVE THAT YOU CAN COMPLETE THE PIPELINE AND WE TRIED TO DO THIS AS EARLY AS POSSIBLE SO WE COULD SEE WHERE WERE FAILING AS EARLY AS POSSIBLE AND CORRECT FOR THEM AND THIS NOT ONLY IMPROVES THE CHANCES OF SUCCESS AND WE HAVE THE FIRST DATA SET IN PHASE 1 IT WAS BY FAR IN THE MOST PERFECT DATA SET BUT ALLOWED US TO GO THROUGH THE PROCESS AND IT RATING THE PIPELINE ALLOWS US TO UNDERSTAND THE COST AND MAKE CORRECTIONS AND DECISIONS PAVE ON THAT AND GET SUPPORT. IT'S IMPORTANT TO HAVE A BACK UP PLAN AND WE HAD MANY THOUGH WE CHOOSE TO SELECT MICROSCOPE BECAUSE OUR SAMPLE PREPARATION WAS EQUALLY EFFECTIVE FOR SCANNING MICROSCOPY AND BEFORE WE DECIDED ON A PRODUCTION SAMPLE WE DID SECTIONING AND THIS IS AN ASPECT AND IT WAS IMPORTANT AND TOOK LONGER THAN WE SHOULD HAVE TAKEN AND CONSISTENT RELIABILITY ARE KEYS AND PROTOCOLS WE USED PROVIDED NOT ONLY MOST THE ANIMALS WERE SUCCESSFUL BUT THE SAMPLES LOOKED SIMILAR AND HERE'S AN EXAMPLE FROM DIFFERENT MICE AND THE PROTOCOL WAS DEVELOPED BY OUR TEAM AND BASED ON THE PROTOC PROTOCOL IS THE MOST RELIABLE THIS ALLOWS US NOT TO HAVE A PIPELINE RELIABLE AND CONSISTENT FOR THE PROJECT BUT ALLOWED US TO REPRODUCE A COLLECTION OF THE DATA SETS ON DIFFERENT REGIONS OF THE MOUSE BRAIN AND ACROSS SPECIES WITH AND SPEAKS TO THE DISCUSSION OF THE WORKSHOP THAT CREATING SOMETHING THAT IS RELIABLE AND CONSISTENT AND ALLOWS US THEN TO MAKE THE SECO SECOND, THIRD AND FOURTH SAMPLES MUCH EASIER. AND WE HAD AN EVALUATION OF SAMPLES WITH THE SAME SOLUTIONS OUR PRODUCTION SAMPLES AND LOOKED AT THE PRODUCTION SAMPLE WITH THE MICRO CT AND EVALUATE THE STAINING. THIS WAS A LONG PROCESS TO MAKE SURE THE PRODUCTION SAMPLES WERE THE QUALITY WE WANTED AND WE WERE ABLE TO IMPROVE THE SAMPLE PREPARATION ITSELF. I'M EXCITED WITH THIS WORK OF THE TEAM USING MICRO CT TO ASSESS STAINING AND YOU SEE THE COMPLETE MOUSE BRAIN BEING SAMPLED WITH THE MICRO CT WHILE BEING OSMUTATED AND WE CAN TUNE IN TO THAT IMPROVEMENT AND EARLY ON KNOW IF THE SAMPLE IS GOOD ENOUGH AND NOT WASTE TIME ON THE PROTOCOLS. THE LAST QUESTIONS ARE THAT THE METHODS ARE SUITABLE FOR ALL MOUSE BRAIN CONNECTOMICS PRIOR TO IMAGING AND THIS MEANS WE HAVE TO BLOCK THE BRAIN IN DIFFERENT PARTS AND WHEN WE BLOCK THERE'S ALWAYS TISSUE THAT IS LOST AND SO WE'LL BE ABLE TO LOOK AT STRATEGIES AND I'M GOING ARGUE ON TOP OF THAT WE SHOULD THINK OF WAYS TO BRIDGE THE GAPS COMPUTATIONALLY BECAUSE AT THE END THE SUCCESS IS MEASURED BY THE RECONSTRUCTION AND NOT THE IMAGES OF THE TISSUE AND MANY TESTED THE CONCEPT OF CELL TYPES AND HAVE KNOWN FOR A LONG TIME THERE'S DIFFERENT MORPHOLOGIES TO START AND AS WE BUILD TO THE BLOCKS IT HAS TO RESEMBLE THE MORPHOLOGY AND THAT'S TRUE OF THE GROSS MORPHOLOGY COMPARING THE NEURON WITH THIS COMPLETE MORPHOLOGY AND THERE'S A DIFFERENT BETWEEN CELL TYPES EVEN AT THE MICRO LEVEL AND ONE EXAMPLE IS THE SEGMENT OF CELLS AND THEY TARGET THE SEGMENTS TO HAVE A CLEAR PATTERN OF CONNECTIVITY SO WHEN THEY CROSS THE BRIDGE FROM BLOCK TO BLOCK WE SHOULD LOOK FOR NEURONS WITH THE SAME PATTERN OF CONNECTIVITY. AND THE NICE THING IS WITH THE MICRORON DATA SETS WE HAVE LARGE RECONSTRUCTION FROM EXCITATORY CELLS AND DIFFERENT CELLS AND SO FORTH THAT ARE LARGE ENOUGH TO GATHER KNOWLEDGE OF THEIR CONNECTIVITY AND MORPHOLOGY BUT WE HAVE A PLAYGROUND WHERE WE CAN REMOVE SECTIONS OF A PARTICULAR TYPE OF DIMENSION, 15 NANO METERS AND SEE WHAT KIND OF BRIDGE ON THE BLOCKS CAN WE ALLOW AND TISSUE CAN WE ALLOW TO STILL BRIDGE AND RECOVER THE RECONSTRUCTION OF NEURONS. I'D LIKE TO FINISH BY THANKING EVERYONE. >> I'M ERIC BUSHONG IN IN THE NATIONAL CENTER FOR MICROSCOPY AND IMAGING RESEARCH. WE STARTED WITH IMAGING SYSTEMS AND HAVE TOMOGRAPHY AND USE X-RAY MICROSCOPY A LOT AND SO WE HAVE DIFFERENT ORGANISMS AND WE SPENT A LOT OF TIME LOOKING AT MOUSE BRAINS AND ASKED TO TALK ABOUT HOW WE PREPARE SPECIMENS TO MAXIMIZE PRESERVATION IN STAINING FOR IMAGING. YOU CAN SEE OTHER THAN A LITTLE BIT OF CHARGING IN THE CELL THE BULK OF THE SPECIMEN HAS NO CHARGING ON EFFECTS AND ALL THE CELLULAR PROCESSES SEEM TO BE EXTRACTED AND AND YOU CAN SEE A TON OF DETAILS HERE AND IN PARTICULAR THE MEMBRANES ARE INTACT AND SMOOTH AND THE MITOCHONDRIA LOOK HAPPY AND YOU SEE THE NUCLEAR PORES AND DETAILS IN THE SPECIMEN DUE TO THE WAY IT WAS PRESERVED AND STAINED AND CAN BE CAPTURED IN THE IMAGING. THIS IS WHAT I CALL GOLD STANDARD FOR A WELL STAINED MOUSE BRAIN. AND I'LL SPEND THE NEXT 10 MINUTES IN TALKING ABOUT THE KEY STEP FOR THE STRUCTURE. AN ELECTROMICROSCOPY IS NOT THE SAME AND I WANT TO POINT OUT THE KE TAIL -- DETAILS AND PERFUSION PRESSURE SHOULD BE HIGH ENOUGH TO MAKE SURE BLOOD IS WASHED OUT AND NOT SO HIGH YOU'RE POPPING BLOOD VESSELS. YOU WANT TO MAKE SURE THE SOLUTION IS OXYGENATED TO HELP WITH THE FLUSHING PROCESS AND AS FOR THE FLUSHING THERE'S ONLY A COUPLE MILLILITERS OF BLOOD IN THE MOUSE THEN WE HAVE CALCIUM AND IT AVOIDS WHOSE -- HOLES THAT CAN DEVELOP IN THE BRAIN. AND WE BEGIN CHILLING THE BRAIN AS SOON AS POSSIBLE SO EVEN DURING THE PERFUSION WHEN YOU TAKE IT OUT OF THE SKULL IT SHOULD BE COLD BECAUSE THAT'S WHEN YOU FIRST START FIXING THE MEMBRANES AND CELLS. AND WE ALWAYS MAKE SURE WE USE THE GLUTARALDEHYDE. AND MOST LABS INCLUDING OUR LAP MOST THE TIME DO THE PROCESS ON ICE BUT IT HAS BEEN SHOWN A PROGRESS LOWERING OF TEMPERATURE PROTOCOL CAN BE ADAPTED AND I'D LIKE TO SHOW THE TISSUE FOR VOLUME IMAGING. THEY DON'T ALLOW THIS AND DON'T ALLOW FOR THE GENETIC CODE. THE PROTOCOL WE DEVELOPED ALLOYS YOU TO DO BOTH. HERE YOU SEE A SLICE OF THE MOUSE BRAIN FIXED WITH ALDEHYDES AND YOU CAN SEE THROUGHOUT THE VOLUME THERE'S AXONS THE MYELIN SHEETS ARE INTACT AND THE SYNAPSES ARE EASY TO DETECT I'LL MOVE TO THE HEAVY METALS AND BEFORE I TALK ABOUT THAT THE STAINING PROTOCOL WILL BE INFLUENCED BY THE IMAGING AND PUT THE TIRB TUE AND IMAGE THE BLOCK AND MAKE SURE IT WAS SUFFICIENTLY STAINED AND THEN YOU HAVE OTHER APPROACHES WHERE WE CUT A SERIES OF SECTIONS FROM THE SPECIMEN AND FOR THESE APPROACHES YOU CAN DO A LOT OF HEAVY METAL STAINING AND YOU DON'T NEED TO ACHIEVE THE HEAVY STAINING OF THE BLOCKED TISSUE AND HAVE HYBRID APPROACHES AND THEY CUT BIGGER SECTIONS AND DO IMAGING ON THEM AND THERE'S OTHER CONSIDERATIONS AND FACTORS DETERMINING WHAT IT SHOULD BE AND I WANT TO POINT OUT YOU CAN STAIN SECTIONS UP TO A FEW MICRONS THICK AND IN DOING BLOCK BASED IMAGING WE STAIN ON BLOCK PRIOR TO IMAGING WE DEVELOPED A PROTOCOL SEVERAL YEARS AGO FOR REDUC REDUCED OSMIUM POE -- POTASSIUM FER FER FERROCYANIDE AND THEN YOU THEY ARE DOWN A THIOCARBOHYDRAZIDE AND THIS IS THE RESULT I GAVE AT THE BEGINNING. THE CON SIDE IS IT ONLY ALLOWS SPECIMENS UP TO A FEW 100 MICRONS THICK AND I START IT SEE HEA HEAVY DEPOSITS OF STAINING. SOME HAVE IMPROVED THE PROTOCOL AND SOME IMPROVED THE STAINING AND WE HAVE A PAPER WHERE THEY SPLIT UP THE REDUCED STEP INTO THE OSMIUM TETROXIDE AND ALLOW UP SO A MILLIMETER THICK TO BE STAINED. I'D LIKE TO POINT OUT AS A SIDE, I GUESS, THE APPROACH WE TAKE IS WITH OPEN SPACES AND SOMETIMES WE HAVE SPECIMENS FOR ONE REASON OR ANOTHER SOMETIMES IT DIDN'T STICK AND PUT IT IN THE MICROSCOPE AND THESE DAYS IT'S NOT AN ISSUE WHERE YOU POINT A SMALL NOZZLE AND CHARGE AND THIS APPROACH HAS BETTER QUALITY AND WE'VE BEEN ABLE TO RUN UP TO SIX MONTHS LONG AND YOU CAN IMAGE EVEN WITH THE FOCUS AND I'D LIKE TO LEARN TO X-RAY MICROSCOPY SPECIMEN SCREENING WE MAKE SURE THEY HAVE SUFFICIENT STAINING AND NO OTHER DE EFFECTS OR ART -- DEFECTS OR ARTIFACTS BEFORE WE INVEST. AND HERE'S A SAMPLE OF BRAIN TISSUE THAT WAS STAINED WITH VARIATIONS I JUST SHOWED. AND IT'S IMMEDIATELY IMPAIRED LOOKING AT THE IMAGES AND WHICH WERE STAINED WELL AND WE CAN SEE SMALL HOLES IN VACULES SHOW CRACKS IN THE TISSUE. IN A TYPICAL LABORATORY IT'S NOT POSSIBLE YET TO IMAGE A SUFFICIENT RESOLUTION TO ASSESS STRUCTURAL RESERVATION IN THE SPECIMENS. AND ALSO THE OPTICS OF MOST LABORATORIES WON'T BUT YOU TO COLLECT A LITTLE LESS THAN ONE MICRON SPACIAL RESOLUTION AND YOU CAN STAIN A WHOLE MOUSE BRAIN WITH A LABORATORY BASED EXOME PLATFORM AND WERE ABLE TO ASSESS THE STAINING QUALITY IN THROUGHOUT THE WHOLE BRAIN AND THEY FORMED THE TOM OWE GRAPHY AND IT'S POSSIBLE TO POTENTIALLY ASSESS THE STRUCTURE IN THE SPECIMEN IN THE MICROSCOPY. AND TO REUSE X-RAY MICROSCOPY IN OUR LAB FIRST TO ASSESS OUR SPECIMEN AND ALSO TARGET TO LIMIT AND CORRELATE THE VOLUME OF THE SETS WITH OTHER IMAGING MODALITIES AND IN VIVO. HERE'S AN EXAMPLE WHERE WE HAVE TE MICROSCOPY AND SPECIFIC CELLS ARE TARGETED AND THAT WAS FOUND IN THE MICROSCOPE AT GUIDED TARGETED ACQUISITION OF A VOLUME OF THAT CELL. THE VOLUME STAINED THE NUCLEI IN THE IMAGING MODALITIES. SO THE REGISTRATION WE ACHIEVE ALLOWS US TO CREATE CELL PROCESSES ANDBURY -- AND BEFORE I WRAP UP I'D LIKE TO POINT TO THE LARGEST PROJECT I WAS INVOLVED IN WE TOOK ADVANTAGE OF SEVERAL ASPECTS OF THE TALK AND THE PROJECT WAS DONE IN COLLABORATION WITH CAL TECH. THIS CONNECTOME WAS PARTICULARLY INTERESTING BECAUSE WE FIRST IMAGED THE BEHAVIORS AND THIS ALLOWS US TO GET MORE UNIFORM STAINING AND USED MICROCT STAINING TO MAKE SURE THERE WERE NO CRACKS AND USED GUIDED THE RUN TO MAKE IT AS TARGETED AS POSSIBLE. SO WE WERE ABLE TO CRE CONSTRUCT THE ENTIRE NEURON AND SEE HOW THE INPUTS OF NEURONS IMPINGED ON THE TARGET NEURON. THIS CONCLUDES MY TALK. >> THANK YOU FOR INVITING ME. I'LL FIRST PUT INTO CONTEXT THE NEED FOR HIGH QUALITY STAINING AND EMBEDDING FOR THE WHOLE MOUSE CONNECTOME PROJECT. THEN I'LL SPECULATE A BIT ON AN UNTRIED PATH I THINK MIGHT GET US THERE. SO FIRST OF ALL AS I PRESENTED AT THE RECENT DOE BIO IMAGING ROUN TABLE THERE'S THE CONNECTOME OF A WHOLE ADULT MOUSE BRAIN AND SCANNING ELECTRON MICROSCOPY. A HYBRID APPROACH USING SERIAL THICK SECTIONING AND BLOCK BASED IMAGING. THE PLASTIC EMBEDDED MOUSE BRAIN WOULD BE CUT INTO RELATIVELY THICK SECTIONS WHICH WOULD THEN BE IMAGED AND SEQUENTIALLY BROUGHT AND THE KEY ADVANTAGES OF SUCH AN APPROACH IS FIRST IT'S COMPATIBLE WITH MULTI BEAM IMAGING AND SECONDLY AND MOST IMPORTANTLY IT DECOUPLES THE Z RESOLUTION FROM THE SECTION THICKNESS YOU CUT OUT. THE IMAGING RESOLUTION CAN BE SET AT WHATEVER IS NEEDED FOR RELIABLE AUTOMATED TRACING WHICH IS TYPICALLY 10 NANOMETERS AND THE SECTION THICKNESS CAN BE SET TO WHAT GIVES THE MOST RELIABLE SECTIONING AND COLLECTION WHICH IN OUR HANDS IS TYPICALLY IN THE RANGE OF 100 NANNEE NANO METERS. AND HERE'S THE DATA SET THAT WAS COLLECTED ON OUR SINGLE BEAM PROTOTYPE AND WE'RE WORKING TO INTEGRATE THE PROCESS AND OUR GOAL IS TO DEMONSTRATE THE TYPE OF FAST, WIDE AREA AUTOMATED IMAGING THAT WOULD BE REQUIRED FOR A WHOLE MOUSE BRAIN CONNECTOME PROJECT. HERE'S IS A FIRST CREWTENT OF THIS WHOLE SYSTEM WITH THE MULTI BEAM -- AND THIS IS THE SECTION OF MOUSE CORTEX. THE IMAGE QUALITY NEEDS IMPROVEMENT CERTAINLY. THERE ARE A LOT OF ALIGNMENT AND STITCHING ISSUES BUT BASICALLY DEMONSTRATE THE WHOLE SYSTEM IS WORKING AND WE'RE MOVING INTO A PARAMETER OPTIMIZATION FRAME RIGHT NOW. HERE'S A CRUDE ESTIMATE IF SUCH A TECHNIQUE WERE USED TO DO AN ENTIRE MOUSE BRAIN. THIS IS PAVE ON THE IDEA -- BASED ON THE IDEA YOU NEED 500 BEAMS AND THE REQUIREMENT OF PICOAMPS AND THE NUMBER NEEDED PER VOXEL WHICH WOULD BE 1,000 ELECTRONS FOR THE EXTREMELY BEST STRAINING ANYBODY HAS PRODUCED TO 10,000 ELECTRONS FOR MORE TYPICAL STAINING LEVELS. IN TURN, THIS SETS THE NUMBER OF MICROSCOPES NEEDED FROM 10 TO 100 MICROSCOPES AND THE TAKE HOME MESSAGE IS THE STAINING LEVEL OF THE TISSUE IS FOR THE CONNECTOME PROJECT. AND TO UNDER SCORE THAT, SUCCESS DEPENDS ON GETTING THE HIGHEST CONTRAST TRAINING THROUGH AN ENTIRE ADULT MOUSE BRAIN. GETTING GOOD RESIN INFILTRATION AND OPTIMIZING THAT RESIN FOR RELIABLE SECTIONING AND QUALITY MILLING. TO MY KNOWLEDGE THE NECESSARY STAIN INTENSITY HAS ONLY BEEN ACHIEVED IN LESS THAN 1 MILLIMETER THICK AND NOT EASY TO REPRODUCE THAT IN THINE -- THIN SECTIONS. I SEE TWO POSSIBLE PATHS NONE OF WHICH HAS BEEN DEMONSTRATED. ONE IS STAINING AND EMBEDDING THE TACT BRAIN PRIOR TO SECTIONING AND THE SECOND I'LL TALK ABOUT IS THE POSSIBILITY FOR ULTRA SMOOTH SECTIONING OF THE BRAIN AFTER ALDEHYDE FIXATION BUT BEFORE THE STAINING SNIPS. THE CLOSEST I'VE SEEN ANYONE GET TO STAINING A WHOLE MOUSE BRAIN WITH THE INTENSITY NEEDED FOR THIS PROJECT IS A METHOD PUBLISHED IN 2015 SHOWN HERE. AND I THIS WOULD BE GOOD TO SHARE WITH YOU GUYS HERE. THE WAY TO READ THE IMAGES, THIS IS THE WHOLE MOUSE BRAIN THAT SHAWN STAINED USING THE PROTOCOL. THE GREEN IS BRAIN AND THIS IS IN THE PERIPHERY OF THE BRAIN. IT LOOKS VERY GOOD. THE STAINING IS EXCELLENT. VERY HIGH CONTRAST AND ALL IN THE PERIPHERY LOOKS GREAT BUT IF YOU GO TO DEPPER -- DEEPER AREAS IN THIS PARTICULAR SAMPLE THE DEEPER STARTS SLOW THE STAINING LEVEL DECREASES. IT'S QUITE DRAMATIC AND ISSUES WITH GETTING THE DEEPER HEAVILY MI MIEL MILE AND THIS MUST BE THE GREATEST POSSIBLE STAIN TO MAKE THIS PRACTICAL FOR THE IMAGING TIMES WE NEED FOR A WHOLE MOUSE BRAIN. I WANT TO MOVE ON TO THE PATH LESS TRAVELED ESSENTIALLY WOULD BE TO TRY TO VIBRATOME THE SECTIONS AND STITCHED BACK TOGETHER. TRADITIONAL VIBRATOMING IT'S UNRELIABLE TO GO ACROSS THEM AND THIS IS A TYPICAL VIBRATOME SECTION THAT I CUT AND THEN RE-EMBED AN THIS IS SHOWING A CROSS-SECTION. I WANT TO WARN YOU I THINK WE CAN GET TO A SMOOTH VIBRATOME SECTION BUT THIS IS PURE SPECULATION ON MY PART. THE IDEAS I'M ABOUT TO PRESENT REQUIRE PRECISION ENGINEERING AND ITERATIVE TESTING AND I HAVE NOT DONE THAT AND NO ONE I KNOW HAS DONE THAT SO TAKE IT WITH A GRAIN OF SALT FROM HERE ON. THINKING ABOUT THE POSSIBILITY OF DOING THESE REALLY SMOOTH PRECISION VIBRATOME SECTIONS. IT SEEMS TO ME FROM THE IMAGES OF REGULAR VIBRATOME CUTS THE TISSUE IS STURDY ENOUGH TO HANG TOGETHER AT THE BOUNDARIES AND THE GLUTARALDEHYDE KEEPS EVERYTHING TOGETHER. WE JUST NEED TO PREVENT RIPPING AND SURFACE TEARING DURING SECTIONING. SO FOR EXAMPLE, IF YOU USE A THICK BLADE WITH THE POORLY CONTROLLED MOVEMENT IT PUSHES THE MOVEMENT TO A POINT OF RIPPING AND TEARING WHEREAS IT SEEMS TO ME IF YOU USED AN EXTREMELY THIN BLADE THAT IS SHARP WITH PRECISE, WELL CONTROLLED VIBRATION YOU SHOULD BE ABLE TO CUT ANY TISSUE AT ANY THICKNESS WITHOUT RIPPING. HOW DO YOU MAKE THIN BLADES? FROM 20 MICRON THICK DIAMOND WAFERS. THEY SELL THEM AT WIDTHS OF 7 MILLIMETERS WIDE THAT SHOULD BE WIDE ENOUGH TO CUT A MOUSE BRAIN AND DIAMOND CAN BE SHARPENED BY MILLING AN THIS IMPORTANT BECAUSE USUALLY THE MECHANICAL POLICY AND THE POLISHING OF DIAMONDITES PUT A LIMIT TO HOW HOW THIN THE BRAIN COULD BE. THE PARAMETERS TO OPTIMIZE COULD BE THE FIXATION IS THE MAXIMUM COMPATIBLE WIN STAINING AND BOTH SIDES UNDER TENSION TO PREVENT COUPLING, SPEED OF CUTS SHOULD BE AS SLOW AS POSSIBLE. THE SIDE TO SIDE MOTION SHOULD BE PRECISE, MICRON SCALE AND ANY LUBRICATION OF THE KNIFE SHOULD BE USED. AND THE SECTION HERE FLOATS AWAY. SO AGAIN THIS IS ALL SPECULATION ON MY PART. IT WILL TAKE A DEDICATED RESEARCH PROJECT TO REALLY TEST WHETHER SUCH ULTRA SMOOTH VIBRATOME SECTIONS IS POSSIBLE AND MAY BE POSSIBLE COMING FROM MY PAST EXPERIENCE FROM GETTING ULTRA THICK SECTION OF TISSUE AND LET ME REVIEW THAT FOR COMPARISON BECAUSE I THINK IT WILL LOOK QUITE SIMILAR. SO THIS IS A VIDEO OF ME SECTIONING PLASTIC EMBEDDED MOUSE BRAIN TISSUE AT 20 MICRON THICKNESS WITH A DIAMOND KNIFE LUBRICATED WITH OIL IN THIS. THIS ONE OF THE SAME SECTIONS THAT WAS JUST CUT 20 MICRONS THICK AND CUT FROM AN ORIGINAL VIE -- VIBRATOME SECTION. AND YOU SEE THE ROUGHNESS. THIS IS VIDEO OF THE CROSS SECTION OF THE SURFACE. IT'S SO SMOOTH WE WERE ABLE TO TAKE MATCHING SURFACES AND FLATTEN THEM COMPUTATIONALLY AND WORK THEM SO TWO MATCHING SLABS CAN BE STITCHED BACK TOGETHER SO WHEN THEY WERE IMAGED SEPARATELY THEY WERE BROUGHT BACK TOGETHER AGAIN AND THEY WERE TRACED BACK THE PROCESSES ACROSS THE GAP. THOSE ARE IMAGES. AND FINALLY, THIS IS A VIDEO THE PROCESS WAS REFINED AND USED TO PRODUCE THE HEMI BRAIN CONNECTOME. THIS IS SHOWING 13 MICRON THICK SLABS CUT WITH THE HOT KNIFE TECHNIQUE AND COMPUTATIONALLY STITCHED BACK TOGETHER AGAIN. THE HOPE IS THIS SAME TYPE OF QUALITY MIGHT BE POSSIBLE BETWEEN VIBRATOME SLICES AS WELL. AGAIN, NOBODY'S SHOWN IT BUT IT WAS KIND OF THE PATH THAT I THINK WE COULD FOLLOW TO GET THE SAME TYPE OF IMAGES WITH THAT. THE TAKE HOME MESSAGE IS THAT WE SHOULD NOT ASSUME THE INTACT MOUSE BRAIN STAINING EFFORTS WILL SUCCESS IN PRODUCING THE HIGH STAINING LEVELS WE THESE FOR A WHOLE MOUSE CONNECTOME PROJECT. INSTEAD, I THINK WE SHOULD HAVE AT LATEST A BACK UP PLAN, PARALLEL EFFORT THAT TRIES TO GET THIS KIND OF ALL ULTRA SMOOTH VIBRATOMY OF TISSUE WORKING. THANK YOU VERY MUCH. >> WE'RE NOW ENTERING THE Q&A SESSION FROM SESSION ONE. WE HAVE QUESTIONS IN THE AUDIENCE NOW. >> INITIAL CRITERIA IS HOW IT IMPACTS THE RESULTING BLOCKS. VARIATIONS OF PROCESSING CAN CAUSE BLOCKS TO BECOME BRITTLE AND DEPOSIT ADDITIONAL MATERIAL ON KNIVES. THE TRADE-OFFS MAY CAUSE NON-DIRECTIVE METHODS. WE CAN'T THINK OF HIGH CONTRAST ALONE AS SOLVING THE PROBLEM. ADDITIONALLY FOR NON-DESTRUCTIVE METHODS CAN GET AROUND IT WITH LINKING METHODS. TO SUMMARIZE THE QUESTION, DAN IS SAYING THAT VERY INTENSE STAINING CAN MAKE THE SAMPLE BRITTLE AND HARD TO SECTION. AND THEN MAYBE THIS COULD BE MITIGATED BY POST STAINING. I INVITE ANY SPEAKERS TO REPLY. >> ONE ASPECT OF THE ANSWER I'D LIKE TO GIVE IS THE NEED OF LOOKING AT THE PROCESS AS A WHOLE AND NOT A SINGLE VARIABLE AND THE IMPORTANCE OF RUNNING THE PIPELINE. AT THE END THE CONTRAST NEEDED FOR THE AUTOMATIC AND THAT'S ONE PART OF MY ANSWER. THE OTHER PART WE DID CONSIDER POST STAINING IN OUR PROCESS AND IT'S A QUESTION OF HOW MUCH RISK DOES THAT BRING TO THE WHOLE PROCESS AND HOW RELIABLE IT IS. WE COULDN'T BRING IT TO A POINT WHERE IT'S RELIABLE ENOUGH FOR THE GAIN WILL BE BETTER THAN THE COST OF DEGREE OR OF ANY OTHER DAMAGE TO OUR SAMPLE BUT IF THE ENGINEERING IT HOLDS POSITIVELY ENOUGH IT WOULD BE SOMETHING WE COULD BRING BACK. >> THERE'S PROS AND CONS TO BOTH. THE RISK IS USUALLY THE LEAD STAINING AND YOU HAVE A TENDENCY TO THROW A BUNCH OF PRECIPITATES AND ARTIFACTS UP YOUR SECTION WHICH COULD BE HARD TO GET RID OF AFTER THE FACT AND HAVING AN ENVIRONMENT CARBON DIOXIDE FREE AND YOU HAVE CONSISTENT SOLUTIONS THAT ARE CARBON DIOXIDE FREE WOULD BE A MORE CONSISTENT ON SECTION STAINING IF THAT'S THE WAY IT'S DECIDED TO GO. ANOTHER ELEMENT WE DIDN'T DISCUSS AT ALL HARDLY IS THE SECTIONING OF THE SPECIMEN IS IN ISSUE WE ARE STILL USING THE SAME RESINS FOREVER AND WE SPENT SOME TIME UP OUR LAB TRYING TO EXPLORE OTHER ALTERNATIVE EMBEDDING MEDIA WE LOOKED AT POLIESTER RESINS AND -- POLYESTER RESINS AND COULD BE IMPROVED IN THE FUTURE. >> WE LOOK AT THE CONTRAST OF THE SAMPLE BUT AS ERIC MENTIONED, RELIABLE POST STAINING FOR A LARGE PROJECT LIKE THE WHOLE MOUSE CONNECTOME IS A PROBLEM WE NEED TO COPE WITH. AND ALSO I OUGHT TO MENTION FOR THE POST STAINING CANNOT SOLVE THE CONTRAST ISSUES ALONE AND FOR INSTANCE IF WE ONLY DO ONE OSMICATION WE ALWAYS FOUND TWO FORM OF ENHANCED OSMIUM STAINING CAN HELP AND THE OTHER POINT I WANT TO BRING IN IS THE OSMIUM STAINING WILL NOT NECESSARILY INCREASE THE BRITTLENESS OF THE TISSUE IF WE SPECIFICALLY STAIN THE MEMBRANES BUT NOT THE WHOLE TISSUE MEANING OSMIUM CAN DEPOSIT SPECIFICALLY TO THE MEMBRANE BUT NOT SPECIFICALLY TO THE WHOLE BLOCK. >> THANK YOU FOR THE QUESTION AND ANSWERS. THE NEXT QUESTION IS FROM BRYAN JONES. THE QUESTION IS DURING OSMIUM REDUCTION THE BRAIN GOT LIGHTER BUT ALSO APPEARED TO SWELL DURING OSMIUM REDUCTION AND DOES IT CHANGE THE RELATIONSHIP OF MEMBRANES? >> ACTUALLY, WE DO SEE THE EXPANSION OF THE TISSUE DURING THE STEP AND 10% TO 20% BUT DID NOT SEE ARTIFACT OF THE MEMBRANES POSSIBLY BECAUSE THE TISSUE WOULD SHRINK BACK TO ITS ORIGINAL SIZE AFTER THE SECOND OSMICATION AND CAN FORM A TINY MICROSCOPIC GAPS BETWEEN THE MEM MEMBRANES BUT COULD PERHAPS HEAL AFTER THE TISSUE RETURNS BACK TO ITS ORIGINAL SIZE. >> FROM BRIAN JONES WE HAVE REGARDING LOW TEMPERATURE HANDLING OF TISSUES PRIOR TO FIXATION KNOW THAT LOW TEMPERATURES ALTER THE SMALL MOLECULE CONCENTRATIONS IN TISSUES AS SMALL MOLECULE ARE DIFFERENTIALLY IMPACTED BY LOW TEMPERATURES. THE SMALL MOLECULES ARE IMPORTANT FOR IDENTIFICATION OF CELL TYPES AND SOME APPROACHES FOR WORK OR FOR IDENTIFYING NEUROTRANSMITTER CONTENT. USING PROBES AND GLUTINATE AND THIS IS MORE OF A POINT THAN A QUESTION BUT IF ANY OF YOU HAVE COMMENTS OR WOULD LIKE ELABORATE THAT WOULD BE VALUABLE I THINK. >> I EMPHASIZED KEEPING THE TISSUE COLD DURING THE PROCESSING STEPS AND DURING THE INITIAL PROFUSION OF THE BRAIN. IN ADDITION [INDISCERNIBLE] COLLAPSE BEFORE IF YOU CHILL THE BRAIN BEFORE IT'S FIXED. I DIDN'T REALLY GO INTO ENOUGH DETAIL. THE FIXATION STARTS OFF AT 37 DEGREES CELSIUS AND OVER THE COURSE OF 10 OR 15 MINUTES YOU'RE PERFUSING THE MOUSE IT'S CHILLED OFF BY THE TIME THE BRAIN COMES OUT. MY HOPE IS ALWAYS THAT THE TISSUE HASN'T STABILIZED ENOUGH BY THE ALDEHYDES THAT HOPEFULLY THE TISSUE'S NOT COLLAPSING DUE TO THE TEMPERATURE AND YOU SEE MICROTU MICROTU MICROTUBE S AND SEEMS TO BE A SAFE CONCLUSION. OR DO HIGH PRESSURE FREEZING LIKE THEY'VE DONE FROM THE '60s AND YOU CAN HAVE A MORE NATIVE STATE REFSHATI -- RESERVATION OF THE TISSUE BUT DOING THAT FOR THE WHOLE BRAIN IS WAY BEYOND WHAT'S POSSIBLE RIGHT NOW. >> THANK YOU, ERIC. NEXT WE HAVE TWO QUESTIONS FOR KEN. ONE IS WHEN YOUR HOT KNIFE CAN BE USED WITH THE WHOLE MOUSE STAINING AND THE SECOND IS COULD YOU COMMENT ON A RELATIONSHIP BETWEEN RESTING EMBEDDING SAM AND MECHANICAL PROPERTIES AND THE -- CUTABILITY BY DIAMOND OR BY [INDISCERNIBLE]. >> FIRST OF ALL THE HOT KNIFE TECHNIQUE I USE THAT BECAUSE WE NEEDED TO GET SECTIONS THAT WERE OPTIMIZED IN SIZE FOR FOCUSSED IMB. SO YES THE HOT KNIFE TECHNIQUE COULD POTENTIALLY BE USED FOR SLICING UP A WHOLE MOUSE BRAIN. IT WOULD PUT EXTRA REQUIREMENTS ON THE CONSISTENCY AND TYPE OF EMBEDDING AND IT MAY PUT REQUIREMENTS ON THE LEVEL OF STAINING IN THE SENSE THAT SOME STAININGS MAKE THE TISSUE TOO BRITTLE TO CUT AT THAT THICKNESS BUT IT'S THEORETICALLY POSSIBLE. THE MAIN QUESTION I'D ASK IS WHY WOULD IT BE USEFUL? IT WAS EXTREMELY USEFUL FOR US BUT THE ONLY USE I'VE SEEN FROM A WHOLE MOUSE BRAIN CONNECTOME POINT OF VIEW WOULD BE TO SUBDIVIDE THE PLASTIC EMBEDDED BRAIN SO ULTRA THIN SECTIONING WOULD BE EASIER TO DO. IN GENERAL I THINK THAT IS PROBABLY NOT A GOOD PATH TO GO DOWN. I THINK IT'S MORE TROUBLE THAN ITS WORTH. IF WE ARE TRYING TO PUSH THE ULTRA THIN SECTIONING, MAYBE THAT IS SOMETHING THAT COULD BE EXPLORED. >> WHAT WAS THE OTHER QUESTION? >> CAN YOU COMMENT ON THE RELATIONSHIP BETWEEN A RESTED EMBEDDED SAMPLES MECHANICAL PROPERTIES AND THE CUTABILITY EITHER BY DIAMOND KNIFE OR BY EM. I THINK IT'S RELATED AS YOU SAY. >> SO THE RESIN EMBEDDED SAMPLES I COULD ANSWER THAT IN TWO DIFFERENT WAYS. FIRST OF ALL THE SAMPLES ARE KIF TO CUT WITH SMOOTH SURFACES BECAUSE EVERYTHING IS HELD TOGETHER BY GOOD RESIN INFILTRATION. AND SO THE SPECULATIVE IDEA OF SMOOTH VIBRATOMY CUTTING OF ALDEHYDE FIXED TISSUE BEFORE THE OSMIUM STEPS, THAT IS ALREADY STARTING OFF MUCH MORE DIFFICULT BECAUSE THE TISSUE IS READY TO RIP AT ANY POINT. LIKE I SPECULATED, I THINK IT COULD BE DONE BUT THE TISSUE PROPERTIES ARE ENORMOUSLY DIFFERENT. ONE THING IS THAT YOU WOULD ALMOST GO DOWN IF YOU HAVE AN ULTRA SONICALLY VIBRATING DIAMOND KNIFE GOING THROUGH FIXED TISSUE I THINK THE FORCES COULD BE BROUGHT TO ALMOST ZERO AS THAT THING IS GOING THROUGH THE TISSUE. I THINK A SEPARATE PART OF THAT QUESTION MIGHT HAVE BEEN TO ADDRESS ION MILLING, WHAT WORKS BEST AND WORSE. THERE ARE DEFINITELY CRITERIA FOR THE RESIN EMBEDDING. SOME WORK BETTER WITH ION MIG AND -- MILLING. IT'S AN EXTRA REQUIREMENT IF WE GET SOMETHING THAT INFILTRATES REALLY WELL AND THEN THAT COULD CAUSE PROBLEMS IN THE OVERALL PIPELINE. THESE TYPES OF CONSTRAINTS AND YOU HAVE TO MAKE SURE THAT EVERYTHING IS DESIGNED WITH THE WHOLE PIPELINE IN PLACE IN MIND. >> I THINK THUNO WANTED TO -- OF >> I THINK NUNO WANTED TO COMMENT AS WELL. >> THE IDEA INTERESTING TO IMPROVE THE VIBRATOME TO DECREASE THE DAMAGE OF LOSS ON SURFACES BECAUSE THAT WOULD BASICALLY MEAN THE HISTOLOGY PART WILL BE BASICALLY SOLD BECAUSE I THINK TO THE LEVEL OF ONE MICRON -- ONE MILLIMETER OF TISSUE THE RELIABILITY NOW OF THE SAMPLE WITH CURRENT METHODS IS NOT PERFECT BUT IT'S REALLY GOOD. SO THAT FOR ME IS A SEPARATE AVENUE IT'S ALMOST LIKE A NO NO-BRAINER AND AN INTERESTING -- IT WILL BE AN ENGINEERING PROBLEM. >> IF WE WANT IT GO BEYOND THE MOUSE BRAIN THAT'S SOMETHING THAT IS GOING TO BE MORE AND MORE DIFFICULT. COULD I THROW IN A QUESTION? HAVE YOU EXPLORED THE POSSIBILITY OF USING PERFUSION IN ANY OF THE STEPS? I KNOW THAT INITIAL OSMIUM PERFUSION AND GIVEN ALL THE TECHNIQUES THAT YOU AND OTHERS HAVE DEVELOPED AND USE METHODS THAT SHOULD BE REVISITED. >> THAT'S A REALLY GOOD POINT. SO WE'RE THINKING ABOUT PERFUSE OSMIUM AND JUST TO FACILITATE THE DIFFUSION BUT THERE'S A LOT OF CONCERN ABOUT THE METHOD BECAUSE YOU NEED TO USE A LOT OF THE [INDISCERNIBLE] AND I DON'T THINK IT'S THE FIRST OSMICATION. IT'S THE REDUCTION AND THE THE SECOND OSMICATION THE LIMITING STEP AND IF WE PERFUSE IT TO THE ANIMAL THAT MAY BE THE FIRST STEP BUT NOT NECESSARILY ISSUES WITH THE REST OF THE STEPS. >> I WONDER HOW MANY STEPS COULD BE DONE BECAUSE THERE'S SOME PROTOCOLS NOW WHERE PEOPLE WANT TO DO LIGHT SHEET IMAGING, FOR EXAMPLE, IN THE BRAIN AND WHEN THEY WANT TO CLEAR THE WHOLE MOUSE AND PERFUSE THE MOUSE WITH A SERIES OF SOLUTIONS TO CLARIFY THE TISSUES AND I WONDERED IF SOMETHING LIKE THAT COULD BE DONE IN PERFUSING MOUSE BRAINS WITH OSMIUM. >> OKAY. IF WE'RE READY TO MOVE ON. THERE'S A QUESTION FOR KEN. HAVE YOU THOUGHT ABOUT YOUR ULTRA SONIC DIAMOND WHEEL FOR SERIAL MICRO TOME BASED IMAGING AND A DIE -- DIAMONNIITE AND THE AREA IS LIMITED BY THE DIAMOND KNIFE AND YOUR ANSWER IS INTERESTING. >> LET ME DOUBLE CHECK I UNDERSTAND THE QUESTION. THE IDEA IS WIDE AREA SO THAT IT IS COMPATIBLE WITH MULTI IMAGING. >> THE WORDS ARE KEN HAVE YOU THOUGHT ABOUT THE DIAMOND WHEEL FOR SERIAL BASED IMAGING? >> I THINK THEY THOUGHT THROUGH THIS BETTER THAN I HAVE SO I'D POINT THEM IN THAT DIRECTION. THERE'S YET ANOTHER PATH NO ONE HAS TRIED SO I'LL THROW IT IN TO THE COMMENT SECTION. THAT'S GRINDING TISSUES AND A DIAMOND PLATE CAN HAVE WHATEVER ROUGHNESS YOU WANT AND SIMPLY GRIND YOU'D HAVE TO MAKE SURE IT'S A SMALL OSCILLATIONS BUT GRIND DOWN THE SURFACE AND I THINK SOME OF THOSE WOULDN'T HAVE THE DIFFICULTY ASSOCIATE WITH THE DIAMOND SCRAPING TECHNIQUE, BECAUSE THE LARGER THE BLOCK IS THE MORE COURSES IT UNDER GOES AND THAT KNIFE HAS TO MAINTAIN IT'S POSITION TO THE EVENTUAL THICKNESS YOU WANT. THAT'S THE PROBLEM OF GETTING THE BLOCK BASED TYPE OF STUFF. IF YOU WERE INSTEAD TO DO GRINDING OF THE SURFACE THAT PARTICULAR PROBLEM WOULD DISAPPEAR. I THINK IT'S A WONDERFUL FIELD BECAUSE THERE'S SO MANY ROUTES PEOPLE HAVE COME UP WITH AND EXPLORING ON IMAGING BRAIN TISSUE. IT'S GREAT TO HAVE DIFFERENT PATHS TO THE ULTIMATE GOAL. >> MAYBE A QUICK FOLLOW-UP ON THAT? KEN, WAS THERE AN IMAGE BETWEEN THE BLADES? >> I'M, SORRY, WHAT? >> GRIND WITH A PROPELLER AND IMAGE BETWEEN THE BLADES. >> OH. I'M GOING TO TAKE THAT SERIOUSLY. THOUGH I SHOULDN'T LAUGH AND THE THING YOU SHOULDN'T DO WHEN GRINDING IS MOVING MORE THAN A FEW MICRONS BACK AND FORTH BECAUSE WHEN YOU DO THE SURFACE BLINDING OF THE TOOL, THE DIAMOND, LET'S SAY, HAS TO BE SUPER WELL MATCHED TO THE ACTUAL SURFACE OF THE TISSUE OF THE NANOMETER SCALE. YOU HAVE TO PUT THAT DOWN ON THE EXACT SAME PLACE AND GRIND AWAY A FEW NANOMETERS AND THEN DO IMAGING. >> MARK NOTED IN POST SCRIPT HE MEANT IT SERIOUSLY. IT'S GOOD YOU TOOK IT SERIOUSLY. CHRIS OHN SAYS WHAT ABOUT MULTI PLEXING AND REIMAGING AND TRADEOFFS AND OPPORTUNITIES, SO BASICALLY IT SOUNDS LIKE BASICALLY LIKE A QUESTION OF SAMPLE PRESERVING VERSUS SAMPLE DESTRUCTIVE IMAGING AND MAYBE BETTER MEANT FOR THE IMAGING SECTION BUT DOES RELATE TO SAMPLE PREP IN THAT IF YOU PRESERVE THE SAMPLE YOU CAN IN INTERROGATE IT USING COMPLIMENTARY METHODS AND ANY COMMENTS WOULD BE APPRECIATED. >> WE DO USE NON-DESTRUCTIVE METHODS. IN OUR HANDS WHAT HAS BEEN USEFUL PRACTICALLY USEFUL IN ONE SITUATION BUT THAT [INDISCERNIBLE] SECOND ONE AND USEFUL IN THE SENSE WHERE IMAGEING DOES NOT GO AS GOOD AND USUALLY NOT A PROBLEM BUT WHEN WE'RE IMAGING AT SCALE FOR HUNDREDS AND MILLIONS OF IMAGES WE'LL ACTUALLY HAPPEN. THE POSSIBILITY OF DOING REIMAGING HAS BEEN SOMETHING WE'RE TAKING ADVANTAGE OF IN OUR PROCESS. THE SECOND THAT DOESN'T REALLY RELATE TO OUR DATA IF YOU WANT TO GO BACK AND LOOK AT SOME CELL ORGANELLES YOU MAY FIND LATER THAT ARE USEFUL, THAT BRINGS BRINGS THAT IN THE CONTEXT OF WHERE WE IMAGED SECTIONS THAT WERE COLLECTED IN THE ORIGINAL [INDISCERNIBLE]. >> IT WILL BE GOOD FOR OTHERS TO COMMENT ON THIS. REIMAGING, ARCHIVABLE AND RE-IMAGEABLE. >> I'D REPEAT WHAT HE SAID. I DO TOMOGRAPHY AND IT'S ALWAYS GREAT TO GO BACK AND RESHOOT SOMETHING OR TAKE A CLOSER LOOK AT SOMETHING THAT LOOKS INTERESTING IN YOUR DATA SET AND ALWAYS FRUSTRATING WHEN SOMETHING GOES WRONG IN THE MIDDLE OF THE NIGHT AND WAKE UP AND REALIZE YOU'VE BEEN COLLECTING GRAY IMAGES ALL NIGHT. IT'S A HUGE ADVANTAGE, DEFINITELY. >> I WOULD AGREE ON THAT. OKAY. NEXT QUESTION FROM TATIANA, FOR ALL THE PANELISTS FOR [INDISCERNIBLE] THAT RUINS A PORTION OF THE TISSUE BUT THE PIERCING ALLOWS DELIVERING OF THE FIXATIVES FASTER IS BENEFICIAL IN YOUR OPINION AT ALL? >> RUINING PART OF THE BRAIN IS A NO-NO AND BRINGS UP SOMETHING I WANTED TO BRING UP IN MY TALK AND DIDN'T FEEL I HAD TIME. THERE ARE PEOPLE WHO TRIED TO RESURRECT A TECHNIQUE WHERE YOU TRY TO PERFUSE THE BRAIN AT HIGH PRESSURE WITH A HIGH SUCROSE SOLUTION AND BY REPLACING THE FLUID WITH SUCROSE AND REPLACING IT WITH THE FIXATIVE SOLUTION YOU CAN PRESERVE THE INTRACELLULAR PLACE OF THE BRAIN BECAUSE IN THE NATIVE BRAIN 20% OF THE BRAIN IS EXTRA CELLULAR AND YOU COULD SEE GAP JUNCTIONS OR WHAT LOOKS LIKE COULD BE A GAP JUNCTION EVEN SYNAPSES MAY BE EASIER TO SEE AND TRACE THE WIRES IF THERE'S STAINING PLUR PLUR -- PLEURAKITTY AND PEOPLE WERE -- PLEURACITY AND YOU POP THE BLOOD VESSELS OPEN TO GET THE SOLUTION IN BUT THERE'S POTENTIALLY ANOXIA. I THINK THAT'S AN AREA I HOPE PEOPLE ARE GOING TO PURSUE AND INVESTIGATE IN THE YEARS TO COME BECAUSE THEY WERE TRYING TO FREEZE TISSUE BEFORE ANOXIA SETS IN AND MIGHT IT'S A TECHNIQUE PEOPLE ARE COMFORTABLE USING IF PEOPLE CAN SHOW IT'S CLOERT -- CLOSER TO THE NATIVE STATE. >> IT LOOKS LIKE WE JUST HAVE A COUPLE MORE MINUTES. I'D LIKE TO THANK THE SPEAKERS AND WE'LL HAVE A 10 MINUTE BREAK AFTER THIS IF I REMIND WHAT THE AGENDA SAID, NO HALF AN HOUR, NO, 10 MINUTES. I WANTED TO GIVE THE SPEAKERS BRIEFLY THUMBS UP, THUMBS DOWN OR SOMEWHERE IN THE MIDDLE DO YOU THINK A WHOLE MOUSE BRAIN FROM A SAMPLE IS FEASIBLE AND LIKELY TO BE ACHIEVED WITH EFFORT OR UNLIKELY TO BE ACHIEVED. JUST CURIOUS. I'M NOT A PROSECUTOR SO I DON'T MIND NOT KNOWING THE ANSWER AHEAD OF TIME YOU'LL GIVE. >> THUMBS UP, NO PROBLEM. >> IT WILL TAKE WORK. >> OKAY. >> I'D LIKE TO GIVE A THUMB UP BECAUSE IT'S SOMETHING I'M WORKING ON AND POSITIVE TO GET A GOOD STAINING PROTOCOL BY THE END. >> I WOULD GIVE A THUMBS UP. I THINK XIAOTANG'S WORK SHOWS PROGRESS AND DOESN'T LOOK LIKE IT WILL DERAIL THE WHOLE PROJECT. IT WILL GIVE A SOLUTION TO A WHOLE MOUSE BRAIN STAINING. >> I THINK IT'S LIKELY TO BE ACHIEVED. IT'S STILL AN ENGINEERING PROBLEM AND WE SHOULD KEEP OUR MINDS OPEN TO THE PATHS WE DESCRIBED INITIALLY BASICALLY USING HISTOLOGY TOO AND KEEP OUR MINDS OPEN TO SOMETHING LIKE KEN DESCRIBED TO HAVE MORE THAN ONE PATH WOULD BE CRUCIAL AND IMPORTANT. >> GREAT. THANK YOU. I THINK WE'RE MOVING TO A BREAK NOW AND REJOIN AT 1:10 P.M. EASTERN. >> WE'VE EXPANDED OUR ABILITY FROM TWO DIMENSION TO THREE DIMENSIONS. AND INSTEAD OF REQUIRING THIS WE USE A LIGHT SHEET AND CAN IMAGE AT HIGHER SPEEDS IN THREE DIMENSION AND IT'S SIMPLE AND AFFORDABLE AND TRIED TO DEVELOP IT AND GET IT INTO COLLABORATORS. I'LL SHOW YOU CAPTURING SPONTANEOUS CAPABILITY AND YOU CAN SEE THIS BEAUTIFUL 3 DIMENSIONAL ACTIVITY AND THE DATA WAS CAPTURED AND COMPARE THE TWO DIMENSIONAL VIEW FROM DENDRITES IN THE IMAGE BELOW AND IT'S DIFFICULT TO VALUE THE CONNECTIVELY BETWEEN THE DIFFERENT REGIONS IF YOU DIDN'T HAVE THE 3-D VIEW AND DIMENSIONALITY AND IF YOU HAVE A PIXEL WITH A PARTICULAR TIME COURSE IF YOU HAVE OTHER PIXEL WITH THE SAME CORRELATED TIME COURSE THEY MAY BELONG IN THE SAME CELL. BY DOING THIS WE CAN TURN THE DATA INTO THIS MATRIX OF THE 3-D VIDEO AND NOW I COLOR CODED THE PIXEL BASED ON THEIR IDENTITY AS BELONGING TO THE SET OF HAVING A SIMIL SIMILAR TEMPORAL PROFILE. THIS WOULD BE INTERESTING TO MEASURE WHEN WE DO A MORE DETAILED CONNECTOME. AND THIS IS A COARSE REPRESENTATION WHERE WE GROUP TOGETHER PICTURES AND THE INFORMATION IS INTERESTING. WE DEVELOPED TWO PHOTON VERSIONS WHICH ALLOWS US TO GO DEEPER IN THE MOUSE BRAIN. WE GET NEAR ISOTOPIC SAM MRIPG MRING AND DID SAMPLING AND LOOKING AT NOT JUST DENDRITES BUT TIME COURSES AND MAYBE PROJECTIONS HERE. I WANT TO POINT OUT BECAUSE THIS SEGMENTATION IS DONE IT CAN ONLY BE DONE WITH REAL TIME 3-D TECHNIQUE AND COULDN'T DO THAT WITH THE PLANES AND SO WE CAN FIND ALL SORTS OF DIFFERENT CELLS IN ONE 3-D VOLUME AND ALLIES THE WAY THE -- ANALYZE THE WAY THE CELLS FIRE AND THIS IS WITH THE ANIMAL NOT DOING MUCH BUT COMPARING THIS TO THE PHYSICAL CONNECTOME WILL BE INTERESTING. THESE TOOLS WE HAVE WE CAN CAPTURE ON THE ENSEMBLE LEVEL AND YOU CAN BASICALLY GET A VIEW OF THE ENTIRE SURFACE OF THE CORTEX. WHAT YOU'RE RECORDING FROM IS THE CELLS YOU'RE INDICATING. YOU SEE AS THE ANIMAL STARTS TO RUN YOU SEE THE SIGNALS THAT CORRESPOND TO IT'S MOVING ITS FEET AND CAN PRESENT SEGMENTATIONS AND THEY IN SOME CASES LAY OVER ON THE STRUCTURAL ATLASES WE ALREADY HAVE AND THOSE ARE BASED ON ANATOMY AND HERE WE'RE LOOKING AT THE DYNAMIC ACTIVITY AND CAN LEARN WHAT ROLE THEY'RE PLAYING IN THE BEHAVIOR BY USING MODEL BASED ANALYSIS OF THE DATA. WE CAN ALSO ANALYZE AND BY SAMPLING THE TIME COURSES WE CAN SEE HOW THEY CORRELATE TO EACH OTHER. THIS IS A SIMILAR MEASURE TO FUNCTIONAL MRI AND SHOWING SYNCHRONY BETWEEN DISTANT BRAIN REGIONS AND IT RELATES TO THE HUMAN SITUATION. THIS IS ANOTHER THING WE CAN ANALYZE AND IN ADDITION WE FIND THE CORRELATIONS CHANGE OVER TIME AND SO UNDERSTANDING ALSO REGIONS THAT REMAIN STRONGLY COORDINATED VERSUS ONES THAT CHANGE CAN BE HELPFUL. THIS DATA COMES FROM THE CELLS WE LABEL AND WE'RE ABLE TO ENCODE DIFFERENT CELL TYPE AND HERE EXCITATORY. YOU CAN ALSO TARGET SPECIFIC SUBSETS AND LOOK AT THEIR CONNECTIVITY. AND THEY'RE MEASURING THE CORTEX. TO GET TO DEEP BRAIN REGIONS I THINK FUNCTIONAL SOUND ARE VALID AND THEY DECODE WHAT IT MEANS HERE. AND THE FASTER AND FASTER WE TRY TO SCAN THE LESS TIME WE HAVE TO LOOK AT EACH INDIVIDUAL PIXEL WHERE WE CAN LOOK OVER A LARGE AREA AT ONCE AND HAS NICE ABILITY AND IT WILL BE SIGNIFICANTLY BETTER AND WE'VE DEMONSTRATED HERE IN A MOUSE WE TRANSLATE THE SAMPLES AND GET BLOCKS OF 3-D DATA AND WE HAVE THIS SECTION HERE IN ABOUT 230 SECONDS AND YOU CAN SEE INEQUALITY OF DATA WE'RE GETTING -- THE QUALITY OF DATA CAN ALLOWS US TO SEE THE COMPLETE PROJECTIONS OF THE CELLS OVER LONG DISTANCES. IF WE DO RESONANT SCANNING WE EXPECT IT WILL BE 34 HOURS OF PURE ACQUISITION TIME AND CONSIDERING WHAT WE DO WITH HUMAN BRAIN AND WE CAN IMAGE SAMPLES THAT ARE 5 OR 6 MILLIMETERS THICK AND WE COULD DO A WHOLE GENOME BRAIN IN A MATTER OF DAYS. THIS CAN REVEAL MORE EXQUISITE DETAIL AND WORKS ON UNCLEAR TISSUE SO THIS IS A THICK BRAIN SLICE AND THIS IS IMPORTANT BECAUSE WE MAY NOT BE ABLE TO DO ELECTROMICROSCOPY ON THINGS CLEARED AND IT'S PLAUSIBLE TO CAPTURE A FRESH OR FIXED TISSUES. AND FINALLY A SHOUT OUT FOR SPECTRAL ENCODING WE'VE BEEN WORKING TO CHARACTERIZE THE WORMS EXPRESSING AND LOOKING TO DRAMATICALLY IMPROVE OUR ABILITY TO USE COLOR AND LOOK AT CONNECTIVITY AND I THINK IT HIGHLIGHTS HOW IMPORTANT WE LOOK HOLISTICALLY AT A BRAIN RATHER THAN BITS AN CHUNKS OF THINGS IN DIFFERENT BRAINS. WITH THAT I'D LIKE TO ACKNOWLEDGE ALL THE HARD WORK OF MY LAB THEY WORKED HARD THIS YEAR DESPITE IT BE DIFFICULT AND MY INSTITUTE AND FUNDING. THANK YOU FOR LISTENING. >> WE STUDY CONNECTIONS FROM THE ENTIRE SYSTEM TO THE SYNAPSE AROUND AND WHY IS THIS INFORMATION USEFUL WE CAN STUDY THE POST-SYNAPTIC FEATURES OF PATHWAYS WITH EXCITATORY AND INHIBITORY NEURONS AND THEN WE'LL SELECT REGIONS OF INTERESTS BECAUSE OF THE PATHWAYS TO PROCESS FOR CRE CONSTRUCTION. SO PATHWAY ENABLING HAS TO BE DONE IN LIVE ANIMALS WITH TRACING INJECTIONS IN ANESTHETIZED ANIMALS AND IN CRYOPROTECTION THE BRAN'S FROZEN AND USING A COUPLE OF PATHWAYS AND POST-SYNAPTIC SITES AT THE SAME TIME. FOR REGIONS OF INTEREST AS WELL WE USE ELECTRON DENSE MATERIALS SUCH AS TMB TO SEE THE PATHWAYS AND POST-SYNAPTIC SITES AND TO VIEW IT IN THE EM. AND HERE'S A BI-DIRECTIONAL TRACER AND IT'S GOING LABEL THEM BACKWARDS. AND FROM THE INJECTION SITE WE CAN USE AND HERE WE USE STEREO LOGICAL ANALYSIS TO SHOW THE FEATURES. AND THERE'S INHIBITORY NEURONS AND IN HIGHER MAGNIFICATION WE SEE THE DIFFERENT TYPES OF NEURONS. THEN WE TAKE A SMALL PART OF THE SYSTEM AND PROCESS AND LABEL IT AS WELL AND LOOK AT ALL THE SECS THROUGH THE SYNAPSE AND CONSTRUCT THEM AS WE SEE HERE THE ACTION TERMINALS ARE SHOWN IN BLUE AND THE EXCITATORY NEURONS IN GREEN AND THE INHIBITORY NEURONS IN PURPLE OR RED. THE CONNECTOME AND HERE WE HAVE TWO SIDES AND HERE IT SHOWS THE TRACER AND THEY'RE COLOR CODED TO SHOW THE DENSE CONNECTIONS IN THE MOUGS QUE SEE TWO INJECTION SITES AND SHOWING THE REGION AND SHOWING THE CONNECTOME. WITH CAN LOOK AT THE MYELINATION AND THE MAJOR ADVANTAGE BECAUSE WE HAVE STUDIED THE SAME FEATURES IN HUMANS AP FEATURES CHANGE IN NEUROLOGICAL DISEASES AND THIS IS AN AREA IN THE RHESUS MONKEY AND SEE THE MYEL AR AND MEASURE THE MYELIN AND SO FORTH. YOU CAN SEE THE DISTRIBUTION OF MYELIN IS IN THIS AREA AND YOU CAB SEE THE BROWN STRANDS HERE IN DIFFERENT AREAS WITH THE SING -- CINGULATE AND WE HAVE AN EXAMPLE TO COMPARE IT IN MY OPERATES AND HUMAN -- PRIMATES AND HUMANS WITH THE AXON DENSITY OF THE WHITE MATTER AGAINST THE GRAY MATTER IN HUMANS AND IN MONKEYS IN THREE AREAS. YOU SEE THE RELATIONSHIP AND WHY WE CAN MAKE PARALLELS AND WE USED POST MORTEM HUMAN TISSUES AND I'LL SHOW YOU A LITTLE BIT OF WHAT WE DID AND ONE IS THE AXONS BLE TO THE ANTERIOR CINGULATE AND NOT TOO MUCH CONNECTIVITY WITH THESE AREAS. AND THIS SHOWS THE SIDE AND THERE'S MANY MORE IN THOSE BRAINS. SO INFORMATION NEEDED FOR SAMPLE SELECTION OF MY -- PRAY MATES IS AGE -- PRIMATES IS AGE, SEX AND SPECIES AND WE DEVELOP NEW METHODS TO PRESERVE THE TIME STRUCTURE OF THE BRAIN WHEN WE ARE USING DOUBLE LABELLING WHICH IS IMPORTANT. TO STUDY CONNECTIONS WE MUST HAVE A ROAD MAP AND IT'S BASED ON THE STRUCTURE WHICH RELATE TO CONNECTIONED AND ALLOWS THE CONNECTIONS AND INHIBITORY NEURONS GIVES SPECIFICITY OF PATHWAYS AND PATHWAYS INTERFACE WITH THIS IN LARGE BRAIN CONNECTOMES AND WE USE AND THIRD WE USE ALTERNATIVE STATES AND STUDY THE MICRON CONNECTION. >> I'D LIKE TO START I TALK BY SHARING AN INTERESTING STUDY WE DID WITH M.I.T. AND THIS IS KNOWN TO CAUSE NEURODE NEURODEGENERATION BUT IT WAS NOT UNDERSTOOD AND WE CREATED A SPECIAL MAP AND THE LAB VIDEO SHOWS THE MOUSE BRAIN AND THE RIGHT SIDE SHOWS A SIX-MONTH BRAIN AND WHITE DOTS SOW THE AFFECTS. IN TWO MONTHS YOU CAN SEE THE AGGREGATED ARE LOCALIZED IB A FEW BRAIN REGIONS AND WOE IDENTIFIED WHERE IT STARTS AND DISCOVERED NEURONS IN ONE OF THE REGIONS SHOWED GREATER EXCITABILITY AND INTERESTINGLY REDUCING THE BRAIN REGION. THERE ARE STILL MANY IMPORTANT QUESTIONS REMAINING TO BE ANSWERED FOR EXAMPLE WE DON'T KNOW WHICH CIRCUITS ARE PARTICULARLY VULNERABLE. ALSO CONSIDERING THE LARGE EVOLUTIONARY GAP BETWEEN MOUSE AND HUMAN IT'S IMPORTANT TO CONDUCT SINGULAR STUDIES WITH HUMAN BRAINS. AND CONDUCTING STRATEGIES WE NEED TO STUDY THE BRAIN ON MULTIPLE SCALES THE WHOLE BRAIN DOWN TO THE [INDISCERNIBLE] AND WE NEED TO STUDY THE COMPLEX CIRCUITS AND PSYCHOLOGICAL FACTORS IN A HOLISTIC WAY AND HOWEVER, IN EXTRACTING THE MULTI SCALE INFORMATION ON THE HUMAN BRAIN WHICH IS 4,000 TIMES LARGER THAN MOUSE BRAIN AND EXTREMELY DENSE AND HETEROGENEOUS REMAINS A MAJOR CHALLENGE. TO TACKLE THE CHALLENGE I HAVE BEEN WORKING WITH A LARGE TEAM OF INCREDIBLY TALENTED ENGINEERS AND SCIENTISTS AT MULTIPLE INSTITUTIONS LISTED HERE. IN THE PAST SEVEN YEARS WE HAVE BEEN WORKING TOGETHER TO DEVELOP A TIME POINT FOR SCALEABLE IMAGING AND PHENOTYPING OF THE HUMAN BRAIN. WE AIM TO DEVELOP TECHNOLOGIES TO ALLOW EXTRACTION OF SPACIAL, MOLECULAR AND MORPHOLOGICAL AND ENVIRONMENTAL AND CONNECTIVITY INFORMATION ALL FROM THE SAME BRAIN. WE WANT THIS TECHNOLOGY PLATFORM TO BE COMPATIBLE WITH TENS OF THOUSANDS OF HUMAN BRAINS CURRENTLY AVAILABLE AT HUMAN BRAIN BANKS WORLDWIDE AND THE NUMBER AT MASS GENERAL HOSPITAL ALONE OVER 400 BRAINS ARE DONATED EVERY YEAR. THIS INCREDIBLY LARGE NUMBER OF BRAINS COVER DIFFERENT SEX, RACE, AGE AND CONDITIONS. MANY OF THEM HAVE ALSO HAD REACHED FUNCTIONAL AND DECADE LONG DEGRADATION AND THEY'RE THE MOST IMPORTANT RESOURCES FOR STUDYING NEUROLOGICAL DISORDER BUT ARE MOSTLY SITTING ON THE SHELVES. WE ALSO WANT IT TO BE COST-EFFECTIVE AND SCALEABLE. SO WE DEVELOPED A SET OF TECHNOLOGIES THAT ENGINEER TISSUE PROPERTIES. AND WE DEVELOPED A SHIELD THAT PRESERVES THE FORMATION WE CARE ABOUT AND ONE EXTENDS TO DO MULTI SCALE IMAGING DOWN TO CONNECTIVITY AND SYNAPSES. WE ALSO DEVELOPED A TECHNIQUE THAT TURNS TISSUE INTO RUBBER LIKE HYDRO GEL WHICH IS STRETCHABLE AND COMPRESSIBLE AND EXPLAIN WHY WE'RE MAKING THIS TRANSFORMATION. USING THE TECHNOLOGY WE CAN PRESERVE ALL THE KEY INFORMATION WE CARE ABOUT AND HERE WE USED ANTIBODIES TO MAP MOLECULES AND CELL TYPES AND STRUCTURES WE'RE INTERESTED IN AND CAN DO THIS MAPPING TO EXTRACT INFORMATION AND MOLECULAR STRUCTURE INFORMATION AND THE DATA SETS CAN BE RAISED. AFTER TREATMENT WE USED THE TECHNIQUE TO TURN THE TISSUE INTO COMPRESSIBLE AND STRETCHABLE HYDRO CELL AND THIS SHOWS THE PROCESS AND YOU CAB SEE IT'S REALLY COMPRESSIBLE AND STRETCHABLE. WITH THIS APPROACH WE CAN BASICALLY MAKE HUMAN TISSUE INDESTRUCTIBLE AND THOUGH THE STRUCTURAL INFORMATION IS PRESERVED SO WE CAN USE HISTOLOGY OR MRNA AND YOU SEE THE STRUCTURE THERE. AND BY COMBINING THIS UNIQUE PROPERTY WITH OTHER TECHNOLOGIES WE DEVELOPED TO ENGINEER THE TRANSPORT WE CAN SIGNIFICANTLY INCREASE THE SCALEABILITY. FOR EXAMPLE, LET'S SAY WE HAVE 5 MILLIMETER TISSUE AND IF YOU TRY TO STAIN IT WITH ANTIBODIES YOU CAN ONLY STAIN THE SURFACE BECAUSE THE PENETRATION IS SLOW. BUT ON THE TIME SCALE CAN BE DESCRIBED LIKE THIS THERE'S A COROLLARY DEPENDENCE BETWEEN TIME AND IF YOU INCREASE THE LENS FIVE TIMES YOU INCREASE THE TIME WHAT WE CAN DO IS COMPRESS THE TISSUE TO AND WE CAN SHORTEN THE PROCESSING PLAN BY SIX TIMES AND COME UP WITH A MORE UNIFORM OR RAPID STAINING AND ONCE THE TISSUE IS STAINED WE CAN REVERSE THE COMPRESSION AND USING THIS WE WERE ABLE TO STAIN EN ONE DAY. AND USING THE APPROACH WE CAN PRESERVE ALL THE INFORMATION AND IMAGE LARGER TISSUE. THIS IS ONE EXAMPLE AND YOU CAN SEE INDIVIDUAL CELLS. IF YOU WANT TO SEE MORE DETAILS, HIGH RESOLUTION STRUCTURES WE NEED HIGH RESOLUTION. SO TO DO HIGH RESOLUTION IMAGING OF THE SAME TISSUE WE EXPAND THE TISSUE AND WHEN IT'S EXPANDED YOU CAN SEE INDIVIDUAL FIBERS AND DETAILS AND SHOWS THE TEMPORAL LOBE STAINED WITH THE ANTIBODIES AND YOU CAN SEE INDIVIDUAL FIBERS AND TRACE THEM. HERE'S OTHER EXAMPLE SHOWING THE FIBERS AND WE HAVE BEEN DEVELOPING TO RECONSTRUCT THE FIBE FIBERS AND THIS SHOWS TRACING IN GRAY MATTER AND TRACING IN WHITE. USING OUR APPROACH WE CAN EXTRACT INFORMATION AND USING OUR PLATFORM IN SUMMARY IT ENABLES MULTI SCALE MOLECULAR PHENOTYPING AND COMPARABLE WITH BRAINS AND CURRENT TISSUE COLLECTION AND POST-EFFECTIVE AND SCALEABLE AN DUE TO THE TIME I'LL SKIP THIS SLIDE I'D LIKE TO THANK THE TEAM WHO MADE PROGRESS IN THE PAST SEVEN YEARS TO ACHIEVE THIS. THANK YOU. >> THANK YOU. >> TODAY I WANT TO FOCUS ON THE CHALLENGE OF MAPPING IN PARTICULAR THE LARGE PRIMATE BRAIN NOT TRANSGENIC AND THEREFORE WE NEED DELIVERING METHODS TO ACCESS IT IN A TRI GENIC FASHION AND ACCESS TO THE OPAQUE MAMMALIAN BRAIN AND ESTABLISH TISSUE CLEARING. IN PARTICULAR I WANT TO HIGHLIGHT THE FACT UNLIKE THE MOUSE BRAIN WHERE WE HAVE A LOT OF TRANSGENIC DRIVER LINES IN THE NON-HUMAN PRIMATE OR HUMAN BRAIN WE ENCOUNTER SIGNIFICANT CHALLENGES HAVING TO DO WITH THE DELIVERY OF GENES AND FOTTONS TO ACCESS FOR -- PHOTONS FOR IMAGING PURPOSES IN TERMS OF LARGE SCALE MACRO SCALE BRAIN CONNECTOMICS. THESE ARE SIGNIFICANT BARRIERS. THE PROBLEM I'LL FOCUS ON FOR THE NEXT FEW MINUTES IS THE GENETIC LABELLING PROBLEM BECAUSE IDEALLY WE WANT TO ACHIEVE GENETIC LABELLING IN THE NON-TRANSGENIC BRAIN BUT IT'S A BIG CHALLENGE FOR THE NON-HUMAN PRIMATE BRAIN AND THE BLOOD BRAIN BARRIERS. THERE'S SIGNIFICANT CHALLENGES TACKLED BY THE SPEAKERS IN THE WORKSHOP HAVING TO DO WITH IMAGING TO CLEARING AND MICROSCOPY AND COMPUTATIONAL RENDERING OF MORPHOLOGY SO I'LL FOCUS ON THIS PART. IN TERMS OF DELIVERING OF GENETIC LABELS TO THE BRAIN, WOULD THE TRI GRENENIC ACCESS WE'RE LEFT WITH VECTORS THAT CODE FOR LABELS TO CIRCUITS INTO THE BRAIN TISSUE AND THIS IS DONE MANY TIMES BY DIRECT INJECTION AS SHOWN HERE. THE CHALLENGE BEING THE SMALL AREA COVERING AND THE RATHER DENSE LABELLING WHICH MAKES MORPHOLOGY RECONSTRUCTION A DIFFICULT TASK. AND WHEN WE WANT TO SCALE THIS HERE I'M SHOWING THE MOUSE BRAIN, WHEN WE WANT TO SCALE TO THE LARGER PRIMATE BRAIN, WE NEED A SIGNIFICANT NUMBER OF HIGHLY INVASIVE INJECTIONS WITH LABELLING AND LACK OF HOMOGENEITY. BECAUSE OF THAT, OVER THE PAST FEW YEAR THERE'S BEEN MANY EFFORTS FROM MANY GROUPS IN TRYING TO ENGINEER BETTER WAYS TO LABEL SUCH CIRCUITS ACROSS THE MAMMALIAN BRAIN. AND THEY'VE TAKEN THE FORM OF EITHER RATIONAL DESIGN OF GENE DELIVERY VECTORS AS SHOWN HERE OR DIRECT REVOLUTION APPROACHES. FOR BOTH METHODS CAPSIDS CAN BE ENGINEERED IN AN ATTEMPT TO TRY TO CROSS THE BLOOD BRAIN BARRIER AND ACCESS THE NEURONAL CIRCUITS IN A WAY THAT'S MACRO SCALE AND GENETICALLY TARGETED. THROUGH THIS KIND OF METHOD ONE CAN TRY TO ACCESS IN A WAY THAT DOES NOT AFFECT ORGANS OR CELL TYPES THAT ARE NOT OF INTEREST FOR BETTER ACCESS TO THE LABELLING. WITH SUCH METHODS AND ACCESS THE MOUSE BRAIN THROUGHOUT. IN A FASHION THAT PROVIDES FOR HIGH OR SPARSE DENSITY LABELLING. BECAUSE ONLY THE CAPSID IS ENGINEERED IN THIS CASE, THERE SAY SIGNIFICANT AMOUNT OF -- IS A SIGNIFICANT AMOUNT OF FLEXIBILITY ONE CAN ACCESS WITH GENETIC SPECIFICITY. FOR TRANSGENIC MICE YOU CAN INTERCEPT WITH DRIVER LINES OR USE PROMOTERS ELEMENTS TO ACCESS AREAS THAT ARE DIFFICULT TO TARGET ANATOMICALLY FOR EXAMPLE THE CEREBELLUM OR ACCESS LARGE AREAS THAT WOULD BE DIFFICULT AND I WANT TO HIGHLIGHT THE LABELLING TO LARGE AREA COVERAGE. ONE CAN ACCESS CIRCUITS IN A WAY THAT'S RENDERED VISIBLE FOR MORPHOLOGY TRACING OR CONNECTIVITY PURPOSES. THIS COULD BE INTERSECTED WITH OTHER METHODS SUCH AS TISSUE CLERG OR SECTIONING OF -- CLEARING OR SECTIONING AND MICROSCOPY CAN TAKE THE FORM OF MICROSCOPY TO ALLOW A LARGE COVERAGE AREA. ONE LABELLING YOU MAY APPRECIATE HERE IN THE IMAGES IS IF YOU DELIVER TOO MUCH LABEL YOU GET OVERLAP IN AREAS THAT ARE UNIFORM IN COLOR WHICH MAKES INTERSECTIONS DIFFICULT TO TELL APART. BECAUSE OF THE REASON THE COMMUNITY IS FAMILIAR WITH THE APPROACH OF TWO COMBINANT VECTOR WHERE HAVE YOU LABELS OR INTERESTS UNDER THE INDUCER. YOU CAN LOOK AT THE CONTROL OF AN INDUCER TO THE TRATTA SYSTEM. WHAT THIS ALLOWS ONE IS TO TURN ON A PARSE SET OF NEURONS THAT RENDERS MULTI COLOR BECAUSE OF THE DELIVERY FOR THE MOUSE BRAIN. HOW IT LOOKS IN PRACTICE AND THIS COULD BE TUNED UP OR DOWN IN ORDER TO GIVE MULTI COLOR PARSE LABELLING OF CIRCUITS THAT ALLOWS RECONSTRUCTION IN A WAY ONE CAN TELL APART ARE FROM DIFFERENT NEURON AND PAIRS WELL WITH TISSUE CLEARING CAN HAVE ACCESS TO THE VOL ULES THIS WAY. -- VOLUMES THIS WAY. THIS IS ALL IN THE MOUSE BRAIN AND YOU MIGHT APPRECIATE THE CHALLENGES WE HAVE IN ORDER TO MOVE SUCH METHODS INTO THE NON-HUMAN PRIMATE BRAIN AND TRANS GENESIS IS ONE BARRIER. FOR THIS WE NEED CELL TYPE SPECIFICITY DELIVERY METHODS THAT DON'T RELY ON TRANS GENESIS AND THERE'S THE SIGNIFICANT ETHICAL NEURON CONSIDERATIONS. ONE MUST MAXIMIZE THE NUMBER OF TRACEABLE CIRCUITS FOR EACH NON-HUMAN PRIMATE BRAIN WE IMAGE. AND OF COURSE THE LOGISTIC CAL CONSIDERATIONS THAT HAVE TO DO WHETHER THE BREEDING IS SEASONAL AND COST AROUND HOUSING AND FEASIBILITY AND THIS HAVING TO DO WITH THE SCALE OF LABELLING REAGENTS. BECAUSE OF THESE REASONS, IT'S IMPORTANT TO TRY TO THEREFORE TOWARDS VECTORS THAT ARE SPECIFIC THAT DOESN'T RELY ON TRANS GENESIS AND EVOLUTION CAN GENERATE A LOT OF DIVERSITY THAT ONE CAN START TO MIND FOR PATTERNS. AND THERE'S MECHANISTIC INSIGHT THAT CAN GUIDE US INTO DEAL HOW TO DEAL WITH THE CELL TYPE SPECIFIC ACCESS ACROSS SPECIES AND THERE'S HOPE FOR THIS BECAUSE AND THE MOUSE COULD BE YERCHK NEAR WHERE CAP SINS TARGETED TOWARDS -- THEY HAVE THROUGHPUT AS IN TRANSCRIPTOMICS ONE CAN START TO BREAK DOWN THE PROPERTIES ON SUB SELL TYPES AND CLASSES OF INTERNEURONS AND EXCITATORY NEURONS. THIS MECHANISTIC INSIGHT CAN GUIDE US OR HOW TO BYPASS THE PROBLEM GENETIC LABELLING IN THE NON-HUMAN PRIMATE BRAIN. AND ONE IN THE MARMOSET BRAIN DESCRIBED HERE AND YOU CAN SEE ACCESS ACROSS THE BLOOD BRAIN BARRIER IS NOW POSSIBLE. AND THERE'S LABELLING TO GET CONNECTOMICS IN THE HUMAN PRIMATE BRAIN. IN TERMS OF THE RHESUS MACAQUE BRAIN SHOWS THE DELIVERY TO ENGINEERED VECTORS CAN BE USED TO COVER A LARGE AREA. YOU SEE HERE THE LABELLING IN THE MACAQUE BRAIN. AND YOU CAN HAVE A VECTOR WITH THE TWO-VECTOR SYSTEM. YOU CAN APPRECIATE FINE AUTHORIZATIONS CAN BE TRACED IN THIS SPACE. IN CLOSING I WANT TO SUMMARIZE AND THIS DECISION IS AVAILABLE AND IN THE MOUSE AND MARMOSET AND FOR FURTHER DEVELOPMENT BY THE COMMUNITY WORKING TOWARDS RELEASES IN HUMAN CELLS. THESE METHODS COMPARE WITH TISSUE CLEARING AND IMAGING THROUGH MICROSCOPY AND I WILL PAUSE IT HERE. THANK YOU. >> HELLO. I'M BOBBY KASTHURI AND PROBABLY THE ONLY EMPIRICAL SCIENTIST COLLECTING THIS DATA AT A NATIONAL LABORATORY. I WANT TO THANK THE ORGANIZERS AND I WANT TO GIVE SOME THOUGHTS ON THIS AND THE SPECIFIC CHARGE I WAS ASKED TO ADDRESS ARE WHAT OTHER TYPES OF TECHNIQUES LIKE MICROSCOPIC TECHNIQUES OR PROENINGS SHOULD BE COLLECTED -- PROENI PROJECTIONS SHOULD BE COLLECTED AND WHAT SHOULD EXTEND TO HUMAN AND NON-HUMAN PRIMATES AND WHAT DO WE NEED IN SAMPLE SELECTION, AGE, SEX, ETCETERA AND WHAT PROJECT OMS SHOULD WE HAVE I THOUGHT IT'D BE USEFUL TO GO BACK TO THE DEFINITION WHATEVER PROJECTOMES MEAN. AND WE TRIED TO DEFINE WHAT A PROJECTION IS WHICH IS THAT FOR CERTAIN INSIGHT IN THE BRAIN, IMAGING IS BETTER. QUESTIONS THAT RELATE TO HOW DIFFERENT REGIONS OF THE NERVOUS SYSTEM MAY NOT NEED PROJECTIONS AND MANY PROJECTIONS ARE PROBABLY POORLY DEFINED AND NEED A WAY TO EXTRACT THE PROJECTIONS AND IT WOULD PROVIDE A ROAD MAP FOR THINKING OF INFORMATION PROCESSING AND ULTIMATELY BE PREPARED FROM A SINGLE CONTROL TO MANY AGES WITH DISEASES AND THERE'S A PIPELINE THAT INCLUDES IN VIVO MRI AND EX VIVO MRI AND AS TO WHAT ANIMALS IT CAN BE APPLIED ON IT'S SPECIES AGNOSTIC AND WORKS ON ALL FROM MICE ALL THE WAY UP TO HUMANS. THE BASIC WORK LOAD IS TO TAKE ANIMALS, FIX THEM WITH ALDEHYDES AND STAIN THEM WITH OSMIUM AND PLACE THEM ON A SINGLE EXTRA ROTATION STAGE AND SOURCE LIKE THE ADVANCED SOURCE WHERE THE SAMPLES ARE ROTATED IN A NON DESTRUCTIVE WAY AND THE IMAGES ARE RECONSTRUCTED. SERIALLY THE SAME SAMPLES CAN BE PUT THROUGH THE PROCESS OF AUTOMATED ELECTRON MICROSCOPY. TO SHOW YOU WHAT THAT CAN LOOK LIKE, THERE'S A PICTURE OF A POST MORTEM MRI AND WE DID MICROSCOPY OF THE BRAIN AT 1 MICRON RESOLUTION AND AND YOU CAN THEN SEE THE DATA SET AND EQUIVALENT BLOOD VESSELS TO REGISTER AND WE CAN IMAGE THE BRAIN OVER SIX ORDERS OF MAGNITUDE OF RESOLUTION. TO SHOW YOU FURTHER WHAT THE EXPERT DATA COULD LOOK LIKE, HERE'S A SMALLER VOLUME OF THE BRAIN THE HIPPOCAMPUS AND I'LL ZOOM IN BRIEFLY TO SHOW YOU THE TYPES OF DATA WE CAN GET. AND AS WE ZOOM IN YOU CAN SEE EVERY WHITE DOT IS A SOW -- SOMA AND YOU SEE THE DENDRITES OF THE NEURONS. THE WHITE SQUIGGLES IS THE BLOOD VESSEL AND THE INDIVIDUAL BLACK BOTS AT THE BOTTOM ARE -- DOTS AT THE BOTTOM ARE INDIVIDUAL DATA. ONE OF THE THINGS WE DID BEFORE WE DID THE X-RAY ANALYSIS IS DO STRUCTURAL MRI AND NOW IN THE PROCESS OF CO-REGISTERING THE DATA SETS. AND YOU CAN FIND INDIVIDUAL NEURONS AND THEIR SHAPES AND DIFFERENT NUCLEI OR IN THE SAME BRAIN YOU CAN GO TO THE HIPPOCAMPUS AND DO CELL BODY COUNTS. AND HERE WE DID ANALYSIS TO THE MRI AND DIFFERENT ORIENTATIONS TO SHOW AGE AND IN J AND K WE PUT THE EXACT AIM MRI AND TRACED THEM TO SHOW ALGORITHMS TO MAKE THEM BETTER. THIS COMBINATION OF X-RAY AND ELECTRON MICROSCOPY WE CALL EXOME MICROSCOPY ALLOWS FOR LONG RANGE AND SHORT RANGE CONNECTIVITY QUESTIONS. INDIVIDUAL VOLUMES CAN BE EXCISED AND SEGMENTS SO YOU CAN LOOK AT MULTI SCALE APPROACHES. AND TO ADDRESS THE THIRD QUESTION ON WHAT LARGER ANIMALS COULD BE APPLIED ON SOMETHING THE LAB COULD BE WORK ON IS GETTING A BETTER UNDERSTANDING OF THE AERIAL AND SYNAPTIC PROJECTIONS AND HERE'S THE MAP OF THE HIERARCHY OF THE CORTEX AND WE WOULD LOVE TO APPLY OUR X-RAY SPECIFICALLY WE'RE IN THE PROCESS OF FIGURING OUT HOW TO SUBDIVIDE THE PRIMATE BRAIN AND STAIN THE VOLUME AND DO X-RAYS AND SELECTIVE CORTICOREGIONS TO GIVE AN EXAMPLE OF THE KIND OF DATA YOU MAY GET WHEN YOU DO ELECTRON MICROSCOPY IN PRIMATE SAMPLES. I'LL SHOW YOU WORK WE'VE DONE IN COMPARISON TO THIS V1. HERE'S THE FIRST PRIMATE CORTEX IN A AND MOUSE AND VISUAL CORTEX IN E, EM SECTIONS THAT SPAN THE CORTICOL CORTICOL CORTICOLEN -- CORTICOLENGTH AND CAN ASK QUESTIONS ABOUT HOW CONNECTIVITY PATTERNS ARE THE SAME AND HOW ARE THEY DIFFERENT. AND WE FIND THAT PRIMATE NEURONS RECEIVE LESS CONNECTIONS AND ALSO TRUE OF INHIBITORY NEURONS FOR A DENDRITIC SENNANCE -- SYNAPSE AND SOMATIC SYNAPSE AND AS WE WORK OUR WAY THROUGH THE CORT CORTIC AL AREAS WE'RE LOOKING AT SYNAPTIC FEATURES AND WE'RE LOOKING AT THE SAME -- SAMPLE SELECTION AND I WOULD SAY ONE THING THAT CONNECTOMICS LARGE VOLUME CONNECTOMICS CAN OFFER IS AN ANSWER TO THE QUESTION OF HOW HUMAN BRAINS WORK. VARIATIONS IN THE CONNECTOME AND WE CAN ANSWER THE QUESTION OF HOW ADULTS LIKE ALBERT EINSTEIN OR JANE GOODALL HOW DO THEIR EXPERIENCE OF THE WORLD LET THEM ENABLE THE WORLD VIEWS THAT THEM TO CHANGE THE WORLD? FINALLY I WANT TO SAY ONE MORE THING ON SAMPLE SELECTION AND THE QUESTION ON LOTS OF SMALL SAMPLES VERSUS ONE LARGE SAMPLE. I HADN'T HEARD THIS EXPLICITLY MENTIONED BUT THINK IT'S WORTH MENTIONING AND A SMALL VOLUME OF BRAIN SAY A MILLIMETER CUBED IN THAT VOLUME WILL BE 100,000 NEURONS AND SOME WHICH YOU'LL IMAGE AND ABOUT A BILLION SYNAPSES. AMONG THOSE BILLION SYNAPSES ONLY 20% OF THEM APPROXIMATELY WE'LL BE ABLE TO IDENTIFY THE PARENT NEURON. WITH 80% OF THE SYNAPSES HOW CAN WE CORRELATE THE ACTIVITY OF NEURONS TO THEIR CONNECTIVITY? IT'S AN INTERESTING QUESTION I'D LIKE TO THINK ABOUT AND ADDRESS WHEN WE THINK ABOUT THE DIFFERENCES BETWEEN SMALL SAMPLES AND LARGE SAMPLES. FINALLY, I'VE THINKING A LOT ABOUT HOW WE IMAGINE THE NEXT BIG THING WE DO IN NEUROSCIENCE RELATIVE TO THE RESOURCES THAT WE HAVE AND I FIND THE ARGUMENTS COMPELLING WE LIMIT BIO RESOURCES AND WOULD LIKE TO END WAY COUNTER EXAMPLE AND FOR ME A COUNTER EXAMPLE IS LITERALLY THE FIRST FLU SHOT. I'D LIKE TO END WITH A QUOTE WHEN IT WAS DECIDED THE UNITED STATES WOULD GO TO THE MOON HE SAYS WE CHOOSE TO GO TO THE MOON IN THIS DECADE AND DO THE OTHER THINGS NOT BECAUSE THEY'RE EASY BUT BECAUSE THEY'RE HARD BECAUSE IT'S A GOAL THAT ORGANIZES US AND MEASURES THE BEST OF US AND BECAUSE IT'S A CHALLENGE WE'RE WILLING TO ACCEPT, UNWILLING TO POSTPONE AND ONE IN WHICH WE INTEND TO WIN AND SOME SAYING THE MOON AND WHY CHOOSE THIS AS OUR GOAL? THEY MAY AS WELL ASK WHY CLIMB THE HIGHEST MOUNTAIN OR THREE THE ATLANTIC AND I IMAGINE THIS IS ALSO A VIEW WE SHOULD THINK ABOUT AND WHEN WE THINK OF A LARGE AMBITIOUS PROJECT ON DOING A MOUSE BRAIN AND SYNAPTIC RESOLUTION. AND A LOT OF THE WORK HAS BEEN ENABLED WITH COLLABORATIONS WITH ARGON SUPER COMPUTING AND USING THE ARGON [INDISCERNIBLE]. THANK YOU FOR YOUR ATTENTION AND I LOOK FORWARD TO YOUR QUESTIONS. >> TODAY I'LL TALK ABOUT CHEMICAL TALKS -- TOOLS AND CLEM AND COLOR EM. WE DEVELOPED A SMALL TAG WHICH COULD LABEL NEWLY SYNTHESIZED PROTEIN AND HERE YOU SEE THE SYNTHESIS OF NEW PROTEIN AND GAP JUNCTIONS AND HOW IT REPLACES OLD PROTEIN. AND YOU MAY SEE THIS HIGH MAG EM IT LAYS DOWN A HIGH LEVEL AND IT'S HARD TO [TECHNICAL DIFFICULTIES] AND IT'S THIS IN WHICH YOU SEE IN THE EM. THIS AMPLIFICATION STEP IS REQUIRED AND THE SMALL MOLECULES CAN FIX INTO SMALL TISSUE NEF BRAIN AND LIMITS IN CONTROLLING THE REACTION AND THERE'S NOT THAT MANY PHOTO SYNTHESIZED. LIGHT IS NOT NEEDED AND THIS IS A SMALL MOLECULE. I'LL GO THROUGH THESE BRIEFLY AND INTRODUCE A SMALL MOLECULE IN COLLABORATION WHERE THE ACTUAL PHOTO SYNTHESIZES IS ONE OF THE DYES WHICH GENERATES THE OM GEN THESE ARE POTENTIALLY USEFUL BECAUSE HE'S SHOWN THEY CAN SELECTIVELY LABEL NEURONS IN LIVE MICE. I WON'T TALK ABOUT THE GENETICALLY ENCODED PEROXIDASES AND IT'S ANOTHER USEFUL TOOL IN THE TOOLBOX. WE INCORPORATED A METAL CHELADE AND THIS HAS MASS SPECTROMETRY SIGNALS AND THERE'S POINTS ABOUT THIS THERE'S LIMITATIONS TO HOW MUCH METAL YOU CAN ADD. WE WANT TO ADD AS MUCH AS POSSIBLE TO INCREASE SENSITIVITY BUT THE MORE YOU ADD THE HARDER IT IS TO PRECIPITATE ONCE IT'S OXIDIZED AND WE LIVE IN TISSUE PENETRATION. ONE IS TO USE MORE METAL COMPLEXES AND THIS IS HOW IT WORKS. WHEN YOU IRRADIATE AN ELECTRON BEAM WITH A FEW OF THE ATOMS IN THE SPECIMEN EIONIZED AND OUT GET THE CHARACTERIZED ELEMENTS IN THE PATH AND DISTINGUISHED DUE TO A METHOD FOR DETECTING WHERE ALMOSTS A ALMOSTS -- ELEMENTS IN THAT SPECIMEN IT CAN BE DISPLAYED IN THE SPECTRUM AND YOU SEE A MINUTE FRACTION OF THE SIGNAL COMES FROM THE SHARP PEAKS. THERE'S OTHERS AS WELL AS WHICH I'LL TALK ABOUT. THIS CAN BE MICROSCOPIC ACOR IMAGE IT TO A -- OR IMAGE IT. THIS IS OUR PROTOCOL FOR MULTI COLOR PRECIPITATION AND I CAN ADD CERIUM DAB OR USE THE GREEN FLUO FLUOROFOR AND YOU CAN HAVE IT OVERLAID ON TOP OF THE ORIGINAL EM. IT ALL HAS THE ORIGINAL RESOLUTION. AND THERE'S SOME LIMITATIONS TO THIS AND THERE'S LIMITED METHODS IN ORTHOGONAL PRECIPITATION OF THE DABs AND IT'S ALSO TIME CONSUMING BECAUSE YOU HAVE TO TAKE MULTIPLE IMAGES AND SENSITIVITY IS LIMITED THOUGH DIRECT [INDISCERNIBLE] GREATLY IMPROVES THIS. AND THIS OTHER SOLUTION THAT WE'RE EXPLORING AND MORE SENSITIVE DETECTION METHODS AND ONE SIMPLE METHOD IS TO LOOK AT THE SINGLE COLOR OR ONE USEFUL THING IS TO LOOK AT THE NEURONS AND SEE HOW ONE COLOR GREATLY DISTINGUISHES SPECIFIC SIGNALLING FROM BACKGROUND HIGH OSMIUM STAINING STRUCTURES FOR EXAMPLE IN THE CELLS OR EVEN AT THE LEVEL OF MAGNIFICATION OF POST-SYNAPTIC DENSITY. ONE WAY TO INCREASE THE SENSITIVITY IS TO MOVE TO A DIFFERENT PART OF THE SPECTRUM. THIS IS STILL VERY SMALL RELATIVE TO THE ELECTED TRON WHICH GO THROUGH BUT SIGNIFICANTLY HIGHER THAN THE HIGH LOSS IT'S A BROADER SPECTRUM AND CAN GET AN INCREASE IN NOISE AND RESOLUTION. THIS IS COMPARED TO THE LOW LOSS AND YOU SEE THE HIGHER SIGNAL TO NOISE AND SHARP PEAKS TO GET DOWN TO THE LEVEL OF A SINGLE PROTON DETERMINATION. THIS SEPARATION ADJACENT ALMOSTS FINALLY ANOTHER METHOD WE'RE STARTING IS TO FOR THE ENERGY USING ELEMENTAL TACT FOR 40 STEM. IN A STEM MICROSCOPE WHEN YOU IRRADIATE THE BEAM ELECTRONS ARE SCATTERED. WE COULD DISTINGUISH BETWEEN SOME OF THE METALS AND THE ENDOGENOUS AND IF WE REPLACE THIS WITH RETHENNIUM THIS IS A MORE ACCESSIBLE HIGH THREE-PUT -- THROUGHPUT AND THROUGH DIRECT DETECTION. I'D LIKE TO STOP THERE. THANK YOU. >> FIRST I'D LIKE TO THANK THE ORGANIZER FOR GIVING ME THE OPPORTUNITY TO SPEAK TODAY. THE TOPIC WE'LL BE DISCUSSING INVOLVES MICROSCOPY APPLIED TO OBTAIN THE MAPS OF ALL CELLS AND TISSUE. A COMMON CHALLENGE IN BIOMARKER RESEARCH IS TO OBSERVE THEM IN THEIR CELLULAR AND TISSUE CONTEXT AND THIS IMPORTANT IN THE COMPLEX ORGANISM WHERE WE LACK THE FUNCTION OF CELLULAR PROTEINS. OUR GOAL AND THERE'S CHEMICAL LABELLING APPROACHES FOR MULTI SCALE, MULTI MODAL IMAGE TO BIOMEDICAL RESEARCH AND WE INTEGRATE INFORMATION ACROSS SCALES AND YOU CAN FOLLOW BIO MOLECULES AND DO SUPER MOLECULAR IMAGING. AND USING THE PROBES WE DEVELOPED AND THEY APLOW -- ALLOW LABELLING AND THE EM PROBE YOU SEE HAS BEEN APPLIED SUCCESSFULLY TO NUMEROUS PROJECTS AND RECENTLY EFFORTS HAVE BEEN MADE TO DEVELOP PROBE THAT ALLOW DETECTION OF PROTEIN DIRECTIONS SUCH AS METHODS FOR EXTRA CELLULAR AND AND USE CHEMIC CHEMICAL LEADING TO THE FORM OF A POLYMER. AND WE GET SPECIFIC INFORMATION AND FOR LINE MICROSCOPY WE CAN OBSERVE THE DYNAMIC BEHAVIOR OF PROTEINS AND WITH THE LATEST ADVANCES IN THE FIELD ONE CAN PUSH THE BOUNDARIES OF OBTAINABLE RESOLUTION AND AND ONE CAN LOOK AT MULTIPLE MODALITIES ACROSS SCALES AND YOU HEARD ABOUT EXAMPLES OF INTEGRATION AND MANY ARE INTEGRATED AND IMPLEMENTS. IT ALLOWS TO RECONSTRUCT AND ONE IS THE TOMOGRAPHY USED TO ACHIEVE HIGH RESOLUTION BIOLOGICAL SPECIMENS AND THE OTHER IS THE SCAN AND THERE'S LARGE 3-D VOLUME AND USES FOR THE STRUCTURE AND FOR CIRCUIT RECONSTRUCTION AND PROVIDES THE 3-D ARCHITECTURE OF ORGANELLES. THE WEALTH OF INFORMATION OBTAINED FROM SPM IS QUITE REMARKABLE. AND THERE'S CONTRIBUTION OF DIFFERENT GLIAL CELLS AND BECAUSE THE RESOLUTION OBTAINED IS IN THE NANO SCALE ONE CAN INTERVENE THE SAMPLES AT VARIOUS ORG ORGANELLES AND CRITICAL IN MANY CELLULAR PROCESSES. AND THERE HAVE BEEN GROUPS IMPLYING DEEP NEURAL NETWORKS. OUR GROUP ALSO FOUND THE SOLUTION AND THIS WAS WORK DONE IN OUR LAB AND WE DEVELOPED WHAT WE CALL A CD3N A CLOUD BASED TOOL. WE APPLIED TO SEGMENTATION TASKS INCLUDING ORGANELLES AND AND AND THERE'S VOL ULES OF SURFACE AREA AND THE DISTRIBUTION OF THE ORGANELLES. WE GET A SET OF METRICS AND CAN LOOK AT THE ROLE OF THE ORGANELLES AND THEIR ORGANIZATION IN DISEASE. THESE TOOLS ALLOW US TO OBTAIN MAPS OF ALL CELLS BUT AND LOOK AT DIFFERENT CELL TYPES IN COMPLEX TISSUE AND TODAY WE CAN INTRODUCE SPECIFIC EM RECORDERS TO DIFFERENTIATE CELL TYPES BY EM OR TARGET PROTEIN SPECIFIC PATHWAYS TO ASSESS THEIR LOCATION AND FUNCTION. I'LL SHOW YOU AN EXAMPLE AND THIS IS A COLLABORATION WITH A GROUP AT UCSD WHERE THE GOAL HAS BEEN TO LOOK AT SYNAPSE IN GIVEN MICROCIRCUITS. TO DO THAT WE COMBINED THE USE OF TRANSGENIC ANIMALS. TO ACHIEVE HIGH RESOLUTION VOLUMES AND SEGMENTATION TOOLS TO EXTRACT VALUABLE INFORMATION FROM THE VOLUMES. THE EMPHASIS OF THE PROJECT WAS THE SYNAPSES WHICH ARE CONNECTS CENTRAL TO UNDERSTANDING OF HOW THE NEURON NETWORK FUNCTIONS AND WE FOCUS ON CHEMICAL NEURAL TRANSMITTERS WHERE THEY RELY ON THE TRANSPORTER KNOWN TO CATALYZE THEIR UPTAKE IN SYNAPTIC VESICLE AND CAN REPRESENT TOOLS TO IMAGE THE INFRASTRUCTURE OF THE NEURAL CONNECTION DEFINED THE NEURAL TRANSMITTERS AND INTRODUCED IT TIED TO THE TRANSPORTER TO THE NEURONS AND SYNAPTIC VESICLES AND WE PRINTED THE LABELLING PATTERN SHOWN HERE WAS A DARK VESICLE. AND WE CONFIRMED THIS AT THE EM LEVEL AND TERMINALS ARE SHOW THIS INTENSE LABELLING AND WE USED TRANSGENIC ANIMALS AND EXPRESSED THE NEURONS BY VIRAL INJECTION AND THE ANTIBODY ONE CAN LOOK AT THE INJECTION SITE AND IT WAS PROPERLY EXPRESSED AND TO VISUALIZE THE LABEL VESICLES AND YOU CAN SEE THE LABEL VESICLE CAN BE LABELLED. WE HAVE THE TOOLS AND LABELLED THE VESICLE SHOWN IN RED FROM THE VESICLES SHOWN IN BLOO BLUE AND THEY'RE WORKING ON TRANSPORTING THEM IN THIS WAY AND WE STARTED COLLECTING DATA FOR THE SYNAPSES WE THINK THESE TOOLS WILL BE USEFUL TO THE NEUROSCIENCE COMMUNITY AND PROVIDE FOR THE FIRST TIME THE DETAILED NANO SCALE ANALYSIS WHERE THE IDENTITY OF THE TERMINALS IS SPECIFIED BY THE NEUROTRANSMITTER. TO CONCLUDE THE COMBINATION OF THE TOOLS ALLOW US TO STUDY THE MORPHOLOGY OF THE EXCITATORY SYNAPSES AND YOU SEE THREE DIFFERENT PROJECTION AREAS AND PROVIDE ANALYSIS OF THE PROFILES AND LOOK AT THE DENSITY AND WE GET THE INFORMATION FROM OTHER ORGANELLES SUCH AS THE SYNAPTIC ALMOSTS AND THIS IS POWERFUL -- AND THIS CAN LEAD TO SYNAPTIC DYSFUNCTION. THE CAPABILITY TO COMBINE IMAGING APPROACHES WITH 3-D AND COMPUTATIONAL POOLS FOR ANALYSIS HAS EXPANDED TO VISUALIZE BIOLOGICAL SPECIMENS AND CONTINUE TO PUSH FORWARD OUR COMPREHENSIVE UNDERSTANDING OF BIOLOGICAL FUNCTIONS. I'D LAKE TO THANK THE FUNDING -- I'D LIKE TO THANK THE FUNDING AGENCIES THAT MADE THE WORK POSSIBLE AND THE WONDERFUL TEAM OF SCIENTISTS AT THE CENTER AS WELL AS OUR COLLABORATORS AND THANK YOU FOR YOUR ATTENTION. >> OKAY. WE HAVE ABOUT 27 MINUTES FOR THE Q&A SECTION. IN THE Q&A WE ALREADY HAVE A NUMBER OF SECTIONS WE ALSO VERY MUCH WELCOME QUESTIONS FROM ATTENDEES AND VIDEOCAST AUDIENCES. AND MAYBE I CAN START WITH AND THE FIRST QUESTION AND THERE'S BRAIN WIDE ACTIVITY MAPS DURING BEHAVIOR. HOW STEREO TYPED ARE THEY? WE'RE DOING AND WE HAVE LONG RANGE SYNAPTIC CONNECTIVITY. >> AND WE HAVEN'T REALLY SAT THERE AND LOOKED AT EXPERIMENTS TO FIND OUT HOW DIFFERENT BECAUSE WE GET IMAGES AND WE LOOK AT AND SO I CAN'T TELL YOU FOR SURE BUT IT'S DEFINITELY NOT EXACTLY THE SAME IN EVERY SINGLE HOUSE NO QUESTIONS ASKED. AND THE CONNECTIVITY AND CORRELATIONS BETWEEN THE DIFFERENT REGIONS CHANGE OVER TIME. THE ANIMALS DO DIFFERENT THINGS OVER TIME AND THE CORRELATION CHANGES AND SOMETHING TO LOOK AT. AND WE ARE TAKING THE MICE NOW AND TRYING TO DO THE CHARACTERIZATION IN VIVO AND DO A CLEAR TISSUE IMAGING TO PUT THEM ON THE FRAMEWORK OF THE ALLEN ATLAS TO REALLY CAREFULLY REGISTER TOGETHER. IN GENERAL THEY'RE SIMILAR ENOUGH TO KNOW WE'RE NOT CRAZY BUT THEY'RE DIFFERENT ENOUGH THEY'RE STRAIGHTFORWARD AND MY PREFERENCE AND THEN I RAISE THE QUESTIONS WHETHER ANY OF THE PRIOR ATLASING APPROACHES HAVE REALLY LOOKED AT HOW STEREOTYPICAL ONE BRAIN IS TO THE NEXT FOR A MOUSE BECAUSE WE KNOW FROM DECADES OF HUMAN BRAIN IMAGING ALL SORTS OF SHAPES AND SIZES COME ALONG. I THINK IT'S ANOTHER POINT. >> WE'VE DONE STRUCTURE AND CONNECTIVITY IMAGING OF MORE THAN 3,000 INDIVIDUAL MICE NOW ALL OF WHICH COME FROM THE C57 BLACK 6 BACKGROUND AND GENETIC BACKGROUND IS VERY HOMOGENEOUS. WE SEE VARIABILITY OF ANATOMICAL REGIONS ACROSS THE DIFFERENT ANIMALS THE VARIABILITY IS NOT BIG BUT WE DON'T KNOW ABOUT THE SPECIFIC CONNECTIVITY PATTERNS YET AND HOW VARIABLE THEY ARE. THAT'S AN IMPORTANT QUESTION TO ADDRESS EVEN FOR INBRED STRENGTH. IT'S GOING TO BE A BIGGER QUESTION WHEN WE GET TO HUMAN AND NON-HUMAN PRIMATES. THAT ARGUES FOR DOING AS MUCH AS WORK FOR THE SAME BRAIN AS POSSIBLE. >> A LOT OF THE QUESTIONS CAN BE CONSOLIDATED INTO ONES YOU KNOW POSES AND EVERY SPEAKER CAN PLAUSIBLY RESPONSE TO WHICH IS HE WRITES WHICH IMAGING MODALITIES OR LABELLING WOULD BE VERY VALUABLE TO BE COLLECTED FOR THE SAME SAMPLE IN WHICH WE ACQUIRE THE WHOLE BRAIN CONNECTOME AND WHICH ARE SUFFICIENT TO CORRELATE ACROSS BRAINS? AND WHERE CROSS BRAIN SUFFICIENT, WHAT ARE THE MOST IMPORTANT ASPECT TO FACILITATE REGISTRATION TO THE BRAIN THAT THE WHOLE BRAIN CONNECTOME IS DONE ON? DANIELLEA SAID SHE'D ANSWERED FIRST. EVERYBODY ELSE CHIME IN. >> WHEN IT COMES TO DEFINING CELL TYPES WHICH HAS BEEN DISCUSSED EVEN IN THE PREVIOUS WORKSHOP WHERE ONE CAN DEFINE THE DIFFERENT CELL TYPES IN THE BRAIN BASED ON THEIR PROJECTOMES AND BASED ON FUNCTIONAL AS PROSPECTS. I THINK THE POSSIBILITY OF INTEGRATING LIGHT MICROSCOPY WHERE ONE CAN COLLECT ACTIVITY INFORMATION AND THEN CORRELATE THEM TO THE EM RECONSTRUCTION AND IT'S VALUABLE AND THEN THERE'S DIFFERENT WAYS TO CORRELATE THE DIFFERENT IMAGING MODALITIES WHETHER ONE IS BY USE IN STRINNIC FEATURES -- INTRINSIC FEATURES IN THE IMAGES LIKE THE VASCULATURES OR OTHER MARKERS THAT CAN BE ADDED TO THE SAMPLES. AND SO I THINK THIS ADDRESSES BASICALLY A VERY IMPORTANT QUESTION REGARDING THE CELL TYPES. AND SPECIFICATION. >> WE'RE LOOKING AT PROJECTIONS FROM PATTERNS IN IN VIVO WE'VE IN THE HAD THE CHANCE TO TAKE THAT SMALL PART OF THE BRAIN AND GO AND DO CONNECTOMICS AND LOOK AT HOW THAT LOOKS IN THE EM BUT THAT COULD BE A GREAT FIRST STEP I THINK. WE COULD INTERPRET HOW TO UNDERSTAND BACK AND FORTH BETWEEN THEM. >> THAT'S A GOOD QUESTION. I THINK IF WE LOOK AT THE CONNECTIONS DONE IN THE PRIMATE BRAIN THERE'S SO FEW REGIONS THAT HAVE BEEN STUDIED BECAUSE IT IS IMPORTANT TO LOOK AT WHAT IS AVAILABLE FROM DIFFERENT INVESTIGATORS AND PERHAPS A DATABASE AND ZERO IN ON THOSE. OF COURSE YOU CANNOT DO EM THE WAY YOU ARE VALIANTLY TRYING TO DO IN THE MOUSE AND I ADMIRE THE EFFORT FOR THE PRIMATE BRAIN AND OF COURSE THE HUMAN BUT THERE'S A LOT OF METHODS WE USE AND PRINCIPLES WE HAVE DERIVED WE CAN USE FOR THE HUMAN. I SHOW THE EXAMPLE THAT WE DID THAT WE SEE PARALLELS BASED ON HOW CONNECTIONS ARE TO PROCEED. WE CAN TAKE THE FEATURES THAT SO MANY OF YOU HAVE BEAUTIFULLY SHOWN TO ADDRESS SPECIFIC QUESTIONS ABOUT WHAT IS VALUABLE IN HUMAN DISEASE AND IN NEUROLOGICAL DISEASES AND WE UNDERSTAND HOW CONNECTIONS CAN BE DISRUPTED. IT'S ONE OF THIS REASONS WHY WE STARTED LOOKING AT THE WHOLE SYSTEM AND MICROSCOPE AND THEN GOING TO SEE WHAT'S HAPPENING. THE REASON WE DO THAT IS SEEING RELIABILITY OF PATHWAYS AND DO WE DEBT -- GET THE SAME THING AS WE LATER DO IN THE SYNAPTIC LEVEL. FOR EACH ONE WE TAKE A LOT OF TIME AND I DON'T KNOW HOW LONG IT WOULD TAKE TO DO A THOUSAND REGIONS. I THINK THE RESOLUTION METHOD IS PHYSICAL AND YOU ZERO IN TO FIGURE OUT WHAT MAY BE HAPPENING AT THE EM LEVEL. >> I'LL AEROSPACE A QUESTION. THANK YOU. >> I CAN ASK ONE IF YOU WANT. >> A COUPLE QUESTIONS WERE TARGETED TO BOBBY. CAN YOU IMAGE THE ENTIRE BRAIN AT THE RESOLUTION SEEN AT HIPPOCAMPUS PART OF THE BRAIN. >> I SUSPECT OUR RESOLUTION ISN'T 600 NANOMETERS THROUGHOUT THE WHOLE MOUSE BRAIN PRIMARILY BECAUSE OF STAINING PROBLEMS AND IT TOOK SEVEN HOURS TO COLLECT THE RESOLUTION OF THE ENTIRE MOUSE BRAIN. I THINK THAT WAS THE ANSWER TO ALL THOSE QUESTIONS BUT TELL ME IF I MISSED IT. >> A MINOR ASPECT DOES IT REQUIRE MONTAGING. MY UNDERSTANDING IS IT DOES. >> WE TRANSLATE THE BRAIN AND TAKE IMAGES. IT'S DIFFERENT IN THE SENSE WE'RE NOT MONTAGING INDIVIDUAL TO THE IMAGES AND IT'S IN FREQUENCY SPACE OPPOSED TO REAL IMAGING SPACE. >> THANKS. I WAS INTERESTED IN THE PROJECT PROJECTOMIC APPROACHES AND ARE THERE WAYS TO COMBINE IT WITH MYELIN AND OTHER THINGS AND WE CAN DO X-RAY IMAGING. YOUR THOUGHTS THERE WOULD BE HELPFUL. >> LET ME START WITH THE PROS AND CONS AND COMBINE WAYS TO COMBINE. THERE'S PROS AND CONS. DOESN'T MEAN I PREFER ONE TO THE OTHER BUT FOR PURPOSES OF CONVERSATION, THE PROS OF X-RAY VERSUS STANDARD LIKE TECHNIQUES WE GET MANY IF NOT ALL OF THE FEATURES IN SINGLE AND THE CELLS AND MYELINATED AXONS AND THE OTHER APPROACH IS THAT THE ONES WE FOCUS ON, MRI, X-RAY AND EM ARE SPECIES AGNOSTIC. WE CAN IMAGINE LOOKING FOR THAT PRIMATE AND HUMAN SAMPLES AND PLACES WHERE WE'RE NOT WAITING FOR GENETIC ACCESS TO EXPRESS CHLOROFORS AND MAYBE THERE'S A WAY GENETIC ACCESS IS DOABLE. THAT BEING SAID IT'S COOL DO EXPERIMENTS THAT COMBINE BOTH AND WE CAN DO IN VIVO IMAGING AND MICROSCOPY AND THERE'S ISSUES WITH FIXATION THAT GIVES GREAT ELECTRON MICROSCOPY OFTEN REMOVES FLUORESCENT LABELS AND OFTEN MAKES THE EM NOT LOOK AT GREAT BUT ABSENT THOSE KINDS OF TECHNICAL SOLUTIONS WHICH I SUSPECT EXIST, WE'D LOVE TO GET IDEAS FOR COMBINING THEM IN SMALLER VOLUMES. >> THANK YOU. THERE'S TWO QUESTIONS HERE FOR STEVEN AND VIVIANA. ARE THERE OPPORTUNITIES TO APPLY THE METHODS DESCRIBED IN THE SAME BRAIN TARGETED FOR A WHOLE MOUSE BRAIN CONNECTOME? IS THIS NECESSARILY COMPLIMENTARY WORK IN SAMPLES FROM SEPARATE BRAINS? THAT WAS MY QUESTION FOR YOU GUYS AS WELL. AND ANOTHER QUESTION LATER. >> I CAN START. AT THE PRESENT STAGE IT'S HARD TO IMAGINE IT BEING APPLIED TO THE WHOLE CONNECTOME BRAIN. THE MOLECULES I'M USING ARE SUBSTANTIALLY LARGER I CAN'T IMAGINE THAT HAPPENING AND WE'RE LIMITED BY THE NUMBER OF COLORS WE CAN DISPLAY SO THE APPLICATIONS I CAN IMAGINE WOULD BE FOR MAPPING SAY SINGLE NEURONS WHICH HAVE BEEN GENETICALLY TAGGED OR TARGETED IN COMBINATION WITH SOME KIND OF ACTIVITY REPORTER. SO WE CAN GET AN EM READOUT. THAT WOULD SUGGEST MORE COMPLIMENTARY APPLICATIONS. TO A SELECTED BRAIN REGION AFTER A WHOLE CONNECTOME. >> I WOULD ADD TO THAT REGARDING WHETHER THIS CAN BE USED FOR THE WHOLE MOUSE BRAIN. SOME OF THE TECHNIQUES ARE TECHNICALLY ABSOLUTELY PARTICULARLY WE AND OTHERS SHOWED THAT WHEN WE LABEL WHOLE CELLS WITH THE PROBES, THIS CAN BE IMAGED NOT ONLY BY EM BUT FOR EXAMPLE THEY ARE VISIBLE BY X-RAY SO THAT ALLOWS US TO CORRELATE AND NAVIGATE ACROSS DIFFERENT IMAGING MODALITIES. THEY ARE AMENABLE FOR WHOLE BRAIN CONNECTOME. >> I WOULD LIKE TO ADD TOO THAT WHAT WE DO WITH THE BRAIN WHEN WE DO A LOT OF THINGS ON THE SAME BRAIN AND THIS IS VALUABLE BECAUSE WE CANNOT IN THE HUMAN BRAIN. YOU CAN SAMPLE IT AND HAVE IT MATCH SO WHEN WE CUT THE BRAIN WE PHOTOGRAPH IT EACH TIME TO KNOW WHERE EACH SECTION COMES FROM. FOR THE MACAQUE BRAIN IT TAKES 1400 TO 1500 SECTIONS OF NEW NAN NANOMETERS AND IN THE BRAIN MORE AND YOU CAN MATCH AND WHAT I WOULD LOVE TO SEE IN THE MOUSE BRAIN AND SMALLER BRAINS YOU'RE USING IS TO LABEL THEM AND FOR THE MYELIN SUGGEST BECAUSE IT CAN IMAGE IN ACUTE LEVELS IN HUMANS. AND PEOPLE HAVE DONE THAT AND FROM ELIZABETH TO BOBBY THERE'S WAYS TO DO CELL TYPE SPECIFIC LABELLING FOR X-RAY IMAGING. BOBBY YOU WROTE SOMETHING IN THE CHAT BUT MAYBE YOU CAN ANSWER LIVE. >> IN FACT I THINK IT WAS NUNO GAVE US SOME TRANSGENIC ANIMALS THAT EXPRESSED ONE OF THESE PER OX DAYSES WE HEARD -- PEROXDASES AND WE CAN SEE THEM AND THEIR D DENDRITE WHEN THEY'RE FILLED WITH OSMIUM IN THE X-RAY AND THEN CAN WE GET COLOR OUT OF THIS? AND I'LL ASK EXPERTS AND THEY'LL PROBABLY E-MAIL ME THAT SAID WEREN'T YOU LISTEN WHEN WE DESCRIBED IT TO YOU ABOUT THE X-RAYS IN THE AIRPORTS BUT I'LL FIND OUT. >> THERE'S A DETAILED QUESTION FROM CHRISTEN HARRIS TO DANIELLA BUT MAY SPEAKS TO ISSUES WITH THE METHOD IN GENERAL. WHY SO FEW VESICLES LABELLED WITH THE [INDISCERNIBLE]. >> THANK YOU FOR ASKING THAT QUESTION. IT WAS SOMETHING THAT SPRAFD US AS WELL. GIVEN THAT ALL THE REAGENT ARE REALLY ACCESSIBLE TO THE TISSUES SO WE WERE SURPRISED TO SEE THE SUBSET OF THE VESICLES ARE LABELLED. AND I GUESS WE CAN POSTULATE DIFFERENT REASONS AND ONE IS WE'RE LOOKING AT THE GLUTAMATE NEURONS AND THE SUBSET ALSO EXPRESS OTHERS LIKE DOPAMINE AND GABA. ONE POSSIBILITY IS ONE OF THE VESICLES MIGHT CONTAIN THE TRANS TRANSPORTER TO THE NEURONS AND THEN THE GAMMAERGIC ONES AND IT'S POSSIBLE SOME OF THE VESICLES DON'T CONTAIN ANY. WE REALLY DON'T KNOW WHAT THAT MEANS IN TERMS OF THE BIOLOGY. BUT IT'S CERTAINLY SOMETHING WE WANT TO FOLLOW UP ON AND FOR EXAMPLE LOOKING AT DIFFERENT BRAIN AREAS TO SEE WHETHER WE OBSERVE A SIMILAR PATTERN. >> IT LOOKS LIKE ONE THING I MAY NOT BE COMPATIBLE WITH EACH OTHER AND I GUESS YOU CANNOT BE COMBINED WITH X-RAY OR [INDISCERNIBLE] BECAUSE THE STRUCTURE ALL DISAPPEARED AFTER IT WAS CLEARED. I WONDER IF YOU GUYS HAVE ANY COMMENTS ON THAT AND WOULD THOSE BE TOTALLY INDEPENDENT APPROACHES AND THEN A SEPARATE COLLECTION FOR XIAOTANG HOW DO YOU ENSURE THE CONTINUOUS TRACING IN YOUR LIGHT MICROSCOPY IN THAT APPROACH? >> TO ANSWER YOUR FIRST QUESTION, YES, THE TECHNIQUES WE USE AROUND WE SHOWED IN 2013 WE CAN STILL SEE SOME MEMBRANE STRUCTURES AND THE QUALITY IS REALLY IMPORTANT. I DON'T THINK THE CURRENT TISSUE TRANSFORMATION CLEARING IS COMPATIBLE BUT MAYBE IN THE FUTURE ONE COULD FIGURE OUT A WAY TO CREATE ENOUGH SUBSET OF LIPIDS TO GET ENOUGH [INDISCERNIBLE] FOR DOWN STREAMING. AND HOW TO TRADE AXONS BETWEEN SLACKS. WE HAVEN'T DEMONSTRATED YET WE DON'T KNOW IF IT'S FEEFBL FEASIBLE OR NOT BUT WORKING ON IT AND MAKE SURE WE MINIMIZE DAMAGE DURING THE SESSION WE DEVELOPED THAT CAN SLICE HUMAN BRAIN SIZED TISSUE WITH NICE PROFILE. WE ARE CURRENTLY CHARACTERIZING THE PROFILE AND GETTING DATA TO SEE IF WE CAN TRACE BETWEEN BLOCKS. WE CURRENTLY DON'T HAVE DATA SHOWING [INDISCERNIBLE]. >> I'LL ADD MY TALK WAS SO FAST IT WAS PROBABLY IMPOSSIBLE TO CATCH WHAT I SAID BUT ONE THING I DID MENTION WAS THAT WE DO TISS TISS TISSUE QUERING AND SHOWED THE SECTION OF MOUSE BRAIN WERE ABLE TO IMAGE THROUGH THAT AND QUICKLY AS WE WERE ABLE TO IMAGE CLEAR TISSUE. I WOULD SAY THAT IF WE DESPERATELY WANT TO BE ABLE TO DO SOME IMAGING OF TISSUE PRIOR TO CONNECTOMICS AND THE REASON FOR CLEARING IS TO GET THAT ABILITY TO IMAGE THESE LARGER VOLUMES WHICH WE DON'T NECESSARILY NEED. AS TO TRACING AXONS, I WOULD SAY WHEN IT COMES TO REALLY LARGE CHUNKS OF SAY HUMAN BRAIN, WE STILL NEED TO THINK CAREFULLY ABOUT HOW MUCH MOTIVATION WE CAN EVEN ATTEMPT TO GET ON THE FIRST TRI. -- TRY. IMAGING AT THE LEVEL YOU'D NEED TO TRACE EVERY SINGLE INDIVIDUAL AXON WILL BE SUCH AN ENORMOUS DATA SET IT WILL TAKE A LONG TIME. I THINK THERE'S OTHER POINTS TO THINK ABOUT THERE PERHAPS. >> SORRY, WE'RE OUT OF TIME. MAYBE WE CAN GET BACK TO IT IN THE DISCUSSION BUT THE NIH ORGANIZERS HAVE ASKED US TO START AT 3:00 EASTERN SHARP IN NINE MINUTES. SORRY TO INTERRUPT BUT LET'S TAKE A QUICK BREAK AND COME BACK OR THE DISCUSSION PANEL. >> WE'LL TALK ABOUT THE SCALE CAMPAIGN THAT'S HAPPENING NOW. IT'S AN OPPORTUNITY FOR EVERYONE TO SEND ME YOUR INPUT REGARDING THE BRAIN CONNECTIVITY AND BRAIN CONNECTOMICS PROJECT. THE PLANNING PROCESS. SO WE STRONGLY ENCOURAGE EVERYBODY TO ENTER THEIR INPUTS. >> AND JUST TO GIVE A LITTLE BIT IF YOU WANT TO DO IT GO TO BRAIN CONNECTIVITY SERIES.com THE NIH/DOE THING AND A LINK CALLED JOIN THE DISCUSSION RIGHT AT THE TOP AND IT TELLS YOU HOW TO PUT YOUR THOUGHTS IN. >> OKAY. LET'S START THE DISCUSSION PANEL. WE HAVE FIVE PANELISTS. WE'LL HEAR THEIR PITCH AND IDEAS FOR FOUR MINUTES AND THEN EACH PANELIST WILL ALSO HAVE AN OPPORTUNITY TO RESPOND TO THE OTHER PANELISTS. WE'LL START WITH JOANN BUCHANAN AND JEFF LICHTMAN AND LISA MILLER AND LINNAEA OS TROUGH AND KLAY REID. >> YOU HAVE TO UNMUTE. >> THANK YOU FOR INVITING ME. I LOOKED OVER THE QUESTIONS WE WERE CHARGED WITH AND THE BIG ONE WAS ON THE LIMITATIONS. YOU HEARD A LOT ABOUT SAMPLE PREPARATION AND HOW IMPORTANT IT IS TO THE WHOLE BRAIN PROJECT. SO ME WE'RE USING OSMIUM AND THEY WERE MADE FOR SMALL TISSUE SAMPLES. ONE OF THE BIG QUESTIONS IS HOW TO SCALE UP AND OVERCOME THE PROBLEMS WE'RE HAVING. TO ME ONE OF THE BIG PROBLEMS IS THE PENETRATION OF OSMIUM INTO THE SAMPLE. SECOND IS GETTING CONSISTENT RESULTS. . IN OUR PHASE 1 PROJECT OUR SAMPLES WERE 200 MICRON THICK SLICES AND WE COULD GET CONSISTENT RESULTS EVERY TIME BUT HOWEVER, WHEN WE MOVED TO LARGER SAMPLES, 1 MILLIMETER OR 2 MILLIMETER THICK SAMPLES WE STARTED ENCOUNTERING PROBLEMS. THIS CENTERS AROUND THE OSMIUM PENETRATION AND AS IT GOES IN THE TISSUE IT BUILDS A BARRIER AND GO TO AN AREA UNSTAINED WE CALL THE CORE. THE MYELIN TAKE UP MORE OSMIUM AND IS PART OF THE CAUSE OF THE CORE FORMATION WE WOULD TAKE A SEMI THIN SECTION AND SEE THERE IS THE CORE. YOU CAN SEE IT IN CROSS SECTION AND WE USED WHAT WAS ADDED AND IT'S A COMBINATION OF ROTO TO MAKE THIS PROTOCOL AND THE SAMPLE HAS SEVEN HOURS OF OSMIUM AND STILL HAS A CORE. WHEN YOU LOOK BY EM AND SEE WHAT THE CORE LOOKS LIKE IT LOOKS PRETTY GOOD AS THE EDGES BUT AS YOU GET DEEPER INSIDE TOWARDS THE MIDDLE EVERYTHING IS DIMINISHED AND SEE THAT IN THE MYELIN. ANOTHER PROBLEM TOO WAS THE RESULTS WERE NOT CONSISTENT FROM TIME TO TIME WE COULD DO THE SAME THING AND JUST COME OUT WITH A COMPLETELY DIFFERENT SAMPLE. THIS IS FRUSTRATING BECAUSE AS WE ALL KNOW EM IS A VERY SLOW AND LABORIOUS PROCESS AND OUR SAMPLES ARE VALUABLE. BEING ABLE TO VAELS -- EVALUATE THE SAMPLES IT WAS VALUABLE IT SEE THE CORE AND NOW WITH THE WORK OF WATCHING LIVE WATCHING THE OSMIUM GO IN THE TISSUE IS A BIG BREAKTHROUGH IN SAMPLE PREP. I'D LIKE TO CHALLENGE PEOPLE TO THINK OUTSIDE THE BOX AND DEVELOPMENT OF NEW CHEMISTRY TO HELP WITH THE STAINING PROBLEM AND WAYS TO IMPROVE THE PENETRATION PHYSICALLY MAYBE USING MICROWAVES OR ULTRASOUND. AND FINALLY THE BIG QUESTION TO ME TOO IS WE HAVE THE WHOLE BRAIN DO WE KEEP IT WHOLE OR DIVIDE AND CONQUER? >> I'LL POINT OUT THE ROAD MAP IS MANY STEPS BEGINNING WITH PRESERVATION AND THEN STAINING WE HEARD A BETTER WAY TO DO THIS IS MAYBE SECTION AND THEN THIN SECTIONING AND THEN IF IT WAS WITHBLOCK FACE WE'D IMAGE IN A ROUND ROBIN AND IMAGE EVALUATION STITCHING, ALIGNMENT, SEGMENTATION AND SYNAPSES AND OTHER ANNOTATIONS AND ANALYSIS AND SHARING. THAT'S BASICALLY WHAT WOULD HAVE TO HAPPEN AT THE MINIMUM TO HAVE A WHOLE MOUSE BRAIN. THE EARLIER STEPS ARE ESSENTIAL. THEY HAVE TO WORK FOR IT TO MAKE SENSE TO GO FURTHER. IT'S MY SENSE THAT LARGE VOLUME OSMIUM STAINING UP TO 250 CUBIC MICRONS WHICH WOULD BE A MOUSE BRAIN WILL BE REALIZED IN THE REASONABLE FUTURE BUT THERE'S PLAUSIBLE BACK UPS. I'D LAKE TO FOCUS IN MY FEW MINUTES THE SUM OUTSTANDING PROBLEMS OR QUESTIONS THAT ARE MOST VEXING AND SHOULD WE BE DOING THIS WITH BLOCK FACE OR PRESERVE TIF APPROACHES WHERE THE SAME SAMPLE CAN BE LOOKED AT MANY TIMES AND I WANT TO ADD ONE ADDITIONAL ISSUE TO CONSIDER WHETHER IT'S IMPORTANT THAT IS IF THE TIME IT TAKES TO DO A WHOLE BRAIN MATTERS. IF IT TAKES 20 YEARS THERE IS ONE ADVANTAGE OF PRESERVATIVE APPROACHES AND PARTS OF THE BRAIN THAT REQUIRE LOW RESOLUTION LIKE WHITE MATTER CAN BE SCANNED QUICKLY WHEREAS THE NERVE FILLED SECTIONS CAN BE SCANNED AT HIGHER RESOLUTION. SO DO THE WHOLE BRAIN AT LOWER RESOLUTIONS SAY A MICRON PER PIXEL AND GO BACK AND DO THE PARTS YOU NEED TO BE HIGHER RESOLUTIONS AND POTENTIALLY SAVE TIME BECAUSE THEY CAN GO ON IN PARALLEL IN DIFFERENT PARTS OF THE DATA SET. THE SECOND QUESTION IS IF YOU SAY YOU HAVE A WHOLE BRAIN LABELLED AND DON'T WANT TO USE THE BLOCK FACE A WHOLE BRAIN BLOCK FACE MAY BE A MILLIMETER AND ONE COULD RELIABLY SECTION A MILLIMETER? WE DON'T HAVE A GREAT DEAL OF DATA WE HAVE CUT ONE BRAIN OF 15 NANO METERS AND WHETHER ONE CAN DO THAT CONSISTENTLY IS A BIG QUESTION. HOW TO DEFEND -- DIVIDE AND CON SER IS -- CONQUER IS AN ACTIVE AREA AND DOING LABELLING WITH MICROSCOPY OR POST EMBEDDING CAN BE DONE ON THE SAME MOUSE. I WANT TO BE EXTREME AND SAY YOU WOULD GET 100 ANSWERS IF YOU ASKED 100 PEOPLE AND ONE POSSIBILITY IS NOT DO ANY MOLECULAR LABELS. I KNOW SOME WOULD DISAGREE AND IT'S UNREALISTIC TO GET THE COMPLETE [INDISCERNIBLE] AND SO WHAT IS REQUIRED FOR THIS PARTICULAR MOUSE? I'D SUBMIT PERHAPS NOTHING. >> THANK YOU FOR THE GREAT ROAD MAP AND QUESTIONS. WE CAN DISCUSS LATER. NEXT IS LISA. >> THANK YOU. I'M LISA MILLER AT BROOK HAVEN LAB. MY RESEARCH GROUP FOCUSSED ON CHEMICAL IMAGING OF BIOLOGICAL TISSUE. I'D LIKE TO EMPHASIS IMAGING THE WHOLE BRAIN REQUIRES A MULTI MODAL APPROACH AND NEW TAGS ARE NEEDED TO AID IN THIS APPROACH. THIS PROVIDES THE RESOLUTION NEEDED AND REQUIRES HEAVY METAL STAINING AND CHALLENGES TO OBTAIN SUFFICIENT CONTRAST. WE'VE SEEN THE MICROCT IS USED TO ASSESS THE QUAL OF STAINING IN THE WHOLE BRAIN AND WITHOUT SECTIONING BUT AND TRADITIONAL MICROCT IS LIMITED TO SPACIAL RESOLUTION OF A MICRON AND HEARD NEW METHODS CAN IMPROVE THE SPACIAL RESOLUTION DOWN TO 20 NANOMETERS OR EVEN BETTER AND ELECTRON X-RAYS WE KNOW GENETICALLY ENCODED TAGS LIKE GFP ARE THE GOLD STANDARD FOR INDIVIDUAL PROTEINS AND CELLS AND PROVIDES AN ADVANTAGE FOR INDIVIDUAL CONNECTIONS. HOWEVER, THE SPACIAL RESOLUTION VISIBLE LIGHT MICROSCOPY IS NOT WHAT YOU CAN GET TO BE SUFFICIENT FOR IMAGING THE BRAIN CONNECTOME. HERE I'D LIKE TO SUGGEST A WAY TO COMBINE THE POSITIVE ASPECTS BY USING GENETICALLY ENCODED TAGS SENSITIVE TO ALL THREE IMAGI IMAGING MODALITIES. I'D LIKE TO PROPOSE IT CAN BE DONE WITH GFP WITH BINDING TAGS AND WE KNOW IT FOR VISIBLE LIGHT AND TAGS ARE SOMETHING YOU'RE PROBABLY LET FAMILIAR WITH AND SHORT PEPTIDES WITH A TIGHT BINDING SITE AND WERE INITIALLY DEVELOPED AT M.I.T. AND BOSTON UNIVERSITY FOR SPECTROSCOPY AND PHASING IN CRYSTALLOGRAPHY AND THEY HAVE THE POTENTIAL FOR SUFFICIENT CONTRAST WITH X-RAYS AND HYPOTHETICALLY IT CAN PERMIT THE IMAGING AT THE SAME TISSUE IN ALL THREE IMAGING MODAL -- MODALITIES. IN THE LAST PANEL I WANT TO SHOW YOU THAT THE BINDING TAGS PRODUCE RESOLUTION USING A SINGLE SOURCE WITH ANTI-FLUORESCENCE DETECTION. YOU SEE THE RED COLOR INDICATES THE BINDING TAG TO LANTHAN IDE TAG AND SINCE X-RAYS ARE MORE PENETRATING THEN ELECTRONS 3-D TOMOGRAPHY CAN BE ANALYZED AND THE YELLOW IS THE MEMBRANE PROTEIN AND IT'S BASICALLY LOCALIZED ON THE OUTER SURFACE OF THE CELL. WHILE THE FUSION TAGS WORK FOR VISIBLE LIGHT THERE'S STILL CHALLENGES THAT REMAIN. FURTHER WORK NEEDS TO BE DONE TO TEST THE APPROACH WITH ELECTRON MICROSCOPY AND MAY REQUIRE FURTHER DEVELOPMENT IN DETECTERS IN ELECTRON MICROSCOPES AND FURTHER REFINING IS NECESSARY TO OPTIMIZE COMBINED VISIBLE AND X-RAY IMAGING APPROACHES INCLUDING THE SPEED AND DETECTION SENSITIVITY BUT I BELIEVE THE GENETICALLY ENCODED TAGS ARE A WAY TO ENHANCE THE IMAGING IN THE BRAIN CONNECTOME. >> I THINK THESE METHODS HAVE A LOT TO BROING -- BRING TO THE TABLE AND DOING RECONSTRUCTION. THERE WERE BEAUTIFUL APPLICATIONS OF USING STANDARD EMO TECHNIQUE TO DO RECONSTRUCTIONS OF CIRCUITS IN THE PRIMATE BUT THERE ARE A COUPLE OTHER OBVIOUS USES. ONE IS LOOKING FOR MARKERS AND CELL TYPES SO IF YOU HAVE AN UNLABELLED BRAIN YOU CAN IDENTIFY USING MORPHOLOGY AND IT'S POTENTIALLY POSSIBLE DEPENDING ON WHAT KIND OF MARKER YOU'RE USING TO USE DEN DRIEDS TO FOLLOW FROM ONE SECTION TO THE NEXT TO FACILITATE ALIGNING ACROSS SECTIONS THAT ARE SMALLER THAN AN ENTIRE BRAIN. THERE'S THE IMPAIRMENT OF MERVOLOGY. -- MORPHOLOGY. THAT'S A CONCERN TO A CERTAIN EXTENT DEPENDING ON WHAT YOU'RE LOOK AT AND A LITTLE BIT OF A RED HERRING. WHAT I WANTED TO DO I WANTED TO MAKE SURE WE'RE ALL AND THE SAME PAGE AND WHAT THE MORPHOLOGY LOOKS LIKE USING STANDARD ANTIBODY TECHNIQUES AND THESE ARE ALL RODENT BRAIN SAMPLES. THEY'VE ALL BEEN PREPARED DIFFERENTLY BUT WE HAVE TRANSGENIC MARKERS YOU CAN BUY OFF THE SHELF. NONE WERE OPTIMIZED FOR MORPHOLOGY OR CONTRAST. AND THERE'S PLENTY OF ROOM FOR IMPROVEMENT AT THE POST-STAINING LEVEL AND YOU CAN SEE THE MOEFSHOLOGY IS PRESERVED. AND THIS IS GABA WHICH IF YOU DO ANY OF THIS YOU KNOW IT'S LOW HANGING FRUIT BUT YOU CAN ALSO DO SOME PEP SIDES AND PRE EMBEDDING LABELLING TENDS TO BE MORE SENSITIVE AND ONE ADVANTAGE IS YOU DON'T HAVE TO HANDLE THE SECTIONS BUZ -- BECAUSE THE ANTIBODIES ARE DONE BEFORE IT'S EMBEDDED AND HAS THE ADVANTAGE IN THIS CONTEXT THE ANTIBODIES DON'T PENETRATE VERY FAR IN THE TISSUE. THE AXONS AN DENDRITES LABELLED OR THE MARKERS ARE ONLY LABELLED A COUPLE MICRONS IN. WE'RE LOOKING AT EDGE. YOU CAN FOLLOW THE S THROUGH INTO THE DEPTH OF THE TISSUE AND THE LABELS DO NOT OCCLUDE ANYTHING. THESE ARE SIMPLE PROTOCOLS I'M NOT ADVOCATING FOR ANY SPECIFIC APPROACH THE POINT IS IT'S PERFECTLY POSSIBLE TO PRESERVE USABLE MORPHOLOGICAL DETAIL TO THE POINT YOU CAN DO A LOT OF RECONSTRUCTION THE TISSUE IN THE REFRIGERATOR FOR A WEEK BEFORE WE REHYDRATED IT AND ANTIBODY LABELLED IT. I'M NOT TRYING TO BE SPECIFIC ABOUT A PROTOCOL BUT CAN RETHINK OUR REFUSAL TO USE DATA FROM THE DATA SETS. >> LAST BUT NOT LEAST, CLAY. >> THANK YOU. THANK YOU FOR INVITING ME AND FOR ORGANIZING THIS. I WANT TO TALK MORE GENERALLY ABOUT THE LARGE ANIMAL PROJECT PROJECTOME WHICH HAS ALMOST GOTTEN EQUAL BILLING TO THE EM CONNECTOME AND THERE'S TWO PROJECTS AND THE FIRST IS WHOLE MOUSE BRAIN EM AND THE SECOND IS ANIMAL PROJECTOMES. TODAY WE HEARD ABOUT WHOLE BRAIN PROJECTOMICS AND WANT TO CALL TENSION THE TALKS ABOUT POINTING TOWARDS THE HUMAN PROJECTOME I'LL SHARE MY SCREEN AND TALK ABOUT THE HUMAN PROJECTOMAL AXIS AND IT'S THE DENSE NAMING OF AXONS WITH EITHER ANTIBODIES OR IMAGING AS XIAOTANG TOLD OR BOBBY DISCUSSED. BOTH ARE HIGHLY VALUABLE BUT ONE CAN ARGUE THE DIFFERENTIAL AND WHAT WE HAVE NOW AND WHAT MIGHT WE HAVE IN A NUMBER OF YEARS IS ARGUABLY HIGHER FOR THE HUMAN PROJECTOME. NEVER HAS A SINGLE AXON BEEN TRACED FROM THE HUMAN AREA NOT ONE. A NUMBER OF US ARE DISCUSSING GOING FROM ZERO TO BILLIONS OF AXO AXONS SIMULTANEOUSLY IN THE SAME HUMAN BRAIN AND WHAT COULD GIVE YOU A MAP OF THE AREA AND INTERCONNECTIVITY AND WILL HAVE LONG TERM CLINICAL IMPORTANCE. THERE'S GREAT VALUE IN NEXT GENERATION NEUROPATHOLOGY. AND DEEP BRAIN STIMULATION BY AXONS. MOST DEGENERATIVE DISEASES HAVE A WHITE MATTER COMPONENT. ALONG FOR THE RIDE IS THE [INDISCERNIBLE] FOR THE BRAIN AND THERE COULD BE MOLECULAR LABELLING. THE HUMAN BRAIN PROBABLY HAS MORE IMPORTANCE FOR A LARGE SCALE MODEL OF COGNITION. ACTUALLY AT THE PROPER RESOLUTION 100 NANO METERS TO GIVE YOU MAYBE 70% OF ALL MYELINATED AXONS IT'S AN EXTREMELY AMBITIOUS GOAL BUT PROBABLY SIGNIFICANTLY LESS EXPENSIVE SO ONE COULD DO BOTH THE EM MOUSE CONNECTOME AND THIS AND WITH LIGHT MICROSCOPES THEY'RE NOT THAT EXPENSIVE IT'S NOT A MUL BE -- MULTI BEAM SEM AND ONE CAN HAVE INTERMEDIATE GOALS OF VARIOUS SIZES AND HAVE A GREAT DEAL OF TECHNIQUE DEVELOPMENT AND IMPORTANT SCIENCE. A MOUSE DENSE PROJECTOME NOT LIKE THE ALLEN TRACE PROJECTOME BUT WHERE YOU LABEL EVERY AXON IS A PETABYTE AND THE SIZE OF SOMETHING THAT OUR LAB OR LICHTMAN'S LAB CAN DO EVERY COUPLE MONTHS. THIS IS IMPORTANT FOR THE MOUSE CONNECTOME AND HAVE IT AS AN AVAILABLE LINKAGE MAP. >> SORRY, WE RAN OUT OF TIME. >> -- IMMEDIATE STEPS. THANK YOU FOR ALLOWING ME TO GO ON. >> THANKS, EVERYBODY FOR THOSE RESPONSES TO THIS WORKSHOP WE'RE GOING TO DO ANOTHER ROUND OF TWO MINUTES MAY CLAY YOU CAN DO YOURS IN ONE MINUTE. BEFORE WE -- CLAY -- BEFORE WE DO THAT WE WERE SUPPOSED TO HAVE ANOTHER DISCUSSANT A GOOD FRIEND AND COLLEAGUE HAD A FAMILY EMERGENCY AND COULDN'T BE HERE BUT I KNOW THAT HE WAS PLANNING TO TALK ABOUT SPINAL CORD AND WAS PLANNING TO ARGUE IN ADDITION TO THE CENTRAL BRAIN WE SHOULD THINK OF THE REST OF THE CENTRAL NERVOUS SYSTEM. I THINK IT RELATES TO JEFF'S IF WE TOOK OUR TIME WE CAN DO A LOT AND IN THE SAMPLE PREPARATION FOR THE PROJECT DO WE KEEP THE OLFACTORY BULBS AND THE RETINAS AND TRY TO GET THE SPINAL CORD ALL FROM THE SAME ANIMAL. I WON'T SAY MORE OTHER THAN TO SAY THAT WAS A TOPIC AREA WE HOPED TO TOUCH ON AND CAN NO GO AROUND IF ANYONE WANTS TO SPEAK TO THAT, THAT WOULD BE WELCOME. NO NEED TO SAY A LOT BUT YOU HAVE TWO MINUTES EACH TO RESPOND. >> I LIKE THE IDEA OF LOOK AT AS MANY THINGS AS POSSIBLE. WE FIRST HAVE TO OVERCOME AND DECIDE HOW THINGS ARE GOING TO BE DONE AND DIFFERENT LABS AT DIFFERENT PLACES WILL FOCUS ON WHOLE BRAIN OR PIECES OF BRAIN OR THE SPINAL CORD. AS PEOPLE DEVELOP THEIR PROTOCOLS AND APPROACHES AND CAN SHARE THEM AND DIFFERENT LABS HAVE DIFFERENT RESOURCES AND COMBINING THINGS IN A WAY TO PUSH FORWARD THE PROJECT WITH WHOLE BRAIN BEING THE GOAL BUT REALIZING ALONG THE WAY THERE'S LOTS OF BENEFICIAL DATA TO COME FROM THESE KINDS OF STUDIES AND LOTS OF LOW HANGING FRUIT EVERYWHERE EVEN IN LOW VOLUMES. >> I'M INTERESTED IN US HAVING A DATA SET WHERE FROM SOME SENSORY MODALITIES YOU CAN TRACE THE WIRING AND OTHER MOVEMENTS CAN PROBABLY BE DONE WITH BRAIN STEP NEURONS BUT IT WOULD BE NICE TO HAVE BEHAVIORS FROM ALPHA TO OMEGA WITHOUT THAT AND IN THE HEMI BRAIN AND OTHER ANIMALS EXCEPT C ELEGANS IT'S HARD TO UNDERSTAND THE PURPOSE OF THE BEHAVIOR IF YOU DON'T HAVE THE OUTPUT OR WHAT'S DRIVING IT AND HAVE YOU THE DISEMBODIED BRAIN WITHOUT THE INPUT AND OUTPUT. IT WOULD BE GREAT TO DO THE WHOLE SPINAL CORD BUT REMEMBER THAT WOULD QUADRUPLE THE EFFORT AND GET MORE BECAUSE IT'S A LONG THING THE RANGE OF A CENTIMETER THAT'S EXTRA TISSUE AND JOHN NYE SAID IN OUR LIFE TIMES AND I LOOK AT HIM AND ME AND WHAT WE MEAN IS SOMETHING QUITE DIFFERENT. IT'S A SLIGHTLY DIFFERENT POINT OF VIEW. >> THE QUESTION BEFORE THE END OF THE BREAK HAD TO DO WITH MULTI COLOR AND X-RAYS BECAUSE I THINK THAT WAS A LITTLE BIT OF A DISCUSSION AND I WANT TO ADD X-RAYS HAVE THE OPPORTUNITY DEPENDING ON THE ENERGY YOU CHOOSE IF YOU IMAGE ABOVE AND BELOW THE OSMIUM AND LEAD AB SORPTION YOU CAN GET DIFFERENT METALS AND I WANT TO EMPHASIZE ABLE ANSWER TO A QUESTION THAT CAME UP BEFORE THE BREAK. X-RAYS CAN DO COLOR YOU CAN DO MULTIPLE COLOR IMAGING. WE'RE USING CHEMISTRY DEVELOPED IN THE 1950s AND '60s. AND IT HAS NOT CHANGED AND IT'S AN EXTRAORDINARILY AGGRESSIVE OXIDIZER AND GOOD LIPID FIXATIVE BUT ALSO USED AS METAL CONTRAST AND IS TO FIND WAYS MUCH FIX LIPIDS AND STAINING THEM LATER THAT DON'T INVOLVE SUCH AGGRESSIVE CHEMISTRY AND I DON'T KNOW IF THIS IS A VIEW POINT OR QUESTION TO THROW OUT THERE, I WONDER WHAT EVERYONE THINKS ABOUT THE RELATIVE VALUE IN TALKING ABOUT ADDING PARTS OFF THE BRAIN AND DOING MAYBE SMALLER PARTS OF MANY BRAINS ACROSS SPECIES. IS IT MORE VALUABLE TO SEE THE ENTIRE SENSORY WIRED INPUT TO OUTPUT OR A PART OF IT BUT ACROSS MAYBE THE STAIN FUNCTIONAL AREA IN FIVE SPECIES. >> I WANT TO USE MY TIME TO QUICKLY ANSWER TWO QUESTIONS POSED OF THE SPEAKERSO -- CAN YOU FOLLOW AXONS BETWEEN PHYSICAL QUESTIONS AND CAN YOU IN THESE TECHNIQUES COMBINE LIGHT MICROSCOPIC TECHNIQUES WITH EM LINKING AXONAL CONNECTOME AND THE ANSWER IS KIND OF THE SAME. ONE IS THAT AT LEAST BY HUMAN TRACERS TO SUCCESSIVE PHYSICAL SECTIONS WITH THE CUT ENDS WHICH WITHOUT TRYING VERY HARD ARE NICE BUT A LITTLE BIT DISTORTED. IT'S QUITE EASY TO TRACE CERTAINLY THE MAJORITY OF AXONS BETWEEN SECTIONS. IT'S SOMETHING THAT NEEDS WORK BUT I THINK IT'S SOMETHING THAT CAN BE OPTIMIZED ON AND DON'T THINK THAT'S THE RIGHT STEP. THE SAME IDEA IS CAN YOU TAKE A SECTION AND DO LIGHT MICROSCOPY UP TO A BORDER AND THEN DO EM. THIS WAS CLEARLY TOO EARLY TO DE FUND IT BUT THE IDEA WAS TO DO A PHYSICAL SECTION POST FIXATION BUT PRIOR TO CLEARING OR ANYTHING AND THEN PUNCH OUT A SECTION FOR HEAVIER FIXATION AND THEN EM. AND CORRELATING AXONS. I HOPE THAT IS POSSIBLE BUT THAT'S ONE WAY TO DO IT. >> THANKS TO EVERYBODY AND THANKS TO DISCUSSANTS AND OPEN IT UP A ROUND TABLE DISCUSSION WITH THE SPEAKERS AND DISCUSSANTS TODAY. I GUESS IT COULD BE FREE FLOWING. >> IT CAN BE BUT AS THE GRATEFUL DEAD SAID DON'T DOMINATE THE RACK, JACK. TRY TO ADD SOMETHING THAT HASN'T BEEN SAID BEFORE OR THINGS THAT DRAW OUT TOPICS WE HAVEN'T HIT BEFORE, IF YOU CAN. WE'LL SEE WHAT POPS UP HERE AND THEN HIT THE Q&A. >> FOR ALL THE SPEAKERS IF YOU WANT TO MAKE A POINT YOU CAN RAISE YOUR HAND AND WE'LL CALL YOU OUT. >> AND ACROSS MULTIPLE AREAS AND ANIMALS IT'S PART OF THE SCALING TO DO CONNECTOME. AND THEREFORE I THINK SOMETIMES THESE THINGS ARE DESCRIBED AS EITHER OR BUT NOT NECESSARILY EITHER/OR CONNECTING MULTIPLE VOLUMES WILL BE PART OF THE SCALING. >> YOU HEARD OF LOUD MUSIC BECAUSE I CLICKED ON THE VIDEO MARK SENT TO US. ANYWAY. BLAME MARK. >> THE MUSIC WAS FILTERED DOWN. >> MAYBE MY ADMONITION WAS TOO SEVERE. I'LL TURN TO THE Q&A THEN. >> ASK THE DISCUSSANTS TO DISCUSS SILOED VERSUS AND SILOED IF WE BOUGHT THE BEST EMs AND PUT THEM TO THE TASK VERSUS HAVING LABS WORK ON MANY ANIMALS IN PARALLEL. I THINK RELATEDLY BRYAN JONES RIGHT A CANONICAL CONNECTOME CAN'T POSSIBLY CONTAIN ALL THE DATA AND WE HAVE TO POPULATE IT WITH ADDITIONAL DATA AND COMPARE THEM TO GET THE MEASURE OF VARIABILITY. THAT'S HOW WE'LL GET AT TRUE MODELLING. THOSE ARE TWO RELATED COMMENTS IN THE Q&A RIGHT NOW PEOPLE CAN RESPOND TO IF THEY CHOSE OR YOU CAN BUBBLE UP SOMETHING ELSE. >> I'D LIKE TO RESPOND TO THE IDEA OF DOING ONE CONNECTOME. IT'S IMPORTANT TO UNDERSTAND THIS. I SAID IT AT THE LAST WORKSHOP BUT I'LL SAY IT AGAIN, IT'S VERY EXPENSIVE, REALLY EXPENSIVE TO DO ONE CONNECT OME BUT REALLY CHEAP TO DO A LOT OF THEM BECAUSE THE INFRASTRUCTURE ONLY HAS TO BE OBTAINED ONCE. IT'S LIKE GENOMICS OR ANYTHING ELSE. I DON'T THINK THERE'S ANY REASON WHY ONE CAN'T ASSUME ONCE YOU'VE DONE ONE, YOU'LL BE ABLE TO DO MANY MORE AND I ASSUME THAT'S AN IMPORTANT IDEA. ANOTHER IDEA IS SILOED. MAYBE TALKING ABOUT DEMOCRATIZATION. IT'S NOT ABSOLUTELY REQUIRED ALL THE MACHINES BE IN THE SAME PLACE ESPECIALLY IF THEY'RE WORKING IN PARALLEL ON THE SAME DATA SET EITHER BY DIVIDING AND CONQUERING OR SOME WAY THE BRAIN HAS BEEN DIVIDED AND IT ALLOWS PEOPLE ALL OVER THE COUNTRY IN BETWEEN RUNS OR WHEN ONE BRAIN IS OVER TO USE THE MACHINES FOR OTHER KINDS OF CONNECTOMICS. I DON'T SEE THIS AS SILOED. THE IDEA OF MANY DOING MANY CONNECTOMES AT THE SAME TIME SEEMS TO ME LIKE A RIVER THAT IS BROKEN INTO SMALLER AND SMALLER TRIBUTARIES AND EACH AT A GLACIALLY SLOW PACE. I WOULDN'T PERSONALLY THINK THAT IS THE BEST IDEA. I'D LIKE TO SECOND BOTH OF THOSE. FOR EM AND FOR THE WHOLE BRAIN PROJECT OMS. I THINK MY FAVORITE SLIDE WAS KWANGHUN'S SLIDE. HAVING LOOKED AT BOTH DATA A LOT LIGHT MICROSCOPY AND AXONAL TRACTS ARE FAR MORE SIMPLER THAN CONNECTOME WITH SYNAPSES AND COMPLICATED BRANCHING STRUCTURES. I THINK THAT ONCE ONE HAS DONE ONE MACAQUE BRAIN, HUMAN BRAIN FOR LIGHT MICROSCOPY OR SYNCO SYNCHOTRON AND ALONG THE DIMENSIONS OF PATHOLOGY IT'S SOMETHING THAT WILL HAPPEN. IT'S JUST SO IMPORTANT MEDICALLY WHY NOT START NOW. ANDDEMOCRATIZING THE NICE THING ABOUT THE LIGHT MICROSCOPY AND ANTIBODY APPROACH ANYONE CAN DO IT. THE BARRIER TO ENTRY IS FAIRLY LOW. I THINK A LOT OF PEOPLE CAN BE IN THIS FIELD EVEN AT A SMALLER SCALE IN PATHOLOGY DEPARTMENTS ETCETERA. >> I SHOULD HAVE MENTIONED WEBINAR ATTENDEES ARE ABLE TO SUBMIT QUESTIONS THERE'S A LINK I THINK IT SAYS LIVE FEEDBACK FOR SOMETHING IF YOU'RE WATCHING THE VIDEO STREAM. WE HAD SOMETHING FROM ANTON ARCHOPOV RELATED TO THE POINT ABOUT MUSCLE OUTPUT FOR A WHOLE BRAIN CONNECTOME IT WOULD BE HIGHLY USEFUL TO HAVE RETINA AND DO PANELISTS THINK IT WILL BE POSSIBLE TO INCLUDE SUCH INPUTS OR ARE THERE BARRIERS AND I'D ASK THIS QUESTION FROM THE PERSPECTIVE OF SAMPLE PREPARATION RATHER THAN IMAGING TYPE. >> I'LL WEIGH IN, OUR WORK ON ZEBRAFISH THE PROTOCOL FOR THE BRAIN DIDN'T WORK FOR THE RETINA BUT IT'S TRUE THE OPTIC NERVE IS MYELINATED IN AN ADULT SO ONE CAN IMAGINE TAKING THE HIGH OUT AND STAINING IT THE WAY YOU WANT AND CONNECTING THE MYELINATED PART AND THE HAIR CELLS -- I DON'T KNOW ENOUGH ABOUT THE AUDITORY SYSTEM BUT MAYBE THAT'S ALSO HARDER TO TAKE OUT BUT THERE'S MYELINATED IN THE AUDITORY SYSTEM EARLY ON AS WELL. >> IF THERE ARE NO ADDITIONAL COMMENTS AT THIS TIME I WOULD LIKE TO SUGGEST WE MAYBE DISCUSS A LITTLE BIT MORE ON THE ROAD MAP AND QUESTIONS THAT JEFF PROPOSED. I LIKE THE SLIDE A LOT. IT SUMMARIZES SOME IMPORTANT POINTS. I DON'T KNOW IF JEFF MINDS SHOWING YOUR SLIDE AGAIN MAYBE WE CAN HEAR WHAT OTHER SPEAKERS THINK ABOUT THE QUESTIONS YOU OPPOSED. MAYBE I CAN START. I DON'T KNOW AS CO-LEAD WHERE WE SUPPOSED TO EXPRESS OUR OPINION OR NOT? >> AS A CO-LEAD I SAY, YES. >> GREAT. AND IN RESPONSE TO WHAT JEFF SAID WHEN HE WAS TALKING WITH THE SLIDE LIKE I MIGHT DISAGREE WITH HIM REGARDING WHETHER WE SHOULD HAVE A CELL TYPE LABELLING IN THE EM VOLUME OR NOT. I AM GLAD TO TELL YOU, JEFF, I AGREE WITH YOU 100%. I LIKE THE IDEA OF TACKLING THE FIRST SAY WHOLE MOUSE BRAIN CONNECTOME OR WHOLE HUMAN BRAIN CONNECTOME BY ITSELF RATHER THAN SETTING UP GOALS FROM THE GET GO. HAVING ONE MINIMIZED GOAL WITHOUT TOO MANY DESCRIPTION ATTACHED TO IT -- STRINGS ATTACHED CAN BE A FOCUSSING EFFORT LIKE WHAT BOBBY SAID QUOTING ON THE WHOLE PROJECT GETTING SOMETHING DONE WILL OPEN THE DOORS TO MANY THINGS TO COME. MANY OTHER THINGS WE CAN DO HOWEVER, FOR THE FIRST PROJECT WE CAN'T MAKE IT TOO COMPLICATED. I FULLY SUPPORT THE WHOLE MOUSE BRAIN PROJECT IN THE BLACK 6 MOUSE WITHOUT FUNCTIONAL MANIPULATIONS OR ANYTHING ELSE. JUST TO GET IT DONE AND FOCUS ON TECHNOLOGY TO GET IT DONE AND DONE WELL AND FROM THAT WE'LL LEARN A LOT AND TO THINK ABOUT CELL TYPE LABELLINGS, ADDING FUNCTIONAL DETE AND THINGS LIKE THAT. IF WE CAN DO LABELLING THAT WOULD BE FANTASTIC, WITHOUT IT THERE'S OTHER APPROACHES WE CAN DO WITH MORPHOLOGIES AND LIKE PEOPLE HAVE DONE IN THE FLY BRAIN AND USE THE INFORMATION AS A ROSETTA STONE TO BRIDGE DATA AND KNOWLEDGE. >> WE HAVE ALL THE QUESTIONS HOW MANY CELLS DO WE NEED AND SHOULD WE LOOK AT THIS OR THAT AND IT'S BECAUSE WE DON'T KNOW WHAT WE'RE GOING TO DO WITH IT? I KNOW A LOT OF PEOPLE HAVE A LOT OF IDEAS ON WHAT WE'RE GOING TO DO BUT I DON'T KNOW THERE'S A CLEAR VISION ON WHAT WE'RE GOING TO DO WITH IT AND I THINK THE SAME IS TRUE FOR THE HUMAN GENOME. WE HAVE TO DO IT ONCE BUT THEN I DO THINK IT'S IMPORTANT AND I REALLY APPRECIATE THE FORMAT OF THE WORK SHOPS AND THE WAY YOU BREAK IT DOWN CHALLENGING PEOPLE TO STOP THINKING ABOUT WHAT WE'RE GOING TO DO AND WHAT ARE THE HIGH IMPACT THINGS WE THINK WILL COME OUT OF DOING THIS AND MAKING SURE WHAT WE DO HAS HE BIGGEST IMPACT. >> THAT'S GOOD TO GET THE MOUSE IN THE STRAIGHTFORWARD WAY THOUGH IT'S COMPLEX. THEY'RE BRED SPECIFICALLY. WE SEE A LOT OF VARIATION IN PRIMATES AND HUMANS AND THEY DEVELOP THE GENETIC MOUSE WOULD HAVE TO LOOK AT THE METHODS SUGGESTED BECAUSE IF WE ARE TO UNDERSTAND AREAS VULNERABLE TO DISEASE WE HAVE TO BE MORE TARGETED AT THE SAME TIME. CAN WE DO THEM AT THE SAME TIME? WE HAVE CHOSEN BECAUSE THERE WERE SPECIFIC QUESTIONS. I DON'T THINK IT HAS TO BE TO THE ROLE. THEREFORE SEVERAL PEOPLE WORKING ON THESE WHOLE CONNECTOME OF THE MOUSE BRAIN AND OTHERS. I THINK WE CAN SUPPLEMENT THAT I LIKE ASKING TO THE LONG FACE OR PRESERVATIVE APPROACH AND CAN YOU KNIFE SECTION A BLOCK FACE? I'D BE CURIOUS IF SPEAKERS FROM WORKSHOP 1 WANT TO COMMENT ON THOSE? >> IF I UNDERSTAND CORRECTLY THE LOW RES FIRST PASS FOR MYELINATED AXONS WOULD IF WE WERE THINKING ABOUT DOING SEMI THIN SECTIONING TO 1 NANOMETER THICK THE FIRST IMAGE WOULD GIVE ALL THE GIVE THE MYELINATED AXON MAP AND WOULD REQUIRE NO DESTRUCTION OF ANY REGION BECAUSE YOU WOULDN'T HAVE DONE ION MILLING OF THE REST. >> BUT YOU WOULD DO THAT AT THE 10 NANOMETER RESOLUTION XY? >> YOU CERTAINLY WOULDN'T HAVE TO AT THAT RESOLUTION BUT MAY BE LIMITED IF THIS IS A MULTI SET. THE STAGE MOVEMENTS THEMSELVES MAY TAKE MORE TIME THEN DOING THE IMAGING THE REGULAR WAY. >> I GUESS THAT'S BASICALLY MY POINT. YOU'RE NOT RESTRICTED TO THE MULTI BEAM. >> YOU CAN USE SINGLE B MACHINES THAT ARE FAST. USE LARGE PIXEL SIZE. >> PICK A MIDDLE VALUE AND WHOLE BRAIN INTO 500 NANOMETER SECTIONS, THOSE WAFERS WOULD BE IMAGED BY WHATEVER MEANS YOU WANT. THAT WOULD GET THE PROJECT OME. >> WE HAVE JUST ONE OR TWO MORE MINUTES IF WE HAVE CLOSING THOUGHTS OR UNHEARD TOPICS, THIS IS YOUR CHANCE. >> FROM CHRIS OME THE MOON SHOT PROJECT WAS DRIVEN BY CATASTROPHIC RISK AND HOW WE AS A COMMUNITY BETTER CONNECT THE CONNECTOME TO CATASTROPHIC RISK AND ADULTS ARE AFFECTED EVERY YEAR AND HOW CONNECTED ARE WE TO DISEASE RISK? FINALLY A COMMENT, A HUMAN GENOME REFERENCE WAS A COMPOSITE OF 10 PARTS -- SORRY, 10 PEOPLE REGARDING THE INPUT. WE TALKED ABOUT MOON SHOTS AND THE RACE TO GET TO THE MOON YIELDED TECHNOLOGIES WE DIDN'T HAVE BEFORE THAT HAVE NOTHING TO DO WITH GOING TO THE MOON AND IMPROVEMENTS IN ALL SORTS OF OTHER THINGS. IT'S WORTH RECOGNIZING THAT IS WHAT THIS EFFORT SHOULD BRING. >> THAT'S A GREAT POINT. >> I'LL ECHO THAT AND TO MAKE A GESTURE OF GRATEFULNESS. ANY VERSION OF THIS IS A GREAT CHALLENGE IN EXO SCALE DATA AND COMPUTING. A GREAT DEAL OF NON-NEUROSCIENCE COMPUTER SCIENCE WILL BENEFIT FROM THIS. >> WELL, I'M GOING TO TAKE THE CO-ORGANIZERS' PREROGATIVE. I WANT TO EMPHASIZE SOMETHING KEN SAID EXPLORING TECHNICAL PADS IN PARALLEL AND REDUNDANCY IS SUPER IMPORTANT TO CONSIDER IN A PROJECT OF THIS SCALE AND THE FEWER CRITICAL PATHS YOU HAVE TO GET JUST RIGHT THE BETTER AND THROWS WHO HAVE DONE THIS KNOW IT'S DAMN HARD THOUGH WE'RE TALKING GLIBLY TODAY. REDUNDANCY AND DIFFERENT APPROACHES AND BEEN INVOLVED IN PROJECTS WHERE TWO APPROACH ARE FUNDED AND THEY BOTH WORKED AND THAT'S A WONDERFUL OUTCOME TOO. THOSE ARE MY CLOSING THOUGHTS. HONGKUI. >> WE MAY HAVE TO HAVE SEVERAL APPROACHES IN PARALLEL. I LIKE WHAT WAS SET AT THE BEGINNING -- SAID AT THE BEGINNING WHEN WE TALK ABOUT PREPARATION WE NEED TO THINK ABOUT THE CONTEXT OF THE PIPELINE FROM SAMPLE TO IMAGES TO THE END AND MAY HAVE TO TAKE PARALLEL APPROACHES TO SEE WHICH CAN BRING US TO THE FINISH LINE. I ALSO WANTED TO SAY WE DO NEED TO ADVOCATE FOR THE IMPORTANCE MUCH THIS PROJECT LIKE THE HUMAN GENOME THOUGH IT'S INCOMPLETE AND AN IMPORTANT REFERENCE AND THE FIRST STEP. HOW DO WE RELAY THAT TO HUMAN DISEASES ULTIMATELY. HOW WILL WE IMPROVE HUMAN HEALTH AS OUR ULTIMATE GOAL. WE NEED TO ARTICULATE THAT WELL FOR THIS KIND OF LARGE SCALE HEROIC EFFORT. WITH THAT WE ARE CLOSING THIS WORKSHOP. THANKS EVERYBODY, ALL THE SPEAKERS AN DISCUSSANTS FOR YOUR WONDERFUL CONTRIBUTION AND STIMULATING DISCUSSIONS. >> THANK YOU.