Project Grant S10OD036344

MINFLUX 3D MICROSCOPE - PROJECT SUMMARY THIS APPLICATION SEEKS FUNDS TO PURCHASE A MINFLUX 3D MICROSCOPE. THIS INSTRUMENT WOULD SUPPORT NINE NIH-FUNDED USERS IN FIVE DEPARTMENTS AND FOUR COLLEGES WITHIN THE TEXAS A & M UNIVERSITY (TAMU) COMMUNITY AND A GROUP OF FOUR NIH-FUNDED INVESTIGATORS AT UT SOUTHWESTERN MEDICAL CENTER. THE MINFLUX MICROSCOPE WOULD BE THE SECOND MAJOR INSTRUMENT IN A RECENTLY ESTABLISHED SHARED USER FACILITY, THE JOINT MICROSCOPY LABORATORY (JML), WHICH IS FOCUSED ON SINGLE MOLECULE FLUORESCENCE APPLICATIONS. THE JML INCLUDES SIGNIFICANT WET-LAB AND TISSUE CULTURE SPACE TO ENCOURAGE USE BY MORE DISTANT LABORATORIES BOTH ON CAMPUS AND EXTERNAL TO THE UNIVERSITY. MINFLUX IS A RELATIVELY NEW STATE-OF-THE-ART POINTILLISTIC IMAGING AND PARTICLE TRACKING STRATEGY THAT IS EXTREMELY THRIFTY WITH THE USE OF PHOTONS, REQUIRING ~10-FOLD LESS PHOTONS THAN THE COMMON PALM/STORM- TYPE POINTILLISTIC SUPER-RESOLUTION APPROACHES. CONSEQUENTLY, MINFLUX CAN ACHIEVE PRECISION LEVELS OF A FEW NANOMETERS ON A SUB-MILLISECOND TIMESCALE WITHIN FUNCTIONALLY ACTIVE CELLULAR SYSTEMS AND LONG SINGLE MOLECULE TRAJECTORIES IN THREE-DIMENSIONS (3D) CAN BE OBTAINED USING SINGLE FLUOROPHORE TAGS. THE REQUESTED MINFLUX 3D SYSTEM WILL ENABLE NUMEROUS MULTI-COLOR STRATEGIES COMBINING BOTH STATIC IMAGING AND MOLECULAR TRACKING APPROACHES. USERS WILL EXAMINE WELL-CONTROLLED IN VITRO SYSTEMS AS WELL AS STABILIZED AND COMPLEX CELLULAR SYSTEMS (FIXED AND PERMEABILIZED CELLS), MANY WITH AN EYE TOWARDS LIVE CELL INVESTIGATIONS. THE MAJOR USERS WILL EXAMINE FUNDAMENTAL AND DIVERSE CELL BIOLOGICAL AND MECHANISTIC BIOCHEMISTRY QUESTIONS FOCUSED ON NUCLEOCYTOPLASMIC TRANSPORT, CONDENSATES, BACTERIAL PILI, NUCLEAR MECHANICAL STRESS AND SYNAPSES. THE MINOR USERS PROJECTS INCLUDE STRUCTURE, FUNCTION AND BIOPHYSICAL STUDIES OF MITOCHONDRIAL RNA EDITING AND KINASE SIGNALING, CHEMOTAXIS, ENDOSOMAL ESCAPE, ENDOCYTIC RECYCLING, ANTIBIOTIC BIOSYNTHESIS, PHAGE INFECTION, AND ADDITIONAL PROJECTS ON CONDENSATES AND SYNAPSES. THE FULL-TIME TECHNICIAN NEEDED TO RUN THE MINFLUX MICROSCOPE WILL BE SUPPORTED BY A COMBINATION OF USER FEES AND ONGOING CONTRIBUTIONS FROM DEPARTMENTS, COLLEGES, TEXAS A & M HEALTH, AND THE VICE PRESIDENT FOR RESEARCH, EMPHASIZING THE WIDESPREAD IMPORTANCE OF THE NEW MICROSCOPE CAPABILITIES TO ADVANCE THE CAPABILITIES AND GROWTH OF CURRENT RESEARCH PROGRAMS. THE INSTRUMENT WILL BE HOUSED IN THE COLLEGE OF MEDICINE BY THE DEPARTMENT OF CELL BIOLOGY AND GENETICS, WHICH HAS DONATED SUBSTANTIAL EQUIPMENT AND SPACE FOR THE NASCENT JML MICROSCOPE FACILITY. ALTOGETHER, THE IDENTIFIED USERS HAVE PLANNED NEW RESEARCH DIRECTIONS THAT ARE EXPECTED TO ULTIMATELY REQUIRE > 90% OF THE TOTAL ACCESSIBLE USER TIME, INDICATING THE SUBSTANTIAL DEMAND FOR BOTH EXISTING AND NEWLY DEVELOPING PROJECTS. IN TOTAL, THE REQUESTED MINFLUX 3D MICROSCOPE WILL PROVIDE SUBSTANTIAL AND FUNDAMENTAL INFRASTRUCTURAL SUPPORT FOR A WIDE RANGE OF PROJECTS IMPORTANT FOR UNDERSTANDING AND IMPROVING HUMAN HEALTH.