Project Grant R01EY032994

IN VIVO IMAGING OF THE HUMAN RETINA AT THE MOLECULAR LEVEL - PROJECT SUMMARY THE FLUORESCENCE LIFETIME MEASURES THE DURATION A FLUOROPHORE REMAINS EXCITED BEFORE RETURNING TO THE GROUND STATE BY EMITTING A PHOTON. IT IS AN INTRINSIC PROPERTY UNIQUE TO EACH FLUORESCENT MOLECULE AND ITS ENVIRONMENTAL STATE. FLUORESCENCE LIFETIME IMAGING OPHTHALMOSCOPY (FLIO) CAN PERFORM IN VIVO LIFETIME MEASUREMENTS OF ENDOGENOUS SIGNALS IN THE RETINA AND RETINAL PIGMENT EPITHELIUM (RPE), RANGING FROM VITAMIN A DERIVATIVES IN LIPOFUSCIN (LF) TO FLAVIN-ADENINE-DINUCLEOTIDE (FAD) IN MITOCHONDRIA. AS MANY FLUORESCENT MOLECULES HAVE BEEN IMPLICATED IN AGING AND AGE-RELATED MACULAR DEGENERATION (AMD), FLIO PROMISES PRECISE MEASUREMENTS OF THE METABOLIC STATUS AND OXIDATIVE STRESS LEVELS IN RETINAL LAYERS/CELLS. WITH THREE SPECIFIC AIMS, WE PROPOSE TO DEVELOP CELLULAR-LEVEL FLIO. FIRST, WE WILL MARRY FLIO WITH STATE-OF-ART ADAPTIVE OPTICS (AO) TO OVERCOME THE EYE'S OPTICAL IMPERFECTIONS AND CREATE ADAPTIVE OPTICS (AO) ENHANCED FLIO (AOFLIO). WE WILL DEVELOP AOFLIO ON AN AO SCANNING LASER OPHTHALMOSCOPE (AOSLO), A RETINAL IMAGING INSTRUMENT WITH PROVEN CELLULAR RESOLUTION. THE AOFLIO INSTRUMENT WILL ACHIEVE DIFFRACTION-LIMITED IMAGE RESOLUTION (~2 MM LATERAL, ~ 30 MM AXIAL) OVER A 7o X 7o FOV IN A SINGLE FRAME; THIS IS ~21 TIMES LARGER THAN THAT OF THE CURRENT AOSLO. FURTHER, WE WILL FORTIFY AOFLIO WITH NOVEL IMAGE-PROCESSING STRATEGIES TO QUANTITATIVELY RESOLVE THE COMPLEX MIX OF COLORS AND LIFETIMES OF RETINAL AND RPE FLUOROPHORES. THE IMPROVED PROCESSING CAPABILITIES OF PHASOR ANALYSES OF FLUORESCENCE LIFETIMES WILL PERMIT SUPERIMPOSED SIGNALS FROM MULTIPLE MOLECULAR SPECIES TO BE UNMIXED AND PERMIT IMAGING WITH A MEAGER LIGHT BUDGET. WE WILL LEVERAGE RECENT ADVANCES IN THE HISTOLOGY OF THE HUMAN RETINA AND RPE FOR IN VIVO ANALYSES USING FLIO TO MEASURE FLUOROPHORES WITHIN INDIVIDUAL CELLS, SPECIFIC LAYERS, AND SUBCELLULAR STRUCTURES. THUS, AOFLIO PROMISES THE COMBINED SPEED AND RESOLUTION WITH AN ADEQUATE FIELD OF VIEW (FOV) TO OFFER A NEEDED WINDOW INTO RETINAL FUNCTION IN NORMAL PHYSIOLOGY AND DISEASE. SECOND, WE WILL CHARACTERIZE AND DISTINGUISH FLUORESCENCE SIGNALS WITH HIGH SPATIAL PRECISION IN NORMAL HUMAN SUBJECTS AND ESTABLISH A NORMATIVE DATABASE OF FLUORESCENCE LIFETIMES IN DIFFERENT RETINAL LAYERS AND INDIVIDUAL (PHOTORECEPTOR AND RPE) CELLS IN HUMAN SUBJECTS OF AGE, GENDER, AND RACE/ETHNICITY, THEREBY FACILITATING CORRELATION WITH LEADING CANDIDATES FOR SIGNAL SOURCES. THIRD, WE WILL USE AOFLIO TO CHARACTERIZE THE METABOLIC FUNCTION OF THE RETINAL PIGMENT EPITHELIUM (RPE) IN AMD, A DISEASE THAT CAUSES VISION LOSS IN OVER 10 MILLION OLDER AMERICANS. OUR STUDY WILL FOCUS ON INTRARETINAL HYPERREFLECTIVE FOCI (IHRF), THE STRONGEST PREDICTOR OF THE DEVELOPMENT OF LATE AMD, AND INVESTIGATE FUNDAMENTAL QUESTIONS OF IHRF PATHOGENESIS AND ITS ROLE IN THE PATHOPHYSIOLOGY OF AMD. THESE AIMS WILL HELP US IMPROVE RETINAL AND RPE METABOLIC FUNCTION ASSESSMENT IN NORMAL AGING AND AMD. THE ACHIEVEMENT WILL PROVIDE A FOUNDATION FOR FUTURE INVESTIGATION OF THE PATHOPHYSIOLOGY OF A BROAD ARRAY OF RETINAL AND CENTRAL NERVOUS SYSTEM DISEASES FOR WHICH AGING IS A RISK FACTOR.