The Investigation of Autofluorescence in Myopes and Emmetropes A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Biology, Medicine and Health 2018 Teresa S L Tee School of Biological Sciences / Division of Neuroscience & Experimental Psychology Contents List of Figures ...................................................................................................... 6 List of Tables ...................................................................................................... 14 List of Abbreviations ......................................................................................... 16 Abstract .............................................................................................................. 18 1. Fundus Autofluorescence (FAF) and Myopia 1.1. Retinal Pigment Epithelium (RPE) Lipofuscin ............................................. 21 1.1.1. The Formation of RPE Lipofuscin .................................................. 23 1.1.2. Autofluorescent Properties of RPE Lipofuscin................................. 27 1.1.3. Biochemical Properties of RPE Lipofuscin ...................................... 28 1.1.4. RPE Lipofuscin and Aging ............................................................ 29 1.1.5. RPE Lipofuscin in Disease ............................................................ 29 1.2. FAF Imaging ........................................................................................... 30 1.2.1. Confocal Scanning Laser Ophthalmoscope (cSLO) ........................ 32 1.2.2. Quantitative Assessment of FAF Images ....................................... 34 1.2.3. Sources of Error and Asymmetry in FAF imaging ........................... 37 1.2.4. Patient-based Factors Affecting the Autofluorescence Image.......... 38 1.2.5. Autofluorescence of the Normal Fundus ........................................ 39 1.2.6. Interpretation of the FAF Image ................................................... 42 1.2.7. Applications of FAF Imaging ......................................................... 42 1.2.7.1. Disease Detection and Diagnosis ........................................ 42 1.2.7.2. Patient Management ......................................................... 45 1.2.7.3. Visual Function.................................................................. 48 1.2.8. Advantages of FAF Imaging ......................................................... 49 1.2.9. Limitations of FAF Imaging .......................................................... 50 1.3. Myopia ................................................................................................... 50 1.3.1. Physical Dimensions of the Eye in Myopia ..................................... 51 1.3.2. Pathological Myopia and FAF ....................................................... 52 1.3.3. Autofluorescence Around the Optic Disc in High Myopia ................. 54 1.3.4. Eye Diseases Associated with Oxidative Stress and High Myopia ... 55 1.4. Contrast Sensitivity .................................................................................. 56 1.4.1. Contrast Sensitivity with Age ........................................................ 58 1.4.2. Contrast Sensitivity in Myopia ...................................................... 60 1.5. Objective of the Thesis ............................................................................. 62 1.6. References .............................................................................................. 63 2. General Methodology 2.1. Subject Recruitment ................................................................................. 73 2.2. Clinical Procedures ................................................................................... 75 2.2.1. Refraction and Keratometry ......................................................... 75 2.2.2. Axial Length Measurement ........................................................... 75 2.2.3. Autofluorescence Imaging ........................................................... 75 2.2.4. Optical Coherence Tomography (OCT) ........................................ 76 2.2.5. Contrast Sensitivity ..................................................................... 76 2.3. Determination of Autofluorescence Image Quality ...................................... 77 2.3.1. FAF Image Quality Analysis .......................................................... 78 2.3.2. Parapapillary Autofluorescence (PAF) Image Quality Analysis ......... 83 2.4. Statistical Analysis .................................................................................... 86 2.5. References .............................................................................................. 88 2 3. Effect Of Image Capture Settings on the Autofluorescence Signal 3.1. Abstract .................................................................................................. 90 3.2. Introduction............................................................................................. 93 3.3. Part A - Effect of Image Averaging (Automatic Real-time Averaging) on FAF Signal ..................................................................................................... 95 3.3.1. Methods ..................................................................................... 95 3.3.1.1. Ethics and Subjects ............................................................ 95 3.3.1.2. Autofluorescence Imaging .................................................. 95 3.3.1.3. Image Sampling ................................................................ 96 3.3.1.4. Statistical Analysis ............................................................. 98 3.3.2. Results ....................................................................................... 98 3.3.2.1. Average Intensity of Sampled FAF from 55° and 30° Images ...................................................................................... 98 3.3.2.2. Standard Deviation (SD) of Sampled FAF from 55° and 30° Images ......................................................................... 100 3.4. Part B - Optimising Detector Sensitivity Level for 55° and 30° Images ....... 103 3.4.1. Methods ................................................................................... 103 3.4.1.1. Ethics and Subjects ......................................................... 103 3.4.1.2. Autofluorescence Imaging ............................................... 103 3.4.2. Results ..................................................................................... 104 3.4.2.1. Autofluorescence Intensity ............................................... 104 3.4.2.2. Caucasian vs Asian Eyes .................................................. 105 3.4.2.3. Effect of Detector Sensitivity on FAF Profile....................... 109 3.5. Part C – Effect of Photobleaching on FAF ................................................. 112 3.5.1. Methods ................................................................................... 112 3.5.1.1. Ethics and Subjects ......................................................... 112 3.5.1.2. Autofluorescence Imaging ............................................... 112 3.5.1.3. Image Analysis................................................................ 112 3.5.1.4. Statistical Analysis ........................................................... 113 3.5.2. Results ..................................................................................... 114 3.5.2.1. FAF Profile ....................................................................... 114 3.5.2.2. Autofluorescence Intensity over Time ............................... 116 3.5.2.3. FAF Peak Location ........................................................... 117 3.6. Discussion and Conclusion ...................................................................... 118 3.6.1. Part A – Effect of Image Averaging (Automatic Real-time Averaging) on FAF Signal ............................................................................ 118 3.6.2. Part B – Effect of Detector Sensitivity on FAF .............................. 118 3.6.3. Part C – Effect of Photobleaching on FAF .................................... 119 3.7. References ............................................................................................ 120 4. The Spatial Profile Of Fundus Autofluorescence (FAF) In Young Healthy Eyes 4.1. Abstract ................................................................................................ 123 4.2. Introduction........................................................................................... 124 4.3. Methods ................................................................................................ 126 4.3.1. Ethics and Subjects ................................................................... 126 4.3.2. Procedure ................................................................................. 127 4.3.3. Image Analysis.......................................................................... 127 4.3.4. Statistical Analysis ..................................................................... 128 4.4. Results .................................................................................................. 130 4.4.1. Intra-session Coefficient of Repeatability (COR) of FAF Peak Location and Intensity ............................................................................ 130 4.4.2. Autofluorescence Intensity Profiles ............................................. 132