# En-Face Optical Coherence Tomography Angiography for Longitudinal Monitoring of Retinal Injury
## 3 Results  
Key findings: Orthogonal OCT allowed precise landmarking and quantification of the lesions (Figure 1) to build a mathematical model of the changes over time. The fundus screening could be  
Figure 1: Orthographic views and lesion measurement. The en-face view (**a**) is aligned with two B-scans (**b**,**c**) to determine the center of the lesion. The red registration lines show corresponding coordinates. (**d**) is the three-dimensional view of the projections illustrating that the linked views are digital slices of the volume from the same scan. The inset is the measurement scheme for the key layers of the lesion. replicated with en-face OCT/OCTA combination that was suited for non-invasive longitudinal studies (Figure 2). The CNV model showed not only neovascularization but loss of perfusion in the Deep Vascular Plexus that can be confirmed with immunofluorescence staining. (Figure 3). In-Vivo Lesion Verification: Each lesion was classified, and measurements were compiled using the following order of assessment: fundus validation, OCT qualitative description, OCT measurements, and OCTA visualization. Following laser irradiation, all animals presented with hyperreflective lesions in the color fundus images. Acute injury dose showed no signs of bleeding or vascular leakage. The initial OCT images confirmed the presence of the lesion for both doses by the presence of a disruption in the ONL CNV dose presented with a clear vaporization bubble in all lesions, and only one lesion had severe bleeding, examples in Figure A1 (Appendix A). The IR imaging tracked the lesions and helped guide the FA. Autofluorescence baseline imaging before FA shows an inconsistent severity of the hyperfluorescent response, some CNV lesions fluorescing brightly while others were not detectable even with maximum gain for detector sensitivity. The maximum fluorescent intensity in either model remains below the detectors threshold with the sensitivity gain used for FA. Because of the low autofluorescence intensity, it is therefore not a major confounding factor when assessing the leakage of the CNV. While the CNV had clear evidence of retinal disruptions, the boundaries of the acute injury lesions were hard to discern from the rest of the retina. Photoreceptor damage observed in the Outer Nuclear Layer (ONL) of the B-scan OCT imaging was often undetectable in the fundus images thus making us unable to determine the extent of the damage of the acute injury lesions. The orthogonal views were used to landmark the optimal B-scans for the lesions; however, any single en-face slice was insufficient to determine the morphology of the lesion. Figure 2: Samples of the multiple imaging views of a single eye. (**a,b**) is the OCTA (Optical Coherence Tomography Angiography) and en-face OCT (Optical Coherence Tomography) of the inner limiting membrane and superficial vascular plexus. (**c,d**) is the OCTA and en-face view of the highlighting Retinal Pigment Epithelium and choroidal disruption. (**a**) through (**d**) are 60 \(\upmu\)m projections of the color OCTA depth map, or maximum intensity projection of the en-face OCT. (**e,f**) are the fluorescein angiography and IR imaging of the retina showing the CNV (Choroidal Neovascularization) leakage. The landmarks in the en-face image (**d**) and fundus f allowed comparisons of the vasculature in the OCTA and FA images. Leakage in the CNV was correlated to the OCTA image in all cases. From day 3, the FA scans verified the vascular leakage in the CNV lesions. The acute injury lesions showed no apparent leakage at any timepoint. In contrast, all but three of the CNV lesions clearly showed signs of leakage at all time points and could be imaged with OCT (Figure 2). The three CNV lesions that were indeterminate were noted as potentially not CNV in the continued evaluation. While three lesions did not show the characteristic pooling in FA, the normal vasculature could not be resolved either (Figure A2). All three lesions were verified as CNV with lectin in the flat mounts.