# En-Face Optical Coherence Tomography Angiography for Longitudinal Monitoring of Retinal Injury
## 2 Materials and Methods
The lesions were then verified with FA to show they were not leaking and OCT for assessment of layer involvement. En-face OCTA Procedure: Implementation of OCTA methods were optimized to either extract functional data, such as blood flow, or widefield high-resolution structural data in-vivo [5]. In this study, for wide field analysis, we were prioritizing analyzing structural data over flow data, as the accuracy of flow rate is currently undetermined. To develop OCTA algorithms for resolving the capillary beds, the nature of the imaging system must be considered. An individual OCT depth profile is the A-scan, produced by the interference pattern of the sampling beam to a reference beam. The image is adjustable to compensate for the curvature of the eye by changing the reference arm length in proportion to the axial distance. The B-scan is a cross-section of the retinal volume produced by shifting consecutive A-scan in a line. The B-Scan pattern can be linear or radial, but for our purposes, we use rectangular volumes of consecutive B-scans. To extract OCTA data from the intensity image, a high-density scan is required. For smooth transitions, the C-scan needs to be isotropic in the lateral plane, i.e. the same number of A-scans per both axes in the C-scan. En-face OCTA can be achieved by using a scan density of \(1000\times 1000\) A-scans, with a lateral resolution of 1.4 \(\upmu\)m, as a single volume. The axial resolution of the system is 1.9 \(\upmu\)m. Averaging or multiple consecutive scans decreases the area to a smaller region of interest, while samples of the lesions were acquired in this manner, a widefield scan was used for analysis. In the en-face scan, the blood flow in the vasculature increases both scatter (phase) and intensity and has been proposed for use in multi-functional OCT imaging to extract angiography and high-resolution structural information from a single scan volume [44]. With the single high-density volume, post-processing produced multiple outputs including high-resolution B-scans and en-face OCTA (Figure 10). Our custom automated image processing routine was developed in ImageJ to handle the raw data Bioptigen OCT reader and generate the registered 3D data cube that compensated for motion artifacts and misalignment of the B-scans. From the data cube, high-resolution B-scans were resliced through the center of lesions for analysis of lesion morphology. The measurements recorded in \(\upmu\)m were self-normalized for each lesion and reported as percent change. The percent change in the dynamics took the first measurements at one hour and normalized the subsequent measurements as expansion (positive values) or reduction (negative values). Additionally, OCTA was generated by further processing with a custom algorithm on the entire data cube or a VOI (Volume of interest, a 3D region of interest). Our algorithm uses structural enhancement and segmentation of the en-face intensity images to produce the 3D angiography. This method is a distinct approach to the implementation of the theory behind the [PERSON], et al method of reflectance-based projection-resolved (rbPR) OCTA algorithm to reduce shadow artifacts [45]. The rbPR algorithm is a probabilistic approach to histogram equalization and segmentation, whereas our approach is a sequential set of localized structural enhancement iterators. Both approaches enhance the capillary network and are robust to rejecting shadow artifacts in the projections of the retinal layers. Lesion Dynamic Descriptors: The longitudinal imaging with the OCT system produced data cube (Figure 10) for each timepoint that was processed for en-face images and B-Scans. The qualitative description of the lesion dynamics defined the critical measurements used to differentiate the influence of the light dose on the degree of retinal injury and identify the extent of retinal layers that were disrupted. The lesions were numbered, and B-Scans that crossed through the lesion midpoint were identified for measurements, exemplars in (Figure 1).