Assessment of different sampling methods for measuring and representing macular cone density using flood- illuminated adaptive optics

PURPOSE. To describe a standardized flood-illuminated adaptive optics (AO) imaging protocol suitable for the clinical setting and to assess sampling methods for measuring cone density. METHODS. Cone density was calculated following three measurement protocols: 50 × 50-µm sampling window values every 0.5º along the horizontal and vertical meridians (fixed-interval method), the mean density of expanding 0.5º-wide arcuate areas in the nasal, temporal, superior, and inferior quadrants (arcuate mean method), and the peak cone density of a 50 × 50-µm sampling window within expanding arcuate areas near the meridian (peak density method). Repeated imaging was performed in nine subjects to determine intersession repeatability of cone density. RESULTS. Cone density montages could be created for 67 of the 74 subjects. Image quality was determined to be adequate for automated cone counting for 35 (52%) of the 67 subjects. We found that cone density varied with different sampling methods and regions tested. In the nasal and temporal quadrants, peak density most closely resembled histological data, whereas the arcuate mean and fixed-interval methods tended to underestimate the density compared with histological data. However, in the inferior and superior quadrants, arcuate mean and fixed-interval methods most closely matched histological data, whereas the peak density method overestimated cone density compared with histological data. Intersession repeat- ability testing showed that repeatability was greatest when sampling by arcuate mean and lowest when sampling by fixed interval. CONCLUSIONS. We show that different methods of sampling can significantly affect cone density measurements. Therefore, care must be taken when interpreting cone density results, even in a normal population.