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To quantify the density, distribution, and depth of invasion of cysts in the corneas of eyes with acanthamoeba keratitis (AK) by in vivo confocal microscopy (IVCM) with a novel scanning pattern.The medical records of patients with AK evaluated at the Doheny Eye Center UCLA between September 2014 and July 2016 were reviewed retrospectively. Patients with clinically diagnosed AK underwent IVCM at various time points during their clinical course. Five corneal locations were scanned at each time point: the central area and 4 standard points on the peripheral cornea corresponding to temporal, nasal, inferior, and superior locations. The IVCM scans were manually graded to quantify the maximum depth of invasion and density of cysts.Twenty-one eyes of 18 patients with visible cysts on IVCM were included. Mean cyst density at presentation was 214.1 ± 120.2/mm2 (range: 64–484 cells/mm2), and the average cyst depth was 164.3 ± 81.2 μm (range: 17–290 μm). In 17 eyes, the average cyst depth was 139.4 ± 68.6 μm (range: 17–245 μm), mean cyst density was 177.9 ± 99.6/mm2, and an average of 1.4 ± 1.3 quadrants was infiltrated at presentation, and reached clinical resolution with medical treatment without surgical intervention. Four eyes that ultimately underwent therapeutic penetrating keratoplasty had cysts in all 4 quadrants and deeper cyst infiltration; the average cyst depth in these corneas was 270.5 ± 17.5 μm (range: 252–290).Eyes with AK requiring therapeutic keratoplasty were more likely to have a deeper and more diffuse penetration of cysts in the cornea compared with those resolving with medical treatment.