Quantifying melanin concentration in retinal pigment epithelium using broadband photoacoustic microscopy

Melanin is the dominant light absorber in retinal pigment epithelium (RPE). The loss of RPE melanin is a sign of ocular senescence and is both a risk factor and a symptom of age-related macular degeneration (AMD). Quantifying the RPE melanin concentration provides insight into the pathological role of RPE in ocular aging and the onset and progression of AMD. The main challenge in accurate quantification of RPE melanin concentration is to distinguish this ten-micrometer-thick cell monolayer from the underlying choroid, which also contains melanin but carries different pathognomonic information. In this work, we investigated a three-dimensional photoacoustic microscopic (PAM) method with high axial resolution, empowered by broad acoustic detection bandwidth, to distinguish RPE from choroid and quantify melanin concentrations in the RPE ex vivo. We first conducted numerical simulation on photoacoustic generation in the RPE, which suggested that a PAM system with at least 100-MHz detection bandwidth provided sufficient axial resolution to distinguish the melanin in RPE from that in choroid. Based on simulation results, we integrated a transparent broadband micro-ring resonator (MRR) based detector in a homebuilt PAM system. We imaged ex vivo RPE-choroid complexes (RCCs) from both porcine and human eyes and quantified the absolute melanin concentrations in the RPE and choroid, respectively. In our study, the measured melanin concentrations were 14.7 mg/mL and 17.0 mg/mL in human and porcine RPE, and 12 mg/mL and 61 mg/mL in human and porcine choroid, respectively. This study suggests that broadband PAM is capable of quantifying the RPE melanin concentration from RCCs ex vivo.