Quantifying melanin concentration in retinal pigment epithelium using broadband photoacoustic microscopy

Abstract

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.

Keywords: (110.5120) Photoacoustic imaging; (170.2655) Functional monitoring and imaging; (170.3880) Medical and biological imaging; (180.6900) Three-dimensional microscopy.

Fig. 1

(a) Structure of RCC and light illumination. (b) Schematic of optical excitation and PA generation by RCC. (c) Simulated energy deposition profile along the depth direction. E. D.: energy deposition. (d) Simulated PA A-line with 110-MHz acoustic detection bandwidth. Norm. Amp.: normalized amplitude. (e) Simulated PA A-line with 70-MHz acoustic detection bandwidth. In PA simulations, RPE thickness was 10 µm; RPE melanin concentration was 20 mg/mL; choroidal melanin concentration was 60 mg/mL.

Fig. 2

(a) Variation of normalized peak amplitudes of RPE (and choroid in cases where axial resolution cannot resolve the boundary between the two layers) PA A-lines with preset RPE melanin concentrations for different acoustic detection bandwidths. M. C.: melanin concentration; choroidal melanin concentration: 15 mg/mL. (b) Variation of relative mean square error with acoustic detection bandwidth if using RPE (and choroid in cases where axial resolution cannot resolve the boundary between the two layers) PA amplitude to estimate RPE melanin concentration. Conditions under different choroidal melanin concentrations were simulated. MSE: mean square error. The relative MSEs were calculated from preset RPE melanin concentration values between 10 mg/mL and 30 mg/mL, which range covers physiological RPE M. C. (c) Variation of choroid to RPE PA signal ratio with choroidal melanin concentrations. Conditions under different RPE melanin concentrations were simulated. Acoustic detection bandwidth: 110 MHz. RPE thickness was 10 µm in all simulations.

Fig. 3

PAM experimental system. Relay lenses are omitted in the dashed box. Piezoelectric transducer and MRR transducer were used to detect PA signal from both sides of the sample. MRR: micro-ring resonator; APD: avalanche photodiode.

Fig. 4

(a) Melanin extinction coefficients spectrum. Shaded area, standard deviation. Ext. Coef.: extinction coefficient. (b) PA signal amplitude generated by phantoms with controlled melanin concentrations. Error bar: standard deviation.

Fig. 5

PA A-lines detected from RCC samples using different ultrasonic detectors. (a) PA A-line of a porcine RCC sample acquired by customized piezoelectric transducer. (b) Amplitude spectrum of the A-line signal in (a). (c) PA A-line of a porcine RCC sample acquired by broadband MRR detector. (d) Amplitude spectrum of the A-line signal in (c). (e) PA A-line of a human RCC sample acquired by broadband MRR detector. (f) Amplitude spectrum of the A-line signal in (e).

Fig. 6

PA images of porcine and human RCC. (a) PA image of porcine RCC acquired by customized piezoelectric transducer. (b)-(c) PA image of porcine RCC acquired by broadband MRR detector. (b) Axially segmented porcine RPE. (c) Axially segmented porcine choroid. (a)-(c) are images of the same area on a single sample. (d) PA image of human RCC acquired by customized piezoelectric transducer. (e)-(f) PA image of human RCC acquired by homemade MRR detector. (b) Axially segmented human RPE. (c) Axially segmented human choroid. (d)-(f) are images of the same area on a single sample. Scale bar, 50 µm.

Fig. 7

(a) PA B-scan of porcine RCC. (b) PA B-scan of human RCC. RPE cells are visualized on top of choroid tissue. Scale bar, 30 µm.

Fig. 8

Comparison of melanin concentration in RPE and choroid between porcine and human samples. M. C.: Melanin concentration. Error bar: standard deviation.