The melanosome translocation experiment was repeated in 10 WT light-adapted zebrafish and 11 WT dark-adapted zebrafish of the same age (
Fig. 4) to confirm the results seen in the preliminary data (
Fig. 3). All zebrafish were imaged on the same day with the same photothermal laser power (2.07 mW). The light-adapted zebrafish reliably display a three-band pattern (
Fig. 4A) seen in previous experiments, while the dark-adapted zebrafish do not have such pattern (
Fig. 4B). LRPs and LAPs were created by averaging the entire OCT and PT-OCT B-scan (400 A-scans) for each zebrafish (
Fig. 4C,
4D). B-scans without major blood vessels were chosen in each eye to optimize phase stability and the PT-OCT signal quality over the entire B-scan. As seen previously, three peaks are seen in the PT-OCT signal for all light-adapted zebrafish (see five example zebrafish in
Fig. 4C), where the central peak corresponds to the RPE and the anterior-most peak overlaps with a photoreceptor band. This pattern was not seen in any of the dark-adapted zebrafish (see five examples in
Fig. 4D) where the PT-OCT signal is limited to the RPE and is composed of a single peak. A Fourier transform of the LAPs seen in
Figures 4C and
4D was performed to analyze the spatial frequency distribution (
Fig. 4E) for all 21 zebrafish. Two regions of spatial frequency are significantly different between the light-adapted group and the dark-adapted group, which correspond to the distance between the anterior-most and posterior-most absorption bands (Δ
x1 = 23.6 μm) and between the anterior-most and middle bands (Δ
x2 = 11.8 μm) in light-adapted zebrafish (
Fig. 4E). This spatial frequency analysis quantitatively highlights the differences in PT-OCT depth profiles between the light- and dark-adapted groups (
P = 0.0001 at 1/Δ
x1 and
P < 0.0001 at 1/Δ
x2, Student's
t-test). Additionally, the average PT-OCT signal intensity per B-scan was calculated for both experimental groups (
Fig. 4F). A significantly higher PT-OCT signal was detected in the light-adapted group compared to the dark-adapted group (
P < 0.0001, Student's
t-test), which indicates higher melanin absorption in light-adapted zebrafish.