In vivo photothermal optical coherence tomography of endogenous and exogenous contrast agents in the eye

Abstract

Optical coherence tomography (OCT) has become a standard-of-care in retinal imaging. OCT allows non-invasive imaging of the tissue structure but lacks specificity to contrast agents that could be used for in vivo molecular imaging. Photothermal OCT (PT-OCT) is a functional OCT-based technique that has been developed to detect absorbers in a sample. We demonstrate in vivo PT-OCT in the eye for the first time on both endogenous (melanin) and exogenous (gold nanorods) absorbers. Pigmented mice and albino mice (n = 6 eyes) were used to isolate the photothermal signal from the melanin in the retina. Pigmented mice with laser-induced choroidal neovascularization lesions (n = 7 eyes) were also imaged after a systemic injection of gold nanorods to observe their passive accumulation in the retina. This experiment demonstrates the feasibility of PT-OCT to image the distribution of both endogenous and exogenous absorbers in the mouse retina.

Figure 1

Photothermal OCT principle and instrumentation. (a) Illustration of the OCT raw signal over time (i) in the absence of a photothermal beam and (ii) in the presence of an amplitude-modulated photothermal beam for a sample containing some gold nanorods (yellow). (b) PT-OCT instrumentation. Imaging beam (SLD) centered at λ = 860 nm is divided between the reference and sample arm using a 50:50 fiber coupler. The photothermal beam (Ti:Sapphire, λ = 750 nm) is amplitude modulated with an acousto-optic modulator (AOM), and coupled to the sample arm. A circulator redirects the light from the sample to the detector. HWP: Half-wave plate. PC: polarization controller.

Figure 2

In vivo PT-OCT of melanin. (a) Average PT-OCT signal from the retina for albino mice (no melanin) and pigmented mice (melanin) cohorts (n = 6 eyes per group). Photothermal laser power at 8 mW. Mean with standard deviation shown. **p < 0.01. (b) Example OCT B-scan of a pigmented mouse retina (grayscale) with PT-OCT signal overlaid (green). (c) Example OCT B-scan of an albino mouse retina (grayscale) with PT-OCT signal overlaid (green, no signal visible). The OCT images show fewer retinal layers than in a typical OCT B-scan of a human retina because of the smaller dimensions of a mouse retina and the axial resolution of standard 860 nm OCT systems, which are also used for human retina imaging. The PT-OCT signal represents the change in optical path length, in units of nm. Scale bar: 100 μm. RPE: retinal pigment epithelium. CO: choroid.

Figure 3

In vivo PT-OCT of melanin for different photothermal laser powers. (a) Example OCT B-scans (grayscale, left) of a pigmented mouse retina with corresponding PT-OCT signal (overlaid in green on the left, signal alone on the right) collected for different photothermal laser powers (1–8 mW). Scale bar: 100 μm. (b) Average PT-OCT signal for one B-scan (400 A-scans) collected at different photothermal laser powers (same eye as presented in (a)). The width at half-maximum of the signal peaks (dashed black lines) corresponds to the location of the RPE in depth. (c) Mean background signal (excludes the dashed region from (b)) and peak-to-background ratio for one example eye at different photothermal laser powers. Error bar: standard error. (d) Mean PT-OCT signal as a function of photothermal laser power for n = 6 eyes. Error bar: standard deviation. Linear fit y = 1.56x with 95%CI on the fit (red dashed). R² = 0.91. *p < 0.05 between 1 mW and 0.4 mW (Mann-Whitney U test).

Figure 4

In vivo PT-OCT of gold nanoparticles. (a,d) OCT B-scan of the retina showing an LCNV lesion (boxed area) in, respectively, a mouse of the control group and a mouse of the gold nanorods group. (b,e) OCT B-scan of the LCNV lesion (grayscale) with PT-OCT signal overlaid (green) in, respectively, a mouse of the control group and a mouse of the gold nanorods group. (c,f) 2D en face mean projection of the OCT (grayscale) and PT-OCT (green) data volume in a control mouse and a mouse injected with gold nanorods. The location of the B-scan shown on the left are indicated by the red dashed line. Scale bar 100 μm. (g) Average PT-OCT signal normalized to the volume of the LCNV lesion for n = 7 eyes per group. Mean with standard deviation shown. *p < 0.05.