3D imaging of aqueous veins and surrounding sclera using a dual-wavelength photoacoustic microscopy

Abstract

Understanding aqueous outflow resistance at the level of aqueous veins has been a challenge to the management of glaucoma. This study investigated resolving the anatomies of aqueous veins and the textures of surrounding sclera using photoacoustic microscopy (PAM). A dual wavelength PAM system was established and validated using imaging phantoms, porcine and human globes perfused with an optical contrast agent ex vivo. The system shows lateral resolution of 8.23 µm and 4.70 µm at 1200 nm and 532 nm, respectively, and an axial resolution of 27.6 µm. The system is able to separately distinguish the aqueous veins and the sclera with high contrast in full circumference of the porcine and human globes.

Fig. 1.

Integrated dual-wavelength PAM system, LPDM: long pass dichroic mirror (cut-off wavelength 1150 nm), SPDM: short pass dichroic mirror (cut-off wavelength 900 nm), SL: scan lens. The green light pass is at the wavelength of 532 nm while the red-light path is centered at the wavelength of 1200 nm.

Fig. 2.

Lateral resolution of dual wavelength PAM system. (a, c) The PAM images of black tape at 1200 nm and 532 nm, respectively. (b, d) Examples of the edge spread functions (ESF) and the corresponding point spread functions (PSF) along the dashed lines in (a,c). The arrows mark the FWHM of the PSF.

Fig. 3.

Axial resolution of dual wavelength PAM System. (a) The PAM image of 6 um diameter microsphere phantom. (b) The example PA signal of one microsphere. The red solid curve is the envelope of the original PA signal plotted in blue dashed curve. The black curve is the Gaussian fitted line of the envelope. (c) Determining the axial resolution. The dashed curves are PA signals generated by the same microsphere with a 27 µm shift along the axial dimension. The solid red line is the summed envelope of the two dashed signals. The signals at the two locations can be separated by their FWHM. (d) The FWHM distribution of microspheres in Fig. 3(a).

Fig. 4.

PAM images of the imaging phantom made of hair, fishing line and red ink. (a) Optical absorption profiles of red ink, melanin in hair fiber [36] and C-H chemical bonds in fishing line [25,33]. (b) The photograph of the three inclusions. (c) Images acquired at the wavelengths of 1200 nm and 532 nm, respectively.

Fig. 5.

Dual-wavelength PAM images of a porcine eye. In all panels, aqueous vasculature was rendered in red, and the textures of the surrounding sclera are rendered in gray. (a) Full circumferential image with both components. (b-c) and (d-e) are magnified regions A and B in (a), respectively, with the two tissue components displayed separately.

Fig. 6.

Dual-wavelength PAM images of a human eye. (a) Full circumferential image with both components. The magnified features of vasculature and sclera tissues of the area A and B in the white boxes in (a) are shown in (b-c) and (d-e), respectively.

Fig. 7.

Resolving the depths of spatial features in a human eye. (a) Aqueous veins image acquired at 532 nm. The depths of individual vessels were encoded in color scale. (b-c) Cross-sectional images of the sclera texture and aqueous veins along the yellow dotted line in (a), respectively.