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. 2013 Mar;18(3):036009.
doi: 10.1117/1.JBO.18.3.036009.

Direct Trabecular Meshwork Imaging in Porcine Eyes Through Multiphoton Gonioscopy

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Free PMC article

Direct Trabecular Meshwork Imaging in Porcine Eyes Through Multiphoton Gonioscopy

Omid Masihzadeh et al. J Biomed Opt. .
Free PMC article

Abstract

The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido-corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans.

Figures

Fig. 1
Fig. 1
Schematic of the custom multiphoton gonioscopy microscope. The pulsed femtosecond laser beam is first sent through a prism compressor and passed through two galvanometric scanning mirrors for raster scanning at the sample. A custom-made lens relay system is used to convert our inverted microscope (Olympus IX71) to an upright microscope for in situ imaging. The multiphoton microscopy (MPM) signal from the sample is collected back through the microscope objective and separated from the excitation laser light with a dichroic mirror (DM1). An additional dichroic mirror (DM2) is used to spectrally separate the second-harmonic-generation (SHG) and two-photon autofluorescence (2PAF) signals, which are subsequently focused on two single-photon counting photodetectors for data acquisition. Pr, prism; HW, half-wave plate; PP, polarizer; Gl, gonioscopic lens; L, focusing lens.
Fig. 2
Fig. 2
Acquired MPM images of the TM of a porcine eye taken through a gonioscopic lens. Panels 2(a)–2(d) are 3×3 tiles of combined MPM images of the porcine trabecular meshwork (TM) at different depths, where 2(a) is closest to the anterior chamber and 2(d) is farthest away from the anterior chamber. The images are 300 µm apart. SHG signal is displayed in green and 2PAF is displayed in red. A collagen-rich mesh structure (SHG) is observed in all images. The mesh structure is located in a gap that separates two distinct regions: a strong SHG region (sclera) and a strong 2PAF region (iris). SC, sclera; I, iris; A, irido-corneal angle.
Fig. 3
Fig. 3
Processed 2.5D pseudo-intensity plot of MPM gonioscopy images. The two-dimensional image of Fig. 2(a) was processed by 2.5D to highlight regions between the collagen fibers lacking signal. The z-axis of the processed 2.5D image is the signal intensity (Sig. Int.) of both the SHG and 2PAF signals (arbitrary units). The dark regions lacking both SHG and 2PAF are assumed to be fluid-filled pores.
Fig. 4
Fig. 4
MPM gonioscopy imaging of the TM region of the porcine eye does not appear to damage ocular structures. A histological section of the region of the porcine eye imaged by MPM (b) shows no distortion or photocoagulation of the tissues near the drainage angle compared to a region from the opposite side of the eye (a). The red line in 4(a) shows the approximate imaging plane of Fig. 2(a); 2(b) and 2(c) would be located parallel to this plane traveling deeper into the scleral/TM region. CB, ciliary body. The scale bar represents 200 µm.

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