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Three-Dimensional Segmentation and Quantitative Measurement of the Aqueous Outflow System of Intact Mouse Eyes Based on Spectral Two-Photon Microscopy Techniques.

Abstract：

PURPOSE:To visualize and quantify the three-dimensional (3D) spatial relationships of the structures of the aqueous outflow system (AOS) within intact enucleated mouse eyes using spectral two-photon microscopy (TPM) techniques. METHODS:Spectral TPM, including two-photon autofluorescence (TPAF) and second-harmonic generation (SHG), were used to image the small structures of the AOS within the limbal region of freshly enucleated mouse eyes. Long infrared excitation wavelengths (930 nm) were used to reduce optical scattering and autofluorescent background. Image stacks were collected for 3D image rendering and structural segmentation. For anatomical reference, vascular perfusion with fluorescent-conjugated dextran (150 KDa) and trans-corneal perfusion with 0.1 μm fluorescent polystyrene beads were separately performed to identify the episcleral veins (EV) and the trabecular meshwork (TM) and Schlemm's canal (SC), respectively. RESULTS:Three-dimensional rendering and segmentation of spectral two-photon images revealed detailed structures of the AOS, including SC, collector channels (CC), and aqueous veins (AV). The collagen of the TM was detected proximal to SC. The long and short axes of the SC were 82.2 ± 22.2 μm and 6.7 ± 1.4 μm. The diameters of the CC averaged 25.6 ± 7.9 μm where they originated from the SC (ostia), enlarged to 34.1 ± 13.1 μm at the midpoint, and narrowed to 18.3 ± 4.8 μm at the junction of the AV. The diameter of the AV averaged 12.5 ± 3.4 μm. CONCLUSIONS:Spectral TPM can be used to reconstruct and measure the spatial relationships of both large and small AOS structures, which will allow for better understanding of distal aqueous outflow dynamics.

journal_name

Invest Ophthalmol Vis Sci

journal_title

Investigative ophthalmology & visual science

authors

Zhang X,Liu N,Mak PU,Pun SH,Vai MI,Masihzadeh O,Kahook MY,Lei TC,Ammar DA

doi

10.1167/iovs.15-18491

subject

Has Abstract

pub_date

2016-06-01 00:00:00

pages

3159-67

issue

eissn

0146-0404

issn

1552-5783

pii

2529376

journal_volume

pub_type

杂志文章

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Three-Dimensional Segmentation and Quantitative Measurement of the Aqueous Outflow System of Intact Mouse Eyes Based on Spectral Two-Photon Microscopy Techniques.