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Trans-scleral imaging of the human trabecular meshwork by two-photon microscopy.

Ammar DA, Lei TC, Masihzadeh O, Gibson EA, Kahook MY - Mol. Vis. (2011)

Bottom Line: The tissue was subsequently fixed, paraffin embedded, and histological sections were photographed for comparison to the 2PM images. 3D analysis of multiple 2PM SHG images revealed an open region deep within the TM consistent with the location of Schlemm's canal (SC).Images of the scleral spur and surrounding tissues were also obtained.This work reveals that 2PM imaging has potential as a new metric for evaluating the aqueous outflow region of the human eye and is worthy of further exploration.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Colorado Denver, Aurora, CO 80045, USA.

ABSTRACT

Purpose: To image the native (unfixed) human trabecular meshwork (TM) through the overlying sclera using a non-invasive, non-destructive technique.

Methods: Two-photon microscopic (2PM) methods, including two-photon autofluorescence (2PAF) and second harmonic generation (SHG), were used to image through the sclera of a human cadaver eye into the TM region. Multiple images were analyzed along the tissue axis (z-axis) to generate a three-dimensional (3D) model of the region. The tissue was subsequently fixed, paraffin embedded, and histological sections were photographed for comparison to the 2PM images.

Results: 3D analysis of multiple 2PM SHG images revealed an open region deep within the TM consistent with the location of Schlemm's canal (SC). Images of the scleral spur and surrounding tissues were also obtained. The SC, TM, scleral spur, and surrounding tissue images obtained with 2PM matched with histologically stained sections of the same tissue.

Conclusions: 2PM imaging of the outflow system of the human eye documented collagenous structures solely from inherent optical properties. 2PM successfully imaged through the sclera into the SC/TM without the need for fixation, embedding, or histological processing. This work reveals that 2PM imaging has potential as a new metric for evaluating the aqueous outflow region of the human eye and is worthy of further exploration.

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Serial sections of the TM region imaged by 2 photon microscopy. Sections shown here are indicated by the distance from the edge of the tissue block (in microns). The Schlemm’s Canal (SC) opening is apparent in many of the sections. Also prevalent is a ~50 µm round structure, representing a collector channel (CC), visible from the 900 µm section through the 2400 µm section. Black bar=100 µm.
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f4: Serial sections of the TM region imaged by 2 photon microscopy. Sections shown here are indicated by the distance from the edge of the tissue block (in microns). The Schlemm’s Canal (SC) opening is apparent in many of the sections. Also prevalent is a ~50 µm round structure, representing a collector channel (CC), visible from the 900 µm section through the 2400 µm section. Black bar=100 µm.

Mentions: A four by four millimeter square region of sclera/TM encompassing the region imaged by 2PM in Figure 2 was excised and prepared for histological analysis. The entire region was cut into ten micron-thick sections and stained with hematoxylin and eosin Y. Sections at regular intervals were photographed and shown in Figure 4. TM and SC are visible in each section. A CC structure is visible from the section at 900 µm through 2400 µm, running perpendicular to the SC for this distance before disappearing from view.


Trans-scleral imaging of the human trabecular meshwork by two-photon microscopy.

Ammar DA, Lei TC, Masihzadeh O, Gibson EA, Kahook MY - Mol. Vis. (2011)

Serial sections of the TM region imaged by 2 photon microscopy. Sections shown here are indicated by the distance from the edge of the tissue block (in microns). The Schlemm’s Canal (SC) opening is apparent in many of the sections. Also prevalent is a ~50 µm round structure, representing a collector channel (CC), visible from the 900 µm section through the 2400 µm section. Black bar=100 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3049734&req=5

f4: Serial sections of the TM region imaged by 2 photon microscopy. Sections shown here are indicated by the distance from the edge of the tissue block (in microns). The Schlemm’s Canal (SC) opening is apparent in many of the sections. Also prevalent is a ~50 µm round structure, representing a collector channel (CC), visible from the 900 µm section through the 2400 µm section. Black bar=100 µm.
Mentions: A four by four millimeter square region of sclera/TM encompassing the region imaged by 2PM in Figure 2 was excised and prepared for histological analysis. The entire region was cut into ten micron-thick sections and stained with hematoxylin and eosin Y. Sections at regular intervals were photographed and shown in Figure 4. TM and SC are visible in each section. A CC structure is visible from the section at 900 µm through 2400 µm, running perpendicular to the SC for this distance before disappearing from view.

Bottom Line: The tissue was subsequently fixed, paraffin embedded, and histological sections were photographed for comparison to the 2PM images. 3D analysis of multiple 2PM SHG images revealed an open region deep within the TM consistent with the location of Schlemm's canal (SC).Images of the scleral spur and surrounding tissues were also obtained.This work reveals that 2PM imaging has potential as a new metric for evaluating the aqueous outflow region of the human eye and is worthy of further exploration.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Colorado Denver, Aurora, CO 80045, USA.

ABSTRACT

Purpose: To image the native (unfixed) human trabecular meshwork (TM) through the overlying sclera using a non-invasive, non-destructive technique.

Methods: Two-photon microscopic (2PM) methods, including two-photon autofluorescence (2PAF) and second harmonic generation (SHG), were used to image through the sclera of a human cadaver eye into the TM region. Multiple images were analyzed along the tissue axis (z-axis) to generate a three-dimensional (3D) model of the region. The tissue was subsequently fixed, paraffin embedded, and histological sections were photographed for comparison to the 2PM images.

Results: 3D analysis of multiple 2PM SHG images revealed an open region deep within the TM consistent with the location of Schlemm's canal (SC). Images of the scleral spur and surrounding tissues were also obtained. The SC, TM, scleral spur, and surrounding tissue images obtained with 2PM matched with histologically stained sections of the same tissue.

Conclusions: 2PM imaging of the outflow system of the human eye documented collagenous structures solely from inherent optical properties. 2PM successfully imaged through the sclera into the SC/TM without the need for fixation, embedding, or histological processing. This work reveals that 2PM imaging has potential as a new metric for evaluating the aqueous outflow region of the human eye and is worthy of further exploration.

Show MeSH