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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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An animation representing a three-dimensional rendering of a 136.3 µm thick z-stack of the TM region of the human eye imaged through the sclera. Rotation is around the y-axis, beginning −60 °C from the original xy-imaging plane through +60 °C. Schlemm’s canal is visible in the lower center. This animation can be viewed in the html version of the article. This image is a representative frame of the animation.
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f3: An animation representing a three-dimensional rendering of a 136.3 µm thick z-stack of the TM region of the human eye imaged through the sclera. Rotation is around the y-axis, beginning −60 °C from the original xy-imaging plane through +60 °C. Schlemm’s canal is visible in the lower center. This animation can be viewed in the html version of the article. This image is a representative frame of the animation.

Mentions: 2PM was performed on a human eye with the sclera facing the 20× objective. Deep tissue imaging was performed by directing the 800 nm infrared laser through the surface of the sclera at a point near the limbal region, focusing toward the TM region of the human eye. Using the META spectral detector, second harmonic generation (SHG: white) and 2-photon autofluorescence (2PAF: blue) were collected as described in the Methods to image the collagen matrix and tissue fluorescence, respectively. Each z-plane consisted of three 450×450 µm individual images stitched together (three images on the y-axis). Twenty z-planes were collected at 7.2 µm intervals for a total inclusive depth of 136.8 µm. This z-stack was computer projected into three-dimensional space and rotated 120° about the y-axis (from −60° to +60° with respect to the surface of the sclera). Radial sections of this animation were taken every 15° and shown in Figure 2. The orientation of these sections should not be confused with the radial sections generated by histology, which are tilted 90° with respect to the surface of the sclera. The scleral spur, iris root and attached TM region can be seen with considerable detail. The SC (red circle) can be seen clearly adjacent to the TM tissue (dotted lines). The complete movie is presented in Figure 3.


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)

An animation representing a three-dimensional rendering of a 136.3 µm thick z-stack of the TM region of the human eye imaged through the sclera. Rotation is around the y-axis, beginning −60 °C from the original xy-imaging plane through +60 °C. Schlemm’s canal is visible in the lower center. This animation can be viewed in the html version of the article. This image is a representative frame of the animation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: An animation representing a three-dimensional rendering of a 136.3 µm thick z-stack of the TM region of the human eye imaged through the sclera. Rotation is around the y-axis, beginning −60 °C from the original xy-imaging plane through +60 °C. Schlemm’s canal is visible in the lower center. This animation can be viewed in the html version of the article. This image is a representative frame of the animation.
Mentions: 2PM was performed on a human eye with the sclera facing the 20× objective. Deep tissue imaging was performed by directing the 800 nm infrared laser through the surface of the sclera at a point near the limbal region, focusing toward the TM region of the human eye. Using the META spectral detector, second harmonic generation (SHG: white) and 2-photon autofluorescence (2PAF: blue) were collected as described in the Methods to image the collagen matrix and tissue fluorescence, respectively. Each z-plane consisted of three 450×450 µm individual images stitched together (three images on the y-axis). Twenty z-planes were collected at 7.2 µm intervals for a total inclusive depth of 136.8 µm. This z-stack was computer projected into three-dimensional space and rotated 120° about the y-axis (from −60° to +60° with respect to the surface of the sclera). Radial sections of this animation were taken every 15° and shown in Figure 2. The orientation of these sections should not be confused with the radial sections generated by histology, which are tilted 90° with respect to the surface of the sclera. The scleral spur, iris root and attached TM region can be seen with considerable detail. The SC (red circle) can be seen clearly adjacent to the TM tissue (dotted lines). The complete movie is presented in Figure 3.

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