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Label-free imaging of trabecular meshwork cells using Coherent Anti-Stokes Raman Scattering (CARS) microscopy.

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

Bottom Line: The signal is predominately from collagen and elastin.Analysis of multiple TPAF images of the TM reveals the characteristic overlapping bundles of collagen of various sizes.Similar images have been obtained with standard histological techniques, however the method described here has the advantage of being performed on unprocessed, unfixed tissue free from the potential distortions of the fine tissue morphology that can occur due to infusion of fixatives and treatment with alcohols.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical Engineering, University of Colorado Denver, Denver, CO, USA.

ABSTRACT

Purpose: To image the human trabecular meshwork (TM) using a non-invasive, non-destructive technique without the application of exogenous label.

Methods: Flat-mounted TM samples from a human cadaver eye were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). In TPAF, two optical photons are simultaneously absorbed and excite molecules in the sample that then emit a higher energy photon. The signal is predominately from collagen and elastin. The CARS technique uses two laser frequencies to specifically excite carbon-hydrogen bonds, allowing the visualization of lipid-rich cell membranes. Multiple images were taken along an axis perpendicular to the surface of the TM for subsequent analysis.

Results: Analysis of multiple TPAF images of the TM reveals the characteristic overlapping bundles of collagen of various sizes. Simultaneous CARS imaging revealed elliptical structures of ~7×10 µm in diameter populating the meshwork which were consistent with TM cells. Irregularly shaped objects of ~4 µm diameter appeared in both the TPAF and CARS channels, and are consistent with melanin granules.

Conclusions: CARS techniques were successful in imaging live TM cells in freshly isolated human TM samples. Similar images have been obtained with standard histological techniques, however the method described here has the advantage of being performed on unprocessed, unfixed tissue free from the potential distortions of the fine tissue morphology that can occur due to infusion of fixatives and treatment with alcohols. CARS imaging of the TM represents a new avenue for exploring details of aqueous outflow and TM cell physiology.

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Related in: MedlinePlus

Label free imaging of the trabecular meshwork of a human cadaver eye using two photon autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS) microscopy. (Green: TPAF, Red: CARS) The arrows indicate the TM cells that reside in the interstitial areas within the collagen extracellular matrix (EM) structure.
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f3: Label free imaging of the trabecular meshwork of a human cadaver eye using two photon autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS) microscopy. (Green: TPAF, Red: CARS) The arrows indicate the TM cells that reside in the interstitial areas within the collagen extracellular matrix (EM) structure.

Mentions: CARS/TPAF images are taken along the TM region in the cadaver coronal rim sample. Figure 3 shows a typical image, displaying the TPAF imaging channel in green and the CARS imaging channel in red. Due to autofluorescence of the collagen molecules, the collagen extracellular matrix shows clearly in the TPAF channel. In these images of the TM, the collagen fibers appear as smooth fiber bundles of various diameters, ranging from 1 and 10 µm. The fibers are straight with a consistent diameter, although the occasional bifurcation is visible. Qualitatively, the fiber structures are similar to those characterized previously using a commercially available two-photon microscope with a tunable femtosecond laser source [3]. In addition, the cell membrane of the TM cells is detected in the CARS channel. These cells are shown residing in the interstitial region between the collagen fiber structure (Figure 3, arrows). The size of the TM cells shown in the image are elliptical shape, with the long axis measured to be 10.4±1.2 µm and the short axis measured to be 6.9±1.1 µm (n=12). We calculate a cell density 281±59 cells/mm2 TM cells (n=5) in the surface region of the filtering TM. These cells have a uniformly smooth and rounded appearance, suggesting that they are healthy. In contrast, the highly autofluorescent pigment granules have a much more irregular shaped and have a considerably smaller diameter at 3.7±0.7 µm (n=12).


Label-free imaging of trabecular meshwork cells using Coherent Anti-Stokes Raman Scattering (CARS) microscopy.

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

Label free imaging of the trabecular meshwork of a human cadaver eye using two photon autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS) microscopy. (Green: TPAF, Red: CARS) The arrows indicate the TM cells that reside in the interstitial areas within the collagen extracellular matrix (EM) structure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198498&req=5

f3: Label free imaging of the trabecular meshwork of a human cadaver eye using two photon autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS) microscopy. (Green: TPAF, Red: CARS) The arrows indicate the TM cells that reside in the interstitial areas within the collagen extracellular matrix (EM) structure.
Mentions: CARS/TPAF images are taken along the TM region in the cadaver coronal rim sample. Figure 3 shows a typical image, displaying the TPAF imaging channel in green and the CARS imaging channel in red. Due to autofluorescence of the collagen molecules, the collagen extracellular matrix shows clearly in the TPAF channel. In these images of the TM, the collagen fibers appear as smooth fiber bundles of various diameters, ranging from 1 and 10 µm. The fibers are straight with a consistent diameter, although the occasional bifurcation is visible. Qualitatively, the fiber structures are similar to those characterized previously using a commercially available two-photon microscope with a tunable femtosecond laser source [3]. In addition, the cell membrane of the TM cells is detected in the CARS channel. These cells are shown residing in the interstitial region between the collagen fiber structure (Figure 3, arrows). The size of the TM cells shown in the image are elliptical shape, with the long axis measured to be 10.4±1.2 µm and the short axis measured to be 6.9±1.1 µm (n=12). We calculate a cell density 281±59 cells/mm2 TM cells (n=5) in the surface region of the filtering TM. These cells have a uniformly smooth and rounded appearance, suggesting that they are healthy. In contrast, the highly autofluorescent pigment granules have a much more irregular shaped and have a considerably smaller diameter at 3.7±0.7 µm (n=12).

Bottom Line: The signal is predominately from collagen and elastin.Analysis of multiple TPAF images of the TM reveals the characteristic overlapping bundles of collagen of various sizes.Similar images have been obtained with standard histological techniques, however the method described here has the advantage of being performed on unprocessed, unfixed tissue free from the potential distortions of the fine tissue morphology that can occur due to infusion of fixatives and treatment with alcohols.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical Engineering, University of Colorado Denver, Denver, CO, USA.

ABSTRACT

Purpose: To image the human trabecular meshwork (TM) using a non-invasive, non-destructive technique without the application of exogenous label.

Methods: Flat-mounted TM samples from a human cadaver eye were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). In TPAF, two optical photons are simultaneously absorbed and excite molecules in the sample that then emit a higher energy photon. The signal is predominately from collagen and elastin. The CARS technique uses two laser frequencies to specifically excite carbon-hydrogen bonds, allowing the visualization of lipid-rich cell membranes. Multiple images were taken along an axis perpendicular to the surface of the TM for subsequent analysis.

Results: Analysis of multiple TPAF images of the TM reveals the characteristic overlapping bundles of collagen of various sizes. Simultaneous CARS imaging revealed elliptical structures of ~7×10 µm in diameter populating the meshwork which were consistent with TM cells. Irregularly shaped objects of ~4 µm diameter appeared in both the TPAF and CARS channels, and are consistent with melanin granules.

Conclusions: CARS techniques were successful in imaging live TM cells in freshly isolated human TM samples. Similar images have been obtained with standard histological techniques, however the method described here has the advantage of being performed on unprocessed, unfixed tissue free from the potential distortions of the fine tissue morphology that can occur due to infusion of fixatives and treatment with alcohols. CARS imaging of the TM represents a new avenue for exploring details of aqueous outflow and TM cell physiology.

Show MeSH
Related in: MedlinePlus