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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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Label-free imaging of TM cells using CARS and collagen extracellular matrix using TPAF. The image is taken using a 60× 1.2 NA water objective with 3× digital zoom. (RED: CARS. Green: TPAF). A: Both CARS and TPAF channels are displayed in the image, clearly showing the TM cells in the CARS channel with arrows indicating the TM cells. B: Only the TPAF channel is displayed and TM cells are not observed, indicating that signal of the TM cells are unique to the CARS signal.
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f4: Label-free imaging of TM cells using CARS and collagen extracellular matrix using TPAF. The image is taken using a 60× 1.2 NA water objective with 3× digital zoom. (RED: CARS. Green: TPAF). A: Both CARS and TPAF channels are displayed in the image, clearly showing the TM cells in the CARS channel with arrows indicating the TM cells. B: Only the TPAF channel is displayed and TM cells are not observed, indicating that signal of the TM cells are unique to the CARS signal.

Mentions: In Figure 4, the scanning magnification of the image has been increased 3 times using the 60× objective (180× final magnification) to show a region with several TM cells in close proximity. At this resolution, the outer cell membrane structure can be clearly observed with no additional intracellular membrane structure. Altogether, the data demonstrates the efficacy of CARS and its ability to selectively visualize the TM cells as the cell membrane structure is only displayed in the CARS channel while only the collagen fiber extracellular matrix structure is shown in the TPAF imaging channel.


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 TM cells using CARS and collagen extracellular matrix using TPAF. The image is taken using a 60× 1.2 NA water objective with 3× digital zoom. (RED: CARS. Green: TPAF). A: Both CARS and TPAF channels are displayed in the image, clearly showing the TM cells in the CARS channel with arrows indicating the TM cells. B: Only the TPAF channel is displayed and TM cells are not observed, indicating that signal of the TM cells are unique to the CARS signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Label-free imaging of TM cells using CARS and collagen extracellular matrix using TPAF. The image is taken using a 60× 1.2 NA water objective with 3× digital zoom. (RED: CARS. Green: TPAF). A: Both CARS and TPAF channels are displayed in the image, clearly showing the TM cells in the CARS channel with arrows indicating the TM cells. B: Only the TPAF channel is displayed and TM cells are not observed, indicating that signal of the TM cells are unique to the CARS signal.
Mentions: In Figure 4, the scanning magnification of the image has been increased 3 times using the 60× objective (180× final magnification) to show a region with several TM cells in close proximity. At this resolution, the outer cell membrane structure can be clearly observed with no additional intracellular membrane structure. Altogether, the data demonstrates the efficacy of CARS and its ability to selectively visualize the TM cells as the cell membrane structure is only displayed in the CARS channel while only the collagen fiber extracellular matrix structure is shown in the TPAF imaging channel.

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