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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

Energy diagrams of the Two-Photon Autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS). A: Energy diagram of TPAF, in which an autofluorescent molecule simultaneously absorbs two optical infrared photons (E2p). After internal-crossing (IC), in which some energy is lost, the fluorescent molecule will emit a fluorescence photon (Eem). B: Energy diagram of CARS, in which two optical photons with the photon energy difference (Epump - EStokes) equaling to the vibrational energy of a molecule (EΩ) is used to excite the vibrational motion of the molecule. A third photon (Eprobe) is subsequently used to interact with the vibtational motion of the molecule, resulting in the emision of an energy-upshifted photon (ECARS). C: A schematic diagram illustrating the CARS process. The pump and the Stokes photons are simultaneously exciting the lipid molecule, with the energy difference between the two photons equal to the vibrational energy of the molecule bond (EΩ). Subsequent interaction of the probe photon coherently interacts with the vibrational motion of the molecule to generate a release of the CARS photon.
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f2: Energy diagrams of the Two-Photon Autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS). A: Energy diagram of TPAF, in which an autofluorescent molecule simultaneously absorbs two optical infrared photons (E2p). After internal-crossing (IC), in which some energy is lost, the fluorescent molecule will emit a fluorescence photon (Eem). B: Energy diagram of CARS, in which two optical photons with the photon energy difference (Epump - EStokes) equaling to the vibrational energy of a molecule (EΩ) is used to excite the vibrational motion of the molecule. A third photon (Eprobe) is subsequently used to interact with the vibtational motion of the molecule, resulting in the emision of an energy-upshifted photon (ECARS). C: A schematic diagram illustrating the CARS process. The pump and the Stokes photons are simultaneously exciting the lipid molecule, with the energy difference between the two photons equal to the vibrational energy of the molecule bond (EΩ). Subsequent interaction of the probe photon coherently interacts with the vibrational motion of the molecule to generate a release of the CARS photon.

Mentions: In human tissue, there are several endogenous molecules (collagen, elastin, and nicotinamide adenine dinucleotide [NADH]) that autofluoresce when excited by photons with the optical energy that matches the absorption spectra. Two-photon autofluorescence microscopy (TPAF) uses a femtosecond or picosecond pulsed laser in the infrared wavelength to excite the endogenous autofluorescent molecules through simultaneous absorption of two infrared photons to have the total photon energy matching the absorption energy of the endogenous molecules [2-5,10-12]. The benefits of using two infrared photons to excite the autofluorescent molecules instead of a single higher energy photon include reduced phototoxicity and much deeper penetration in biologic tissues [13]. As shown in Figure 2A, two optical photons are simultaneously absorbed by an autofluorescent molecule and the autofluorescent molecule subsequently emits an optical photon with reduced photon energy than the combined excitation energy due to energy loss through the internal conversion (IC) process. Since the TM region of the eye is mainly composed of collagen molecules in the extracellular network, capturing the fluorescent photons with the proper optical filters matching the emission spectrum of collagen in front of the photo-detector allows us to image the collagen extracellular matrix around the TM region intrinsically without the need for exogenous labeling.


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)

Energy diagrams of the Two-Photon Autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS). A: Energy diagram of TPAF, in which an autofluorescent molecule simultaneously absorbs two optical infrared photons (E2p). After internal-crossing (IC), in which some energy is lost, the fluorescent molecule will emit a fluorescence photon (Eem). B: Energy diagram of CARS, in which two optical photons with the photon energy difference (Epump - EStokes) equaling to the vibrational energy of a molecule (EΩ) is used to excite the vibrational motion of the molecule. A third photon (Eprobe) is subsequently used to interact with the vibtational motion of the molecule, resulting in the emision of an energy-upshifted photon (ECARS). C: A schematic diagram illustrating the CARS process. The pump and the Stokes photons are simultaneously exciting the lipid molecule, with the energy difference between the two photons equal to the vibrational energy of the molecule bond (EΩ). Subsequent interaction of the probe photon coherently interacts with the vibrational motion of the molecule to generate a release of the CARS photon.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Energy diagrams of the Two-Photon Autofluorescence (TPAF) and Coherent Anti-Stokes Raman Scattering (CARS). A: Energy diagram of TPAF, in which an autofluorescent molecule simultaneously absorbs two optical infrared photons (E2p). After internal-crossing (IC), in which some energy is lost, the fluorescent molecule will emit a fluorescence photon (Eem). B: Energy diagram of CARS, in which two optical photons with the photon energy difference (Epump - EStokes) equaling to the vibrational energy of a molecule (EΩ) is used to excite the vibrational motion of the molecule. A third photon (Eprobe) is subsequently used to interact with the vibtational motion of the molecule, resulting in the emision of an energy-upshifted photon (ECARS). C: A schematic diagram illustrating the CARS process. The pump and the Stokes photons are simultaneously exciting the lipid molecule, with the energy difference between the two photons equal to the vibrational energy of the molecule bond (EΩ). Subsequent interaction of the probe photon coherently interacts with the vibrational motion of the molecule to generate a release of the CARS photon.
Mentions: In human tissue, there are several endogenous molecules (collagen, elastin, and nicotinamide adenine dinucleotide [NADH]) that autofluoresce when excited by photons with the optical energy that matches the absorption spectra. Two-photon autofluorescence microscopy (TPAF) uses a femtosecond or picosecond pulsed laser in the infrared wavelength to excite the endogenous autofluorescent molecules through simultaneous absorption of two infrared photons to have the total photon energy matching the absorption energy of the endogenous molecules [2-5,10-12]. The benefits of using two infrared photons to excite the autofluorescent molecules instead of a single higher energy photon include reduced phototoxicity and much deeper penetration in biologic tissues [13]. As shown in Figure 2A, two optical photons are simultaneously absorbed by an autofluorescent molecule and the autofluorescent molecule subsequently emits an optical photon with reduced photon energy than the combined excitation energy due to energy loss through the internal conversion (IC) process. Since the TM region of the eye is mainly composed of collagen molecules in the extracellular network, capturing the fluorescent photons with the proper optical filters matching the emission spectrum of collagen in front of the photo-detector allows us to image the collagen extracellular matrix around the TM region intrinsically without the need for exogenous labeling.

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