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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

Schematic diagram of the Coherent Anti-Stokes Raman Scattering (CARS)/Two-Photon Autofluorescence (TPAF) microscope. The system consists of a Nd:Vanadate picosceond laser and an optical parametric oscillator. The scanning microscope is based on the Olympus FV-1000 confocal microscope system with two non-descanned external detectors in the epi-direction. EM1 is an emission filter to detect the CARS signal at 662 nm by the CARS PMT detector and EM2 is an emission filter to allow TPAF signal from 420 to 520 nm to be detected by the TPAF photomultiplier tube (PMT).
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f1: Schematic diagram of the Coherent Anti-Stokes Raman Scattering (CARS)/Two-Photon Autofluorescence (TPAF) microscope. The system consists of a Nd:Vanadate picosceond laser and an optical parametric oscillator. The scanning microscope is based on the Olympus FV-1000 confocal microscope system with two non-descanned external detectors in the epi-direction. EM1 is an emission filter to detect the CARS signal at 662 nm by the CARS PMT detector and EM2 is an emission filter to allow TPAF signal from 420 to 520 nm to be detected by the TPAF photomultiplier tube (PMT).

Mentions: The CARS/TPAF images of the TM cells were acquired with a custom-built multiphoton microscopy platform optimized for CARS and TPAF imaging as shown in Figure 1. The system consists of a diode-pumped Nd:Vanadate (Nd:YVO4) picoseconds (ps) laser (picoTRAIN, HighQ Laser, Austria) which is capable of generating 10 Watt at 1064 nm of ~7.5ps optical pulses at a repetition rate of 80 MHz. Inside the laser, 9 Watt of the generated 1064 nm laser beam is redirected to a frequency doubling crystal to produce 4 Watt of 532 nm light with ~6 ps optical pulsewidth. The 4 Watt 532 nm laser beam is subsequently sent into an optical parametric oscillator (Levante Emerald, APE Angewandte Physik & Elektronik GmbH, Berlin, Germany) to convert the 532nm laser beam into a 1 Watt, ~6ps, 816 nm laser beam through the nonlinear optical process of difference frequency generation. The remaining 1W 1064 nm beam (Stokes) from the Nd:Vanadate laser is then optically recombined with the 816nm optical beam (Pump and Probe) and the combined laser beam is sent into an Olympus FV-1000 confocal microscope platform (Olympus, Center Valley, PA) for CARS and TPAF imaging. The optical power at the objective is 40 mW for the 816 nm laser beam and 20 mW for the 1064 nm laser beam.


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)

Schematic diagram of the Coherent Anti-Stokes Raman Scattering (CARS)/Two-Photon Autofluorescence (TPAF) microscope. The system consists of a Nd:Vanadate picosceond laser and an optical parametric oscillator. The scanning microscope is based on the Olympus FV-1000 confocal microscope system with two non-descanned external detectors in the epi-direction. EM1 is an emission filter to detect the CARS signal at 662 nm by the CARS PMT detector and EM2 is an emission filter to allow TPAF signal from 420 to 520 nm to be detected by the TPAF photomultiplier tube (PMT).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Schematic diagram of the Coherent Anti-Stokes Raman Scattering (CARS)/Two-Photon Autofluorescence (TPAF) microscope. The system consists of a Nd:Vanadate picosceond laser and an optical parametric oscillator. The scanning microscope is based on the Olympus FV-1000 confocal microscope system with two non-descanned external detectors in the epi-direction. EM1 is an emission filter to detect the CARS signal at 662 nm by the CARS PMT detector and EM2 is an emission filter to allow TPAF signal from 420 to 520 nm to be detected by the TPAF photomultiplier tube (PMT).
Mentions: The CARS/TPAF images of the TM cells were acquired with a custom-built multiphoton microscopy platform optimized for CARS and TPAF imaging as shown in Figure 1. The system consists of a diode-pumped Nd:Vanadate (Nd:YVO4) picoseconds (ps) laser (picoTRAIN, HighQ Laser, Austria) which is capable of generating 10 Watt at 1064 nm of ~7.5ps optical pulses at a repetition rate of 80 MHz. Inside the laser, 9 Watt of the generated 1064 nm laser beam is redirected to a frequency doubling crystal to produce 4 Watt of 532 nm light with ~6 ps optical pulsewidth. The 4 Watt 532 nm laser beam is subsequently sent into an optical parametric oscillator (Levante Emerald, APE Angewandte Physik & Elektronik GmbH, Berlin, Germany) to convert the 532nm laser beam into a 1 Watt, ~6ps, 816 nm laser beam through the nonlinear optical process of difference frequency generation. The remaining 1W 1064 nm beam (Stokes) from the Nd:Vanadate laser is then optically recombined with the 816nm optical beam (Pump and Probe) and the combined laser beam is sent into an Olympus FV-1000 confocal microscope platform (Olympus, Center Valley, PA) for CARS and TPAF imaging. The optical power at the objective is 40 mW for the 816 nm laser beam and 20 mW for the 1064 nm laser beam.

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