Label-free imaging of trabecular meshwork cells using Coherent Anti-Stokes Raman Scattering (CARS) microscopy.
OBJECTIVE
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.
Department of Electrical Engineering, University of Colorado Denver, Denver, CO, USA.
Tim C
TC
Lei
David A
DA
Ammar
Omid
O
Masihzadeh
Emily A
EA
Gibson
Malik Y
MY
Kahook
D000369:Aged, 80 and over; D001344:Autopsy; D003094:Collagen / Q000032:analysis; D004549:Elastin / Q000032:analysis; D005453:Fluorescence; D006652:Histological Techniques / Q000379:methods; D006801:Humans; D007834:Lasers; D008543:Melanins / Q000032:analysis; D008853:Microscopy / Q000379:methods*; D017785:Photons; D019591:Pseudophakia / Q000473:pathology*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014129:Trabecular Meshwork / Q000648:ultrastructure*
D008543:Melanins; D003094:Collagen; D004549:Elastin