Multimodal nonlinear imaging of the human cornea.
OBJECTIVE
To evaluate the potential of third-harmonic generation (THG) microscopy combined with second-harmonic generation (SHG) and two-photon excited fluorescence (2PEF) microscopies for visualizing the microstructure of the human cornea and trabecular meshwork based on their intrinsic nonlinear properties.


METHODS
Fresh human corneal buttons and corneoscleral discs from an eye bank were observed under a multiphoton microscope incorporating a titanium-sapphire laser and an optical parametric oscillator for the excitation, and equipped with detection channels in the forward and backward directions.


RESULTS
Original contrast mechanisms of THG signals in cornea with physiological relevance were elucidated. THG microscopy with circular incident polarization detected microscopic anisotropy and revealed the stacking and distribution of stromal collagen lamellae. THG imaging with linear incident polarization also revealed cellular and anchoring structures with micrometer resolution. In edematous tissue, a strong THG signal around cells indicated the local presence of water. Additionally, SHG signals reflected the distribution of fibrillar collagen, and 2PEF imaging revealed the elastic component of the trabecular meshwork and the fluorescence of metabolically active cells.


CONCLUSIONS
The combined imaging modalities of THG, SHG, and 2PEF provide key information about the physiological state and microstructure of the anterior segment over its entire thickness with remarkable contrast and specificity. This imaging method should prove particularly useful for assessing glaucoma and corneal physiopathologies.
Aptel
Florent
F
Laboratoire d'Optique Appliquée, ENSTA ParisTech, Ecole Polytechnique, Palaiseau, France.
Olivier
Nicolas
N
Deniset-Besseau
Ariane
A
Legeais
Jean-Marc
JM
Plamann
Karsten
K
Schanne-Klein
Marie-Claire
MC
Beaurepaire
Emmanuel
E
D003094:Collagen / Q000032:analysis; D003315:Cornea / Q000166:cytology*; D015715:Corneal Edema / Q000473:pathology; D003319:Corneal Stroma / Q000166:cytology; D004728:Endothelium, Corneal / Q000166:cytology; D019573:Epithelium, Corneal / Q000166:cytology; D005125:Eye Banks; D005454:Fluorescence Polarization; D006801:Humans; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D012590:Sclera / Q000166:cytology; D014019:Tissue Donors; D014129:Trabecular Meshwork / Q000166:cytology
D003094:Collagen