Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye.
OBJECTIVE
The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes.


METHODS
The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye.


RESULTS
Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes.


CONCLUSIONS
Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye.
Teng
Shu-Wen
SW
Microscopic Biophysics Laboratory, Department of Physics, National Taiwan University, Teipei.
Tan
Hsin-Yuan
HY
Peng
Ju-Li
JL
Lin
Huei-Hsing
HH
Kim
Ki Hean
KH
Lo
Wen
W
Sun
Yen
Y
Lin
Wei-Chou
WC
Lin
Sung-Jan
SJ
Jee
Shiou-Hwa
SH
So
Peter T C
PT
Dong
Chen-Yuan
CY
D000818:Animals; D003094:Collagen / Q000648:ultrastructure*; D003228:Conjunctiva / Q000737:chemistry / Q000648:ultrastructure*; D003315:Cornea / Q000737:chemistry / Q000648:ultrastructure*; D007089:Image Enhancement / Q000379:methods; D016850:Limbus Corneae / Q000737:chemistry / Q000648:ultrastructure; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D012590:Sclera / Q000737:chemistry / Q000648:ultrastructure*; D013552:Swine
D003094:Collagen