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1From the Microscopic Biophysics Laboratory, Department of Physics, the 3Institute of Biomedical Engineering, College of Medicine and College of Engineering, and the 4Department of Life Science, National Taiwan University, Taipai, Taiwan; the 6Departments of Pathology, and 7Dermatology, National Taiwan University Hospital, Taipei Taiwan; the 8Department of Dermatology, College of Medicine, National Taiwan University Hospital, Taipei, Taiwan; the 2Department of Ophthalmology, Chang Gung Memorial Hospital, Linko, Taiwan; and the 5Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
PURPOSE. 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.
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