Multiphoton fluorescence and second harmonic generation microscopy for imaging keratoconus

Yen Sun; Wen Lo; Sung-Jan Lin; Wei-Chou Lin; Shiou-Hwa Jee; Hsin-Yuan Tan; Chen-Yuan Dong

doi:10.1117/12.646909

7 March 2006 Multiphoton fluorescence and second harmonic generation microscopy for imaging keratoconus

Yen Sun, Wen Lo, Sung-Jan Lin, Wei-Chou Lin, Shiou-Hwa Jee, Hsin-Yuan Tan, Chen-Yuan Dong

Proceedings Volume 6138, Ophthalmic Technologies XVI; 61380N (2006) https://doi.org/10.1117/12.646909
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover, increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area. In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.

Citation Download Citation

Yen Sun, Wen Lo, Sung-Jan Lin, Wei-Chou Lin, Shiou-Hwa Jee, Hsin-Yuan Tan, and Chen-Yuan Dong "Multiphoton fluorescence and second harmonic generation microscopy for imaging keratoconus", Proc. SPIE 6138, Ophthalmic Technologies XVI, 61380N (7 March 2006); https://doi.org/10.1117/12.646909

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KEYWORDS

Second-harmonic generation

Cornea

Collagen

Multiphoton fluorescence microscopy

Multiphoton microscopy

Microscopy

Harmonic generation

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