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Biophysical Journal 91:4665-4677 (2006)
© 2006 The Biophysical Society

Second Harmonic Generation Confocal Microscopy of Collagen Type I from Rat Tendon Cryosections

Theodossis A. Theodossiou *, Christopher Thrasivoulou {dagger}, Chidi Ekwobi {dagger} and David L. Becker {dagger}

* Department of Medicine, The Rayne Institute, University College London, London, United Kingdom; and {dagger} Centre for Cell and Molecular Dynamics, Department of Anatomy and Developmental Biology, University College London, London, United Kingdom

Correspondence: Address reprint requests to Theodossis A Theodossiou, Dept. of Medicine, The Rayne Institute, 5 University St., University College London, London WC1E 6JJ, UK. Tel.: 30-69-48-922-616; E-mail: t.theodossiou{at}gmail.com.

We performed second harmonic generation (SHG) imaging of collagen in rat-tendon cryosections, using femtosecond laser scanning confocal microscopy, both in backscattering and transmission geometries. SHG transmission images of collagen fibers were spatially resolved due to a coherent, directional SHG component. This effect was enhanced with the use of an index-matching fluid (ni = 1.52). The average SHG intensity oscillated with wavelength in the backscattered geometry (isotropic SHG component), whereas the spectral profile was consistent with quasi-phase-matching conditions in transmission geometry (forward propagating, coherent SHG component) around 440 nm ({lambda}p = 880 nm). Collagen type I from bovine Achilles tendon was imaged for SHG in the backscattered geometry and its first-order effective nonlinear coefficient was determined (Formula) by comparison to samples of inorganic materials with known effective nonlinear coefficients (LiNbO3 and LiIO3). The SHG spectral response of collagen type I from bovine Achilles tendon matched that of the rat-tendon cryosections in backscattered geometry. Collagen types I, II, and VI powders (nonfibrous) did not show any detectable SHG, indicating a lack of noncentrosymmetric crystalline structure at the molecular level. The various stages of collagen thermal denaturation were investigated in rat-tendon cryosections using SHG and bright-field imaging. Thermal denaturation resulted in the gradual destruction of the SHG signal.




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