Interpreting Second Harmonic Generation Images of Collagen I Fibrils

¹ Cornell University

^* To whom correspondence should be addressed. E-mail: rw36{at}cornell.edu.

Submitted on June 8, 2004
Revised on July 30, 2004
Accepted on 26 October 2004

	Abstract

Fibrillar collagen, being highly non-centrosymmetric, possesses a tremendous nonlinear susceptibility. As a result, second-harmonic generation (SHG) microscopy of collagen produces extremely bright and robust signals, providing an invaluable tool for imaging tissue structure with submicron resolution. Here we discuss fundamental principles governing SHG phase-matching with the tightly-focusing optics used in microscopy. Their application to collagen imaging yields several biophysical features characteristic of native collagen structure: SHG radiates from the shell of a collagen fibril, rather than from its bulk. This SHG shell may correspond to the supporting element of the fibril. Physiologically relevant changes in solution ionic strength alter the ratio of forward-to-backward propagating SHG, implying a resulting change in the SHG shell thickness. Fibrillogenesis can be resolved in immature tissue by directly imaging backward-propagating SHG. Such findings are crucial to the design and development of forthcoming diagnostic and research tools.

Key Words: Collagen, Multiphoton Microscopy, Nonlinear Scattering, SHG Microscopy, Second Harmonic Generation

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