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Abstract
Fibrillar collagen, being highly noncentrosymmetric, 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.