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Second-harmonic generation (SHG) microscopy is a recently developed nonlinear optical imaging modality for imaging tissue structures with submicron resolution and is a potent tool for visualizing pathological effects of diseases. In this letter, we present our investigation on the influence of van Gieson's (VG) alcoholic picrofuchsin staining on SHG in type I collagen (from tendon-rich C57BL/6). Multi-channel imaging and spectra analysis show that the strong SHG signal produced in fresh collagen type I fiber has been greatly suppressed after VG staining, which indicates that staining may induce the structural or characteristic changes of SHG-dependent crystal formed by collagen constituents, such as glycine, proline, and hydroxyproline.