Point-scanning sum-frequency generation (SFG) microscopy enables the generation of images of collagen I fibers in tissues by tuning into specific vibrational resonances of the polypeptide. It is shown that when collagen-rich tissues are visualized near the 2954 cm(-1) stretching vibration of methylene groups, the SFG image contrast is higher compared to the contrast seen in nonresonant second-harmonic generation (SHG) imaging. Polarization and spectrally resolved analysis of the SFG signal as a function of fiber orientation in the CH-stretching range of the vibrational spectrum enabled a comparative characterization of the achiral tensor elements of collagens second-order susceptibility. This analysis reveals that selected on-resonance tensor elements are enhanced over other elements, giving rise to a much stronger anisotropy ? of the signal for SFG (p approximate to 15) compared to SHG (p approximate to 3). The improved anisotropy of the vibrationally resonant signal contributes to the higher contrast seen in the SFG tissue images.