Chirality is a fundamental property of biochemical molecules and often dictates their functionality. Conventionally, molecular chirality is studied by linear optical activity effects. However, poor contrast and artifacts due to anisotropy limit such studies to purified molecules not in their original microenvironments, potentially modifying their conformations. Here, we demonstrate that resonant second-harmonic-generation circular dichroism (SHG-CD) microscopy provides not only tissue imaging with improved chiral contrast, but also molecular chirality information of collagen, the most abundant protein in mammals, at its native state. Gradual protein denaturation shows that the resonant SHG-CD is dominated by the microscopic chirality related to collagen structures smaller than the spatial resolution of the microscope, i.e. to the protein conformation and microfibril organization, while the effects due to fiber orientation/anisotropy are mostly responsible of the non-resonant part. This result agrees well with a simple and intuitive model we propose to explain the resonant behavior and the consequent numerical SHG-CD simulations. Our results demonstrate the possibility to study molecular chirality in intact bio-tissues with nearly-unity contrast and sub-micrometer resolution, which will be useful in a broad range of biological and biochemical applications.
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