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Chirality plays a fundamental role in biomedical fields; many drugs, enzymes, and biomolecules cannot function unless their chiralities are correct. Since the conformation of a molecule, as well as the chirality, is very sensitive to the local microenvironment, it is vital to characterize molecular chirality without altering the surrounding conditions. To determine the chirality in materials, optical activity is the most common way. In linear optics, optical rotatory dispersion and circular-dichroism are the two well-developed methods for probing chirality. However, their weak contrast, poor optical sectioning, and low penetration depth constrain its application to study chirality in tissues and real bio-samples. Therefore, previous research has been mostly limited to surfaces or solutions.
In contrast to linear optics, there are several nonlinear optical activity effects in chiral materials, such as vibrational circular dichroism, Raman optical activity, two-photon absorption circular dichroism, and second-harmonic generation circular-dichroism (SHG-CD). The last one is the most studied nonlinear chiral effect since it shows significantly improved chiral contrast. An additional advantage of SHG-CD is its intrinsic optical sectioning due to nonlinearity. When combined with an infrared excitation, SHG-CD has been demonstrated to provide high penetration depth for three-dimensional imaging. However, in recent studies, the signal origin of SHG-CD in biological tissue is ambiguous, since not only chirality, but also the anisotropy of molecules contribute to SHG-CD response. It will be of great importance to find an experimental skill that can distinguish the contribution between these two mechanisms.
Here we studied SHG-CD of collagen, which is the most abundant protein in human body. Inspired by linear CD where resonant wavelength is required to reveal chirality, we have carried out nonlinear microspectroscopy measurement and shown that when the excitation meets the resonant band of collagen, chirality-induced SHG-CD is strongly enhanced and can be easily identified versus the anisotropy-induced contribution. By slowly heating up the sample, we have further verified that there is a wavelength-independent anisotropy contribution of SHG-CD vanishing at around 40 – 50 degree Celsius, while the resonance-enhanced chirality component of SHG-CD remains until temperature rise to 60 degree. Our results feature the first quantitative identification of chirality-induced SHG-CD in an intact biological tissue, and will be a critical step toward nonlinear chiral microscopy.
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