Superresolution (SR) optical microscopy has allowed the investigation of many biological structures below the diffraction limit; however, most of the techniques are hampered by the need for fluorescent labels. Nonlinear label-free techniques such as second-harmonic generation (SHG) provide structurally specific contrast without the addition of exogenous labels, allowing observation of unperturbed biological systems. We use the photonic nanojet (PNJ) phenomena to achieve SR-SHG. A resolution of ${\sim}\lambda /{{6}}$ with respect to the fundamental wavelength, that is, a ${\sim}{2.3}$-fold improvement over conventional or diffraction-limited SHG under the same imaging conditions is achieved. Crucially we find that the polarization properties of excitation are maintained in a PNJ. This is observed in experiment and simulations. This may have widespread implications to increase sensitivity by detection of polarization-resolved SHG by observing anisotropy in signals. These new, to the best of our knowledge, findings allowed us to visualize biological SHG-active structures such as collagen at an unprecedented and previously unresolvable spatial scale. Moreover, we demonstrate that the use of an array of self-assembled high-index spheres overcomes the issue of a limited field of view for such a method, allowing PNJ-assisted SR-SHG to be used over a large area. Dysregulation of collagen at the nanoscale occurs in many diseases and is an underlying cause in diseases such as lung fibrosis. Here we demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to nondestructively image SHG-active biological structures without labels at the nanoscale with a relatively simple optical method heralds the promise of a new tool to understand biological phenomena and drive drug discovery.

中文翻译：

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超分辨偏振增强二次谐波产生，用于胶原结构纳米级变化的直接成像


超分辨率（SR）光学显微镜可以在衍射极限以下研究许多生物结构；然而，大多数技术都因需要荧光标记而受到阻碍。二次谐波产生 (SHG) 等非线性无标记技术可提供结构特异性对比度，无需添加外源标记，从而可以观察未受干扰的生物系统。我们利用光子纳米喷射（PNJ）现象来实现 SR-SHG。相对于基波波长的分辨率为${\sim}\lambda /{{6}}$ ，即在相同条件下比传统或衍射限制的 SHG 提高${\sim}{2.3}$倍达到成像条件。至关重要的是，我们发现 PNJ 中保持了激发的偏振特性。这是在实验和模拟中观察到的。这可能对通过观察信号中的各向异性来检测偏振分辨二次谐波来提高灵敏度具有广泛的影响。据我们所知，这些新发现使我们能够在前所未有的、以前无法解析的空间尺度上可视化生物二次谐波活性结构，例如胶原蛋白。此外，我们证明使用自组装高折射率球体阵列克服了这种方法的视场有限的问题，允许 PNJ 辅助的 SR-SHG 在大面积上使用。纳米级胶原蛋白的失调发生在许多疾病中，并且是肺纤维化等疾病的根本原因。在这里，我们证明 pSR-SHG 可以前所未有地观察到传统衍射限制 SHG 成像无法看到的纳米级变化。 利用相对简单的光学方法在纳米尺度上对二次谐波活性生物结构进行非破坏性成像而无需标记的能力预示着一种理解生物现象和推动药物发现的新工具的前景。