Superresolved polarization-enhanced second-harmonic generation for direct imaging of nanoscale changes in collagen architecture.
Superresolved polarization-enhanced second-harmonic generation for direct imaging of nanoscale changes in collagen architecture.
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DOI：
10.1364/optica.411325
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发表时间：
2021-05-20
期刊：
影响因子：
10.4
通讯作者：
Mahajan S
中科院分区：
文献类型：
--
作者：
Johnson PB;Karvounis A;Singh HJ;Brereton CJ;Bourdakos KN;Lunn K;Roberts JJW;Davies DE;Muskens OL;Jones MG;Mahajan S
超分辨率（SR）光学显微镜已经允许在衍射极限以下的许多生物结构的调查，然而，大多数的技术是由荧光标记的需要受到阻碍。非线性无标记技术，如二次谐波产生（SHG）提供结构特异性对比，而无需添加外源性标记，允许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，即在相同的成像条件下，比传统的或衍射受限的SHG提高了一倍。关键的是，我们发现，激发的偏振特性保持在PNJ。这在实验和模拟中观察到。这可能具有广泛的影响，以增加灵敏度的偏振分辨的SHG检测通过观察信号中的各向异性。据我们所知，这些新的发现使我们能够在前所未有的和以前无法解决的空间尺度上可视化生物SHG活性结构，如胶原蛋白。此外，我们证明了使用自组装高折射率球阵列克服了这种方法的视场有限的问题，允许PNJ辅助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，并且是诸如肺纤维化等疾病的根本原因。在这里，我们证明了pSR-SHG允许前所未有的观察纳米级的变化，这是不可见的常规衍射限制SHG成像。用相对简单的光学方法在纳米级无标记的情况下对SHG活性生物结构进行非破坏性成像的能力预示着一种新工具的前景，以了解生物现象并推动药物发现。
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 with respect to the fundamental wavelength, that is, a -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.