Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture
Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture
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DOI：
10.1101/2020.02.07.934000
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发表时间：
2020-02
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通讯作者：
P. Johnson;A. Karvounis;H. J. Singh;C. Brereton;K. Bourdakos;Kerry Lunn;J. Roberts;D. Davies;O. Muskens;Mark G. Jones;S. Mahajan
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作者：
P. Johnson;A. Karvounis;H. J. Singh;C. Brereton;K. Bourdakos;Kerry Lunn;J. Roberts;D. Davies;O. Muskens;Mark G. Jones;S. Mahajan
超分辨率（SR）光学显微镜已经允许研究许多低于衍射极限的生物结构，然而，大多数技术都受到荧光标记的需要的阻碍。非线性无标签技术，如二次谐波生成（SHG）提供结构特异性对比，而无需添加外源标签，允许观察未受干扰的生物系统。本文首次实现了超分辨率SHG （SR-SHG）。我们利用光子纳米射流（PNJ）现象实现了相对于基本波长的~λ/6的分辨率，在相同成像条件下，比衍射受限的SHG提高了~2.7倍。关键的是，我们发现PNJ中激发的偏振特性保持不变，通过观察信号的各向异性来检测偏振分辨SHG (p-SHG)，从而进一步提高了分辨率。这些新发现使我们能够在前所未有的、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的高折射率球体阵列克服了这种方法的视野有限的问题，使pnj辅助的SR-SHG能够在更大的区域内使用。纳米尺度的胶原蛋白失调发生在许多疾病中，是肺纤维化等疾病的潜在原因。在这里，我们能够证明pSR-SHG可以在纳米尺度上前所未有地观察到传统衍射受限SHG成像无法观察到的变化。利用一种相对简单的光学方法在纳米尺度上无标记地对shg活性生物结构进行无损成像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。
Super-resolution (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. Non-linear 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. Here we achieve super-resolution SHG (SR-SHG) for the first time. We use the photonic nanojet (PNJ) phenomena to achieve a resolution of ~λ/6 with respect to the fundamental wavelength, a ~2.7-fold improvement over diffraction-limited SHG under the same imaging conditions. Crucially we find that the polarisation properties of excitation are maintained in a PNJ allowing the resolution to be further enhanced by detection of polarisation-resolved SHG (p-SHG) by observing anisotropy in signals. These new findings allowed us to visualise 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 are able to demonstrate that pSR-SHG allows unprecedented observation of changes at the nanoscale that are invisible by conventional diffraction-limited SHG imaging. The ability to non-destructively 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.