Superresolved polarization-enhanced second-harmonic generation for direct imaging of nanoscale changes in collagen architecture

• Published in: Optica 2021-05, Vol.8 (5), p.674-685
• Main Authors: Johnson, Peter B, Karvounis, Artemios, Singh, H Johnson, Brereton, Christopher J, Bourdakos, Konstantinos N, Lunn, Kerry, Roberts, James J W, Davies, Donna E, Muskens, Otto L, Jones, Mark G, Mahajan, Sumeet
• Format: Article
• Language: English
• ISSN: 2334-2536; 2334-2536
• DOI: 10.1364/OPTICA.411325

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.