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

Abstract

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.

Microlens‐assisted microscopy for biology and medicine

“…The polarization dependence of SHG can be used to differentiate different types of collagen and to determine the degree of collagen ordering in tissues. The authors [82] showed that the polarization of light is retained in the PNJ, which makes it possible to carry out polarization-sensitive SHG microscopy with superresolution.…”

“…In article [82], microlenses were used to obtain superresolution in second‐harmonic generation microscopy (SHG microscopy) of collagen fibers in lung tissue samples and spheroid sections. A resolution of 125 nm ( λ /6.4) was achieved using BTG microspheres (5–22 μm and 53–63 μm) with a RI of 1.9 in water immersion liquid.…”

Self-Sensing Scanning Superlens for Three-Dimensional Noninvasive Visible-Light Nanoscale Imaging on Complex Surfaces

“…Microspheres positioned on the surface of a sample can transform near-field evanescent waves into far-field waves, allowing the detection of higher-frequency information. The most unique advantage is the label-free and real-time imaging, which has potential applications in materials research , and life science, − , for example, observation of a 75 nm adenovirus under white light, , direct observation of the Brownian motion of 300 nm transparent particles in water, and high-throughput crack and defect analysis in wafers. , Many attempts have been made to increase the resolution and image quality of superlenses based on microspheres; − when these superlenses are combined with scanning laser confocal microscopy, the resolution can be attained at 25 nm . However, the fundamental disadvantage of microsphere-based superlens imaging is that the imaging region is fixed and imaging area constrained by the size of the microsphere .…”

Super-Resolution Second-Harmonic Generation Imaging with Multifocal Structured Illumination Microscopy

“…12−14 However, the development of super-resolution techniques for SHG imaging is rare. Johnson et al employed dielectric spheres placed on a sample's surface to focus light into the photonic nanojet (PNJ) phenomena for achieving SHG imaging beyond the diffraction limit, 15 but optimal resolution and field of view were realized only at a specific Z-axis position. Based on image scanning microscopy, Gregor et al achieved super-resolved SHG images through digital image processing on excessively large image recordings by single-focus scanning.…”

“…Super-resolution techniques such as stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM) have been developed to exceed the conventional diffraction limit in fluorescence microscopy. − However, the development of super-resolution techniques for SHG imaging is rare. Johnson et al employed dielectric spheres placed on a sample’s surface to focus light into the photonic nanojet (PNJ) phenomena for achieving SHG imaging beyond the diffraction limit, but optimal resolution and field of view were realized only at a specific Z -axis position. Based on image scanning microscopy, Gregor et al achieved super-resolved SHG images through digital image processing on excessively large image recordings by single-focus scanning .…”