Super-resolved polarisation-enhanced second harmonic generation for direct imaging of nanoscale changes in collagen architecture

Abstract

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. List of abbreviationsSecond Harmonic Generation (SHG), Super-resolution (SR), Barium Titanate Glass (BTG), Photonic Nanojet (PNJ), Point Spread Function (PSF) Calculation of Polarisation anisotropyPolarisation anisotropy images were created using a home written Fiji macro in which the key steps are as follows: A difference image was generated by (Ipar -Iperp), this was divided by a total intensity image (Ipar + 2Iperp). The total intensity image was used to select the region of the image containing signal and for display all pixels outside of this region were set to 0. Only the regions containing signal were used for further anisotropy analysis. Collagen fibre analysisCollagen fibre analysis was performed using CT-FIRE V1.3. SHG intensity images were converted to 8bit format before being batch processed using default parameters. Four parameters were measured to describe fibre morphology, width, length, straightness and angle.

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition

“…Polarisation-resolved second-harmonic generation (p-SHG) is a powerful non-destructive multiphoton modality that has previously been utilised for the study of collagen architecture and fibrillar orientation ( Stoller et al, 2002 ). This technique has been used to inform and assess the pathological consequence of disease on extracellular matrix (ECM) structure and conformation in a variety of cancers ( Campbell et al, 2018 ; Chen et al, 2012 ; Tilbury et al, 2014b ; Tokarz et al, 2019 ), fibrosis ( James et al, 2019 ; Johnson et al, 2020 preprint; Lin et al, 2013 ; Rouède et al, 2017 ; Tilbury et al, 2014a ) and in skin ( Ducourthial et al, 2019 ; Fukushima et al, 2018 ; Su et al, 2009 ). Furthermore, p-SHG has been applied to investigate collagenous alterations that accompany ageing ( Van Gulick et al, 2019 ), wound repair and scar formation ( Mostaço-Guidolin et al, 2017 ).…”

Barium Titanate Nanostructures and Thin Films for Photonics

“…BaTiO 3 nanoparticles, as part of the more general category of harmonic nanoparticles (HNP), are a promising candidate as markers for bioimaging. [181][182][183] In comparison to quantum dots or molecular dyes, the SHG signal from harmonic nanoparticles is nonblinking and nonbleaching. SHG has a narrower bandwidth that the excitation, which allows for excellent separation between the excitation and emission.…”