Biology subjects

Johnson, P. B.

Publications and source records attributed to Johnson, P. B..

3 recordsLinked to original sources

bioRxiv2025

Characterising Type I Collagen Architecture in Human Bone and Osteosarcoma with Second Harmonic Generation Microscopy

Aberrant collagen matrix production is a hallmark of many cancers, including osteosarcoma (OS), the most common primary cancer of bone which remains poorly diagnosed. Here, the phenotyping potential of second harmonic generation (SHG) microscopy has been demonstrated through label-free imaging of pathologically altered collagen matrices within clinical biopsies. This work examined SHG imaging in quantifying OS-specific collagen (type I) signatures - focused on fibre length-based parameters - across clinical biopsies. We describe a novel SHG analysis workflow, following systematic optimisation of image resolution and field of view (FOV) size to enable robust quantification of collagen fibre-length metrics in human bone and OS. This included analyses of SHG images with a FOV spanning 350 {micro}m x 350 {micro}m to 1400 {micro}m x 1400 {micro}m, with the latter enabling whole-biopsy visualisation via stitching of up to 64 regions. In normal cortical (rib) bone biopsies, average fibre lengths did not significantly differ across increasing FOV size (26.44 {+/-} 1.75 {micro}m, 26.34 {+/-} 2.01 {micro}m, 27.25 {+/-} 2.47 {micro}m, 28.78 {+/-} 3.58 {micro}m). However, analysis of the top 50th percentile of collagen fibres revealed significant differences between the smallest FOVs; 350 {micro}m x 350 {micro}m and 466 {micro}m x 466 {micro}m, versus the largest 1400 {micro}m x 1400 {micro}m (p = 0.03 and p = 0.04 respectively), highlighting the requirement for larger FOVs for optimal characterisation of human bone collagen. Comparison of normal bone and OS (stage IIB) biopsies, the largest FOV revealed the largest differences, with OS exhibiting significantly reduced collagen fibre lengths (female OS: 23.18 {+/-} 0.56 {micro}m vs. bone: 29.39 {+/-} 2.17 {micro}m, p < 0.001; male OS: 24.39 {+/-} 1.32 {micro}m vs. bone: 27.87 {+/-} 1.05 {micro}m, p = 0.02) versus normal bone. Subsequent analyses across OS stages (IB - IVB) showed further evidence of pathological collagen signatures amplified with OS progression. These findings demonstrate that SHG microscopy enables robust, quantitative phenotyping of human bone and osteosarcoma collagen matrices, offering new potential for improved diagnosis and disease staging.

cancer biology↗

bioRxiv2023

Mechanisms of SARS-CoV-2 Inactivation using UVC Laser Radiation

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has had a tremendous impact on humanity. Prevention of transmission by disinfection of surfaces and aerosols through a chemical-free method is highly desirable. Ultraviolet C (UVC) light is uniquely positioned to achieve inactivation of pathogens. We report the inactivation of SARS-CoV-2 virus by UVC radiation and explore its mechanisms. A dose of 50mJ/cm2 using a UVC laser at 266nm achieved an inactivation efficiency of 99.89%, whilst infectious virions were undetectable at 75mJ/cm2 indicating >99.99% inactivation. Infection by SARS-CoV-2 involves viral entry mediated by the spike glycoprotein (S), and viral reproduction, reliant on translation of its genome. We demonstrate that UVC radiation damages ribonucleic acid (RNA) and provide in-depth characterisation of UVC-induced damage of the S protein. We find that UVC severely impacts SARS-CoV-2 spike proteins ability to bind human angiotensin-converting enzyme 2 (hACE2) and this correlates with loss of native protein conformation and aromatic amino acid integrity. This report has important implications for the design and development of rapid and effective disinfection systems against the SARS-CoV-2 virus and other pathogens.

microbiology↗

bioRxiv2020

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

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 ~{lambda}/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.

biophysics↗