Basic and Translational Science

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.

Microplastic pollution is an urgent global issue. While spectroscopic techniques have been widely used for the identification of plastics collected from aquatic environments, these techniques are often labor-intensive and time-consuming due to sample collection, preparation, and long measurement times. In this study, a method for the two-dimensional detection and classification of flowing microplastic and organic biotic particles with high spatial and temporal resolutions has been proposed based on the simultaneous detection of coherent anti-Stokes Raman scattering (CARS) and two-photon excited autofluorescence (TPEAF) signals. Poly(methyl methacrylate) (PMMA), polystyrene (PS), and low-density polyethylene (LDPE) particles with sizes ranging from several tens to hundreds of micrometers were selectively detected in flow with an average velocity of 4.17 mm/s by CARS line scanning. With the same flow velocity, flowing PMMA and alga particles were measured using a multimodal system of CARS and TPEAF signals. The average intensities of both PMMA and alga particles in the CARS signals at a frequency of 2940 cm were higher than the background level, while only algae emitted TPEAF signals. This allowed the classification of PMMA and alga particles to be successfully performed in flow by the simultaneous detection of CARS and TPEAF signals. With the proposed method, the monitoring of microplastics in a continuous water flow without collection or extraction is possible, which is game-changing for the current sampling-based microplastic analysis.

Collagen assembly during development is essential for successful matrix mineralisation, which determines bone quality and mechanocompetence. However, the biochemical and structural perturbations that drive pathological skeletal collagen configuration remain unclear. Deletion of vascular endothelial growth factor (VEGF) in bone forming osteoblasts (OBs) induces sex-specific alterations in extracellular matrix (ECM) conformation and mineralisation coupled to vascular changes, which are augmented in males. Whether this phenotypic dimorphism arises as a result of the divergent control of ECM composition and its subsequent arrangement is unknown and is the focus of this study. Herein, we have used a murine osteocalcin-specific knockout (OcnVEGFKO) and performed multiscale analysis at the tibiofibular junction of both sexes. Furthermore, we also deleted in OBs extracted from male and female mice in an attempt to link sex-specific matrix signatures to deviations in gene expression. Label-free and non-destructive polarisation-resolved second harmonic generation microscopy (p-SHG) revealed a reduction in collagen fibre number in males following the loss of VEGF, complemented by observable defects in matrix organisation by backscattered electron scanning electron microscopy. This was accompanied only in males by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-stand stability, in addition to matrix carbonation and mineralisation. V deletion in male and female murine OB cultures further highlighted divergence in genes regulating local ECM structure including , , and The current results demonstrate the utility of macromolecular imaging and spectroscopic modalities for the detection of collagen arrangement and ECM composition in pathological bone. Linking the sex-specific genetic regulators to matrix signatures could be important for treatment of dimorphic bone disorders which clinically manifest in both pathological nano and macro-level disorganisation.

We demonstrate a continuous wave (CW) seeded synchronization-free optical parametric amplifier (OPA) pumped by a picosecond, 1 µm laser and show its performance when used as a simple yet powerful source for label-free coherent anti-Stokes Raman scattering (CARS), concurrent second harmonic generation (SHG), and two-photon fluorescence microscopy in an epi-detection geometry. The average power level of above 175 mW, spectral resolution of 8 cm, and 2 ps pulse duration are well optimized for CARS microscopy in bio-science and bio-medical imaging systems. Our OPA is a much simpler setup than either the "gold-standard" laser and optical parametric oscillator (OPO) combination traditionally used for CARS imaging, or the more recently developed OPA systems pumped with femtosecond pulses [1]. Rapid and accurate tuning between resonances was achieved by changing the poled channels and temperature of the periodically-poled lithium niobate (PPLN) OPA crystal together with the OPA seed wavelength. The Pump-Stokes frequency detuning range fully covered the C-H stretching band used for the imaging of lipids. By enabling three multiphoton techniques using a compact, synchronization free laser source, our work paves the way for the translation of label-free multi-photon microscopy imaging from biomedical research to an imaging based diagnostic tool for use in the healthcare arena.

Tauopathies are a group of disorders in which the deposition of abnormally folded tau protein accompanies neurodegeneration. The development of methods for detection and classification of pathological changes in protein conformation are desirable for understanding the factors that influence the structural polymorphism of aggregates in tauopathies. We have previously demonstrated the utility of Raman spectroscopy for the characterization and discrimination of different protein aggregates, including tau, based on their unique conformational signatures. Building on this, in the present study, we assess the utility of Raman spectroscopy for characterizing and distinguishing different conformers of the same protein which in the case of tau are unique tau strains generated . We now investigate the impact of aggregation environment, cofactors, post-translational modification and primary sequence on the Raman fingerprint of tau fibrils. Using quantitative conformational fingerprinting and multivariate statistical analysis, we found that the aggregation of tau in different buffer conditions resulted in the formation of distinct fibril strains. Unique spectral markers were identified for tau fibrils generated using heparin or RNA cofactors, as well as for phosphorylated tau. We also determined that the primary sequence of the tau monomer influenced the conformational signature of the resulting tau fibril, including 2N4R, 0N3R, K18 and P301S tau variants. These results highlight the conformational polymorphism of tau fibrils, which is reflected in the wide range of associated neurological disorders. Furthermore, the analyses presented in this study provide a benchmark for the Raman spectroscopic characterization of tau strains, which may shed light on how the aggregation environment, cofactors and post-translational modifications influence tau conformation in future studies.