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Tauopathies including Alzheimer's disease, are characterized by progressive cognitive decline, neurodegeneration, and intraneuronal aggregates comprised largely of the axonal protein Tau. It has been unclear whether cognitive deficits are a consequence of aggregate accumulation thought to compromise neuronal health and eventually lead to neurodegeneration. We use theDrosophilatauopathy model and mixed-sex populations to reveal an adult onset pan-neuronal Tau accumulation-dependent decline in learning efficacy and a specific defect in protein synthesis-dependent memory (PSD-M), but not in its protein synthesis-independent variant. We demonstrate that these neuroplasticity defects are reversible on suppression of new transgenic human Tau expression and surprisingly correlate with an increase in Tau aggregates. Inhibition of aggregate formation via acute oral administration of methylene blue results in re-emergence of deficient memory in animals with suppressed human Tau (hTau)0N4Rexpression. Significantly, aggregate inhibition results in PSD-M deficits in hTau0N3R-expressing animals, which present elevated aggregates and normal memory if untreated with methylene blue. Moreover, methylene blue–dependent hTau0N4Raggregate suppression within adult mushroom body neurons also resulted in emergence of memory deficits. Therefore, deficient PSD-M on human Tau expression in theDrosophilaCNS is not a consequence of toxicity and neuronal loss because it is reversible. Furthermore, PSD-M deficits do not result from aggregate accumulation, which appears permissive, if not protective of processes underlying this memory variant.SIGNIFICANCE STATEMENTIntraneuronal Tau aggregate accumulation has been proposed to underlie the cognitive decline and eventual neurotoxicity that characterizes the neurodegenerative dementias known as tauopathies. However, we show in three experimental settings that Tau aggregates in theDrosophilaCNS do not impair but rather appear to facilitate processes underlying protein synthesis-dependent memory within affected neurons.

We have developed a method to combine morphological and chemical information for the accurate identification of different particle types using optical measurement techniques that require no sample preparation. A combined holographic imaging and Raman spectroscopy setup is used to gather data from six different types of marine particles suspended in a large volume of seawater. Unsupervised feature learning is performed on the images and the spectral data using convolutional and single-layer autoencoders. The learned features are combined, where we demonstrate that non-linear dimensional reduction of the combined multimodal features can achieve a high clustering macro F1 score of 0.88, compared to a maximum of 0.61 when only image or spectral features are used. The method can be applied to long-term monitoring of particles in the ocean without the need for sample collection. In addition, it can be applied to data from different types of sensor measurements without significant modifications.

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/cm2using a UVC laser at 266nm achieved an inactivation efficiency of 99.89%, whilst infectious virions were undetectable at 75mJ/cm2indicating >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 protein’s 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.

Multiphoton microscopies are an invaluable tool in biomedical imaging given their inherent capabilities for label free imaging, optical sectioning, chemical and structural specificity. They comprise various types of Coherent Raman microscopies (CR), such as Coherent Anti-Stokes Raman Scattering (CARS), Stimulated Raman Loss (SRL) or Stimulated Raman Gain, different kinds of Harmonic Generation imaging (HG) such as Second and Third Harmonic Generation (SHG and THG respectively), and Multiphoton Autofluorescence imaging (MA) such as Two and Three Photon Excited Autofluorescence (TPEAF and ThPEAF respectively). Despite their significant advantages, multiphoton microscopies, comparably to all other types of optical microscopies, exhibit limited penetration depth in tissue due to absorption and scattering. In this work we explore the advantages of multiphoton microscopies in hard and soft deep tissue imaging when using excitation wavelengths in the range of Short-Wavelength Infrared (SWIR) windows which occur between 1000 nm and 2500 nm. These spectral windows have notable merits including longer attenuation lengths and none or very low signal absorption observed for almost all kinds of multiphoton microscopy. We show results of using excitations in the SWIR windows, generated by standard as well as novel sources, such as a thulium fibre laser, in different types of multiphoton microscopy on a variety of hard and soft tissue samples (bone, cartilage and other tissue types) and demonstrate the advantages of using excitations in this wavelength range, including longer penetration depth and high resolution for deep tissue imaging.

In this paper we describe our recent work in multi-excitation surface enhanced Raman spectroscopy (MX-SERS), and its application for robust strain-level bacteria identification. The development of MX-SERS follows directly from our previous work in rapid bacterial identification using multi-excitation Raman spectroscopy (MX-Raman), which enabled highly accurate (up to 99.75%) strain-level distinction of bacteria, including antibiotic resistant strains of bacteria and from within complex media. In this work we use the strong wavelength dependence of both the Raman scattering cross-section and the surface plasmon to demonstrate a novel capability in bacteria identification. Compared to MX-Raman, MX-SERS has up to 8x faster data acquisition speed as well as up to 4000x lower laser power incident on the sample. Furthermore, we fabricate SERS-active substrates with a simple and low-cost fabrication method that can be adapted to fit a chosen wavelength regime. This combination of strain-level sensitivity and high-speed detection, combined with a low-cost SERS substrate, has strong potential applications in clinical diagnostics, and could be integrated within a real-world pathogen detection workflow. This presents new capabilities in label-free bacterial detection including novel culture-free investigation capabilities, and an improved methodology for sample handling with minimal preparation and processing.

We present a topological method for the detection and quantification of bone microstructure from non-linear microscopy images. Specifically, we analyse second harmonic generation (SHG) and two photon excited autofluorescence (TPaF) images of bone tissue which capture the distribution of matrix (fibrillar collagen) structure and autofluorescent molecules, respectively. Using persistent homology statistics with a signed Euclidean distance transform filtration on binary patches of images, we are able to quantify the number, size, distribution, and crowding of holes within and across samples imaged at the microscale. We apply our methodology to a previously characterized murine model of skeletal pathology whereby vascular endothelial growth factor expression was deleted in osteocalcin-expressing cells (OcnVEGFKO) presenting increased cortical porosity, compared to wild type (WT) littermate controls. We show significant differences in topological statistics between the OcnVEGFKO and WT groups and, when classifying the males, or females respectively, into OcnVEGFKO or WT groups, we obtain high prediction accuracies of 98.7% (74.2%) and 77.8% (65.8%) respectively for SHG (TPaF) images. The persistence statistics that we use are fully interpretable, can highlight regions of abnormality within an image and identify features at different spatial scales.

Background Scrotal defects in developing countries are common challenges for the reconstructive surgeon and hence this work has been done with the aim to compare the outcome, advantages and disadvantages of different modalities of scrotal reconstruction. Methods The prospective observational hospital-based study of reconstruction of scrotal defects following trauma and Fournier's gangrene was done over a period of three years. Scrotal defect reconstruction was done in 35 patients by scrotal advancement flap, split thickness skin grafting, medial thigh flap, anterolateral thigh flap and groin flap keeping in mind the various indication of different modalities. The reconstructed scrotums were observed for flap survival and skin graft intake for seven to 10 days in the hospital and then were followed for three months in a follow-up clinic. Results and observations The mean age of our patients was 48.57±5.01 years. Most of the soft tissue defects of the scrotum were post-traumatic (83%). Scrotal reconstruction was done often by flaps and more frequently used flap for reconstruction of scrotum was scrotal advancement flap. All flaps and grafts survived well. Mean hospitalization time was highest for groin flap cover whereas mean operative time was highest for anterolateral thigh flap cover. Conclusion Every case of scrotal defect needs an individual approach for scrotal reconstruction depending upon patient age, general condition of the patient, wound status, and the patient's requirement.

Current methods for diagnosing acute and complex infections mostly rely on culture-based methods and, for biofilms, fluorescencein-situhybridization. These techniques are labor-intensive and can take 2-4 days to return a test result, especially considering an extra culturing step required for the antibiotic susceptibility testing (AST). This places a significant burden on healthcare providers, delaying treatment and leading to adverse patient outcomes. Here, we report the complementary use of our newly developed multi-excitation Raman spectroscopy (ME-RS) method with whole-genome sequencing (WGS). Four WHO priority pathogens are AST phenotyped and their antimicrobial resistance (AMR) profile determined by WGS. On application of ME-RS method we find high correlation with the WGS characterization. Highly accurate classification based on the species (98.93%), wild-type/non-wild type (99.45%), and presence or absence of thick peptidoglycan layers in cell walls (100%), as well as at the individual strain level (99.29%). These results clearly demonstrate the potential of ME-RS as a rapid and first-stage tool for species, resistance and strain-level classification which can be followed up by WGS for confirmation. Such a workflow can facilitate efficient antimicrobial stewardship to handle and prevent the spread of AMR.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. The current methods for diagnosis of acute and chronic infections are complex and skill-intensive. For complex clinical biofilm infections, it can take days from collecting and processing a patient’s sample to achieving a result. These aspects place a significant burden on healthcare providers, delay treatment, and can lead to adverse patient outcomes. We report the development and application of a novel multi-excitation Raman spectroscopy-based methodology for the label-free and non-invasive detection of microbial pathogens that can be used with unprocessed clinical samples directly and provide rapid data to inform diagnosis by a medical professional. The method relies on the differential excitation of non-resonant and resonant molecular components in bacterial cells to enhance the molecular finger-printing capability to obtain strain-level distinction in bacterial species. Here, we use this strategy to detect and characterize the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus as typical infectious agents associated with cystic fibrosis. Planktonic specimens were analyzed both in isolation and in artificial sputum media. The resonance Raman components, excited at different wavelengths, were characterized as carotenoids and porphyrins. By combining the more informative multi-excitation Raman spectra with multivariate analysis (support vector machine) the accuracy was found to be 99.75% for both species (across all strains), including 100% accuracy for drug-sensitive and drug-resistant S. aureus. The results demonstrate that our methodology based on multi-excitation Raman spectroscopy can underpin the development of a powerful platform for the rapid and reagentless detection of clinical pathogens to support diagnosis by a medical expert, in this case relevant to cystic fibrosis. Such a platform could provide translatable diagnostic solutions in a variety of disease areas and also be utilized for the rapid detection of anti-microbial resistance.

Melioidosis caused by the facultative intracellular pathogen Burkholderia pseudomallei is difficult to treat due to poor intracellular bioavailability of antibiotics and antibiotic resistance. In the absence of novel compounds, polymersome (PM)-encapsulation may increase the efficacy of existing antibiotics and reduce antibiotic resistance by promoting targeted, infection-specific intracellular uptake. In this study we developed PMs composed of widely available polyethylene oxide-polycaprolactone (PEO-PCL) block co-polymers and demonstrated their delivery to intracellular B. thailandensis infection using multispectral imaging flow cytometry (IFC) and coherent anti-Stokes Raman scattering (CARS) microscopy. Antibiotics were tightly sequestered in PMs and did not inhibit the growth of free-living B. thailandensis. However, on uptake of antibiotic-loaded PMs by infected macrophages, IFC demonstrated PM co-localisation with intracellular B. thailandensis and a significant inhibition of their growth. We conclude that PMs are a viable approach for the targeted antibiotic treatment of persistent intracellular Burkholderia infection.

Consistently raised levels of inflammatory marker - Neopterin, in urine is linked to increased risk of progression of age-related disease and poorer prognosis. We have developed colloidal SERS sensors and demonstrate their ability, and ease of use, for quantification of neopterin in human urine samples. Results with the sensors are comparable and in agreement with those obtained by HPLC and allow individuals to be stratified into ‘risk’ categories based on their results. With an average 17.85% difference in results between the two analytical approaches, SERS with colloidal sensors, demonstrates an alternative method that is rapid, inexpensive, requires minimal sample treatment, can be performed on a portable instrument with little need for complex data analysis, whilst having the analytical strength to reliably demonstrate an individual’s risk category based on inflammatory load.

Multiphoton imaging methods such as Coherent Raman Scattering (CRS) microscopy which also comprises Second Harmonic Generation (SHG) and Two Photon Excited Auto-Fluorescence (TPEAF) imaging (termed as multimodal Coherent Raman microscopy), have greatly facilitated the advancement of biomedical research due to their unique features. Multimodal CRS microscopy, is label free, chemically specific, inherently ‘confocal’ offering three independent contrast mechanisms which can be associated in a composite image comprising a wide range of chemical and structural information about the interrogated sample. The standard light source for multimodal CRS microscopy is a picosecond pumped Optical Parametric Oscillator (OPO) which has exhibited excellent performance but due to its associated high cost, maintenance, complexity and requirement of a dedicated optics laboratory, has hindered the wider adoption of multimodal CRS microscopy and especially its deployment in clinical applications. Here we present a novel, low cost Optical Parametric Amplifier (OPA) based on a MgO doped Periodically Poled Lithium Niobate (PPLN) crystal seeded by a continuous wave (CW) laser source and pumped by a picosecond laser at 1031nm, which removes any synchronisation requirements. We show that this OPA is a versatile light source module that can be tailored to the tunability and affordability requirements of the specific application. We demonstrate that it can be used either in association with an OPO or on its own as a light source for multimodal CRS microscopy and we show its performance by imaging a variety of standards and biological samples.

This paper describes the effect of basement excavation and foundation loading on tunnel in sand. Typical tunnel geometry of Delhi Metro and soil conditions in part of Delhi (Yamuna Sand) are considered for modelling. The construction sequences of tunnel excavations, basement footing with retaining wall and the loading on footing have been numerically simulated using plane strain assumption in PLAXIS 2D. Basement foundation, retaining wall, sheet pile and support struts are modelled simulating the construction sequence that is normally adopted in practice. The soil is assumed as elastic-perfectly plastic material, and its failure is governed by Mohr–Coulomb criterion. The tunnel lining, footing, sheet pile, wall and struts are assumed as linear elastic materials. The loading on the footing is modelled in several stages simulating different numbers of storeys for studying the effect of basement excavation and structure/foundation loading. The results indicate that the response of tunnel lining is significantly affected by various stages of basement excavation and foundation loading. Based on the several response parameters of tunnel lining, it is concluded that the critical distance between the tunnel edge and basement footing is found as 2.5 times the tunnel diameter within which the basement excavation and foundation loading have shown significant influence on tunnel response.

Microplastic pollution is an urgent global issue. While spectroscopic techniques have been widely used for identification of plastics collected from aquatic environments, these techniques are often labour-intensive and time-consuming due to sample collection, preparation and long measurement times. In this study, a method for two-dimensional detection and classification of flowing microplastic and organic particles with high spatial and temporal resolutions has been proposed based on 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 the size of several tens to hundreds of µm were selectively detected in flow with an average velocity of 4.17 mm/s by CARS line scanning. With the same velocity of flow, flowing PMMA and alga particles were measured using a multimodal system of CARS and TPEAF signals. The average intensity of both PMMA and alga particles in the CARS signals at the frequency of 2940 cm−1 were higher than the background level, while only algae emit TPEAF signals. Therefore, classification of PMMA and alga particles in flow has been successfully performed by simultaneous detection of CARS and TPEAF signals. With the proposed method, monitoring of microplastics in continuous water flow without collection or extraction is possible, which is game-changing for 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; also known as VEGFA) 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 used murine osteocalcin-specific Vegf knockout (OcnVEGFKO) and performed ex vivo multiscale analysis at the tibiofibular junction of both sexes. Label-free and non-destructive polarisation-resolved second-harmonic generation (p-SHG) microscopy 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 by localised divergence in collagen orientation, determined by p-SHG anisotropy measurements, as a result of OcnVEGFKO. Raman spectroscopy confirmed that the effect on collagen was linked to molecular dimorphic VEGF effects on collagen-specific proline and hydroxyproline, and collagen intra-strand stability, in addition to matrix carbonation and mineralisation. Vegf deletion in male and female murine OB cultures in vitro further highlighted divergence in genes regulating local ECM structure, including Adamts2, Spp1, Mmp9 and Lama1. Our 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 that clinically manifest in pathological nano- and macro-level disorganisation. This article has an associated First Person interview with the first author of the paper., Summary: Combined application of polarisation-resolved second-harmonic generation microscopy and Raman spectroscopy identified unique, sexually dimorphic extracellular matrix signatures linking pathological matrix disorganisation with composition.

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 in vitro. 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 vivo in future studies., We report that the physiochemical properties of the aggregation environment dictate the conformation of tau strains, which can be characterized and distinguished using Raman spectroscopy.

An optical parametric amplifier (OPA) seeded by a continuous wave laser diode and pumped by a picosecond laser at 1031 nm is presented. The performance of the OPA in multi- photonic microscopy is demonstrated.

Funder: Engineering and Physical Sciences Research Council (EPSRC), Structured metamaterials are periodically arranged nanostructures in which the dielectric constant is periodically modulated on a length‐scale comparable to the desired wavelength of operation. Interactions of the electric fields of light waves with the sub‐wavelength unit structures can produce effects that are impossible in natural materials. Here, a technique to construct three‐dimensional (3D) metamaterials using self‐assembling M13 viral building‐blocks as templates which are then replicated into a metal quasi‐3D nanostructure is developed. By correct fit of virus fragments, it is possible to employ them in a LEGO‐like way to build up well‐defined structures on the nanoscale. The virus blocks are designed to spontaneously assemble into 3D‐periodic network structures with interesting optical properties. Subsequently, templating of these nanostructures into inorganic materials allows the replication of their network into an inverse periodic metal structure, which has the appropriate architecture for optical metamaterials. Establishing such a technique provides an important link toward the realization of applied metamaterials potentially heralding a new era for developing novel types of bio‐synthetic optical materials. These materials have a wide range of potential uses including cloaking materials, light‐storage devices, high‐speed optical computers and nano‐lasers, and will offer numerous applications in transformation optics.

Light sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

A noncontact method to identify sparsely distributed plastic pellets is proposed by integrating holography and Raman spectroscopy in this study. Polystyrene and poly(methyl methacrylate) resin pellets with a size of 3 mm located in a 20 cm water channel were illuminated using a collimated continuous wave laser beam with a diameter of 4 mm and wavelength of 785 nm. The same laser beam was used to take a holographic image and Raman spectrum of a pellet to identify the shape, size, and composition of material. Using the compact system, the morphological and chemical analysis of pellets in a large volume of water was performed. The reported method demonstrates the potential for noncontact continuous in situ monitoring of microplastics in water without collection and separation.

Distinction between closely related and morphologically similar cells is difficult by conventional methods especially without labeling. Using nuclear-targeted gold nanoparticles (AuNPs) as intracellular probes we demonstrate the ability to distinguish between progenitor and differentiated cell types in a human neuroblastoma cell line using surface-enhanced Raman spectroscopy (SERS). SERS spectra from the whole cell area as well as only the nucleus were analyzed using principal component analysis that allowed unambiguous distinction of the different cell types. SERS spectra from the nuclear region showed the developments during cellular differentiation by identifying an increase in DNA/RNA ratio and proteins transcribed. Our approach using nuclear-targeted AuNPs and SERS imaging provides label-free and noninvasive characterization that can play a vital role in identifying cell types in biomedical stem cell research.

Huntington's disease is an inherited fatal progressive neurodegenerative disorder. A possible new Raman ‘spectral’ biomarker was identified that uses a tiny drop of patients' blood serum; thus can have immense diagnostic and therapeutic implications., Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal CAG expansion in exon 1 of the huntingtin (HTT) gene. Given its genetic basis it is possible to study patients both in the pre-manifest and manifest stages of the condition. While disease onset can be modelled using CAG repeat size, there are no easily accessible biomarkers that can objectively track disease progression. Here, we employed a holistic approach using spectral profiles generated using both surface-enhanced Raman spectroscopy (SERS) and Raman Spectroscopy (RS), on the serum of healthy participants and HD patients covering a wide spectrum of disease stages. We found that there was both genotype- and gender-specific segregation on using the full range in the fingerprint region with both SERS and RS. On a more detailed interrogation using specific spectral intervals, SERS revealed significant correlations with disease progression, in particular progression from pre-manifest through to advanced HD was associated with serum molecules related to protein misfolding and nucleotide catabolism. Thus, this study shows the potential of Raman spectroscopy-based techniques for stratification of patients and, of SERS, in particular, to track disease status through provision of ‘spectral’ biomarkers in HD, with clinical applications for other diseases and trials looking at disease modifying therapies.

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. We use the photonic nanojet (PNJ) phenomena to achieve super-resolution SHG (SR-SHG). A resolution of ~λ/6 with respect to the fundamental wavelength, that is, a ~2.3-fold improvement over conventional or diffraction-limited SHG under the same imaging conditions is achieved. Crucially we find that the polarisation 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 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 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.

Principal component analysis of BSA monomer, oligomer and fibril amide I spectra.

Mineralization of bone is achieved by the sequential maturation of the immature amorphous calcium phase to mature hydroxyapatite (HA) and is central in the process of bone development and repair. To study normal and dysregulated mineralization in vitro, substrates are often coated with poly-l-lysine (PLL) which facilitates cell attachment. This study has used Raman spectroscopy to investigate the effect of PLL coating on osteoblast (OB) matrix composition during differentiation, with a focus on collagen specific proline and hydroxyproline and precursors of HA. Deconvolution analysis of murine derived long bone OB Raman spectra revealed collagen species were 4.01-fold higher in OBs grown on PLL. Further, an increase of 1.91-fold in immature mineral species (amorphous calcium phosphate) was coupled with a 9.32-fold reduction in mature mineral species (carbonated apatite) on PLL versus controls. These unique low mineral signatures identified in OBs were linked with reduced alkaline phosphatase enzymatic activity, reduced Alizarin Red staining and altered osteogenic gene expression. The promotion of immature mineral species and restriction of mature mineral species of OB grown on PLL were linked to increased cell viability and pro-angiogenic vascular endothelial growth factor (VEGF) production. These results demonstrate the utility of Raman spectroscopy to link distinct matrix signatures with OB maturation and VEGF release. Importantly, Raman spectroscopy could provide a label-free approach to clinically assess the angiogenic potential of bone during fracture repair or degenerative bone loss., Highlights • Raman spectroscopy directly links distinct osteoblast (OB) extracellular matrix signatures to differentiation status in vitro. • Poly-l-lysine coating restricts primary OB differentiation and mineralization. • Restriction of OB differentiation is associated with a unique matrix signature and enhanced pro-angiogenic VEGF production. • Characterization of a pro-angiogenic signature may be exploited clinically to assess pathological bone matrix composition.

Gold nanoparticles prepared by two different methodologies, wet-chemical- citrate reduction synthesis and femtosecond laser ablation, are studied for optical nonlinearities. Femtosecond laser pulses at 800 nm wavelength are employed to study the optical nonlinear refraction and absorption through the Z-scan technique, up to 23.37 GW/cm2 laser intensities. Here we demonstrate the distinct crossovers of nonlinear absorption, from varieties of nonlinear absorption mechanisms, saturation of linear absorption, reverse saturation of absorption. The reverse saturation of absorption is primarily contributed by an initial two-photon absorption followed by three-photon absorption process. We could observe that in laser-ablated gold nanoparticles, the transition from saturable to reverse saturable absorption takes place at a lower excitation threshold compared to that in chemically produced samples. The nonlinear refraction data depicted a pronounced asymmetry for chemically synthesized samples, which could possibly be due to the dominance of absorption phenomenon. We observed that nonlinear refractive index of chemically produced samples is higher by one order of magnitude compared to those produced by laser ablation. An appealing application in optical limiting is studied, and we see that the laser-produced samples, due to their strong nonlinear absorption, serve as a suitable choice for the optical limiting application. This study provides an insight into the selection of synthesis method towards their optimized utilization in nonlinear optical domain given that we achieve particle size tunability in the two methods.

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 ∼ λ / 6 with respect to the fundamental wavelength, that is, a ∼ 2.3 -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.

Maintenance of tissue-specific organ lipid compositions characterizes mammalian lipid homeostasis. The lungs and liver synthesize mixed phosphatidylcholine (PC) molecular species that are subsequently tailored for function. The lungs progressively enrich disaturated PC directed to lamellar body surfactant stores before secretion. The liver accumulates polyunsaturated PC directed to very-low-density lipoprotein assembly and secretion, or to triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested for potential coordination between these spatially separated but possibly complementary phenomena under a major derangement of lung PC metabolism, pulmonary alveolar proteinosis (PAP), which overwhelms homeostasis and leads to excessive surfactant accumulation. Using static and dynamic lipidomics techniques, we compared (1) tissue PC compositions and contents, and (2) in lungs, the absolute rates of synthesis in both control mice and the granulocyte–macrophage colony-stimulating factor knockout model of PAP. Significant disaturated PC accumulation in bronchoalveolar lavage fluid, alveolar macrophage, and lavaged lung tissue occurred alongside increased PC synthesis, consistent with reported defects in alveolar macrophage surfactant turnover. However, microscopy using oil red O staining, coherent anti-Stokes Raman scattering, second harmonic generation, and transmission electron microscopy also revealed neutral-lipid droplet accumulations in alveolar lipofibroblasts of granular macrophage colony-stimulating factor knockout animals, suggesting that lipid homeostasis deficits extend beyond alveolar macrophages. PAP plasma PC composition was significantly polyunsaturated fatty acid enriched, but the content was unchanged and hepatic polyunsaturated fatty acid–enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with nonalcoholic fatty liver disease. These data suggest a hepatopulmonary axis of PC metabolism coordination, with wider implications for understanding and managing lipid pathologies in which compromise of one organ has unexpected consequences for another.

We report a case of 7 years old male who was referred from Eye OPD with chronic discharge from right periorbita near inner canthus with suspected foreign body after 3 months of injury.

[Image: see text] Surface-enhanced Raman spectroscopy (SERS) pushes past the boundaries and inherent weaknesses of Raman spectroscopy, with a great potential for a broad range of applications particularly, for sensing. Yet, current real world applications are limited due to poor reproducibility, low-throughput, and stability issues. Here, we present the design and fabrication of self-assembly guided structures based on adjustable block co-polymer (BCP) nanomorphologies and demonstrate reproducible SERS enhancement across large areas. Golden three-dimensional (3D) nanostructured morphologies with controllable dimensions and morphologies exhibit high chemical stability, enhanced plasmonic properties and are highly suitable for SERS substrates due to the strong enhancement of the electromagnetic field. Adjustable, free standing porous nanostructures, continuous in 3D space are achieved by removal of selected BCP constituents. Four BCP morphologies and the corresponding achievable enhancement factors are investigated at 633 and 785 nm excitation wavelengths. The choice of excitation laser is shown to greatly affect the observed signal enhancement, highlighting the sensitivity of the technique to the underlying surface architecture and length scales. By using BCP assemblies, it is possible to reliably tune these parameters to match specific applications, thus bridging the gap toward the realization of applied metamaterials. The fabricated SERS platforms via three-dimensional block co-polymer-based nanoarchitectures provide a recipe for intelligent engineering and design of optimized SERS-active substrates for utilization in the Raman spectroscopy-based devices toward enabling the next-generation technologies fulfilling a multitude of criteria.

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin‐expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High‐resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone–derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone‐derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) are non-linear techniques that allow label-free, non-destructive and non-invasive imaging for cellular and tissue analysis. Although live-imaging studies have been performed previously, concerns that they do not cause any changes at the molecular level in sensitive biological samples have not been addressed. This is important especially for stem cell differentiation and tissue engineering, if CARS/SHG microscopy is to be used as a non-invasive, label-free tool for assessment of the developing neo-tissue. In this work, we monitored the differentiation of human fetal-femur derived skeletal cells into cartilage in three-dimensional cultures using CARS and SHG microscopy and demonstrate the live-imaging of the same developing neo-tissue over time. Our work conclusively establishes that non-linear label-free imaging does not alter the phenotype or the gene expression at the different stages of differentiation and has no adverse effect on human skeletal cell growth and behaviour. Additionally, we show that CARS microscopy allows imaging of different molecules of interest, including lipids, proteins and glycosaminoglycans, in the bioengineered neo-cartilage. These studies demonstrate the label-free and truly non-invasive nature of live CARS and SHG imaging and their value and translation potential in skeletal research, regeneration medicine and tissue engineering.

In recent years, single-molecule sensitivity achievable by surface-enhanced Raman spectroscopy (SERS) has been widely reported. We use this to investigate supramolecular host-guest chemistry with the macrocyclic host cucurbit[7]uril, on a few-to-single-molecule level. A nanogap geometry, comprising individual gold nanoparticles on a planar gold surface spaced by a single layer of molecules, gives intense SERS signals. Plasmonic coupling between the particle and the surface leads to strongly enhanced optical fields in the gap between them, with single-molecule sensitivity established using a modification of the well-known bianalyte method. Changes in the Raman modes of the host molecule are observed when single guests included inside its cavity internally stretch it. Anisotropic intermolecular interactions with the guest are found which show additional distinct features in the Raman modes of the host molecule.

Specific proteins and their aggregates form toxic amyloid plaques and neurofibrillary tangles in the brains of people suffering from neurodegenerative diseases such as Alzheimer’s and Parkinson’s. It is important to study these conformational changes to identify and differentiate these diseases at an early stage so that timely medication is provided to patients. Mid-infrared spectroscopy can be used to monitor these changes by studying the line-shapes and the relative absorbances of amide bands present in proteins. This work focusses on the spectroscopy of the protein, Bovine Serum Albumin as an exemplar, and its aggregates using germanium on silicon waveguides in the 1900–1000 cm−1 (5.3–10.0 µm) spectral region.