Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm−1, is demonstrated.

The unique properties of skeletal stem cells have attracted significant attention in the development of strategies for skeletal regeneration. However, there remains a crucial unmet need to develop quantitative tools to elucidate skeletal cell development and monitor the formation of regenerated tissues using non-destructive techniques in 3D. Label-free methods such as coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG) and two-photon excited auto-fluorescence (TPEAF) microscopy are minimally invasive, non-destructive, and present new powerful alternatives to conventional imaging techniques. Here we report a combination of these techniques in a single multimodal system for the temporal assessment of cartilage formation by human skeletal cells. The evaluation of bioengineered cartilage, with a new parameter measuring the amount of collagen per cell, collagen fibre structure and chondrocyte distribution, was performed using the 3D non-destructive platform. Such 3D label-free temporal quantification paves the way for tracking skeletal cell development in real-time and offers a paradigm shift in tissue engineering and regenerative medicine applications.

Maintenance of tissue-specific organ lipid compositions characterises mammalian lipid homeostasis. Lung and liver synthesise mixed phosphatidylcholine (PC) molecular species subsequently "tailored" for function. Lungs progressively enrich disaturated PC (DSPC) directed to lamellar body (LB) surfactant stores prior to secretion. Liver accumulates polyunsaturated PC directed to VLDL assembly and secretion, or triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested 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 leading to excessive surfactant accumulation. Using static and dynamic lipidomics techniques we compared (i) tissue PC compositions and contents and (ii) in lungs, the absolute rates of synthesis from both control mice and the GM-CSF knockout model of PAP. Significant DSPC accumulation in BALF, Alveolar Macrophage (AM) and lavaged lung tissue occurred alongside increased PC synthesis consistent with reported defects in AM surfactant turnover. However, microscopy using oil red O staining, CARS, SHG and TEM also revealed neutral lipid droplet accumulations in alveolar lipofibroblasts of GM-CSF KO animals suggesting lipid homeostasis deficits extend beyond AMs. PAP plasma PC composition was significantly PUFA-enriched but content was unchanged and hepatic PUFA-enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with NAFLD. These data suggest a hepato-pulmonary axis of PC metabolism coordination with wider implications for understanding and managing lipid pathologies where compromise of one organ has unexpected consequences for another.

BACKGROUNDMacrocyclic lactones are arguably the most successful chemical class with efficacy against parasitic nematodes. Here we investigated the effect of the macrocyclic lactone ivermectin on lipid homeostasis in the plant parasitic nematode Globodera pallida and provide new insight into its mode of action.RESULTSA non‐invasive, non‐destructive, label‐free and chemically selective technique called Coherent anti‐Stokes Raman scattering (CARS) spectroscopy was used to study lipid stores in G. pallida. We optimised the protocol using the free‐living nematode Caenorhabditis elegans and then used CARS to quantify lipid stores in the pre‐parasitic, non‐feeding J2 stage of G. pallida. This revealed a concentration of lipid stores in the posterior region of J2 s within 24 h of hatching which decreased to undetectable levels over the course of 28 days. We tested the effect of ivermectin on J2 viability and lipid stores. Within 24 h, ivermectin paralysed J2 s. Counterintuitively, over the same time‐course ivermectin increased the rate of depletion of J2 lipid, suggesting that in ivermectin‐treated J2 s there is a disconnection between the energy requirements for motility and metabolic rate. This decrease in lipid stores would be predicted to negatively impact on J2 infective potential.CONCLUSIONThese data suggest that the benefit of macrocyclic lactones as seed treatments may be underpinned by a multilevel effect involving both neuromuscular inhibition and acceleration of lipid metabolism. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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