Goal change and goal achievement for emerging adults across the pilot FAMS-T1D intervention for type 1 diabetes.

Objective: Interventions for emerging adults (EAs) with type 1 diabetes (T1D) focus on goal setting, but little is known about how goal achievement relates to intervention outcomes. We examined how goals change, how goal achievement relates to diabetes outcomes, and identified barriers and facilitators to goal achievement. Method: EAs with T1D (N=29, M age=21.6 years, 57% female) were coached monthly to set a behavioral goal across a 3-month feasibility trial. Coaching notes were qualitatively coded regarding type, complexity, and changes in goals. Goal achievement was measured via daily responses to texts. HbA1c, self-efficacy, diabetes distress, and self-care were assessed pre- and post-intervention. Results: EAs frequently set food goals (79%) in combination with other goals. EAs overwhelmingly changed their goals (90%), with most increasing goal complexity. Goal achievement was high (79% of days) and not affected by goal change or goal complexity. Goal achievement was associated with increases in self-efficacy and self-care across time. Qualitative themes revealed that aspects of self-regulation and social-regulation were important for goal achievement. Conclusion: Meeting daily diabetes goals may enhance self-efficacy and self-care for diabetes. Practice Implications: Assisting EAs to reduce self-regulation challenges and enhance social support for goals may lead to better diabetes outcomes.

Exploration of utility of combined optical photothermal infrared and Raman imaging for investigating the chemical composition of microcalcifications in breast cancer.

Microcalcifications play an important role in cancer detection. They are evaluated by their radiological and histological characteristics but it is challenging to find a link between their morphology, their composition and the nature of a specific type of breast lesion. Whilst there are some mammographic features that are either typically benign or typically malignant often the appearances are indeterminate. Here, we explore a large range of vibrational spectroscopic and multiphoton imaging techniques in order to gain more information about the composition of the microcalcifications. For the first time, we validated the presence of carbonate ions in the microcalcifications by O-PTIR and Raman spectroscopy at the same time, the same location and the same high resolution (0.5 µm). Furthermore, the use of multiphoton imaging allowed us to create stimulated Raman histology (SRH) images which mimic histological images with all chemical information. In conclusion, we established a protocol for efficiently analysing the microcalcifications by iteratively refining the area of interest.

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

Matrix-Bound Growth Factors are Released upon Cartilage Compression by an Aggrecan-Dependent Sodium Flux that is Lost in Osteoarthritis.

Articular cartilage is a dense extracellular matrix-rich tissue that degrades following chronic mechanical stress, resulting in osteoarthritis (OA). The tissue has low intrinsic repair especially in aged and osteoarthritic joints. Here, we describe three pro-regenerative factors; fibroblast growth factor 2 (FGF2), connective tissue growth factor, bound to transforming growth factor-beta (CTGF-TGFß), and hepatoma-derived growth factor (HDGF), that are rapidly released from the pericellular matrix (PCM) of articular cartilage upon mechanical injury. All three growth factors bound heparan sulfate, and were displaced by exogenous NaCl. We hypothesised that sodium, sequestered within the aggrecan-rich matrix, was freed by injurious compression, thereby enhancing the bioavailability of pericellular growth factors. Indeed, growth factor release was abrogated when cartilage aggrecan was depleted by IL-1 treatment, and in severely damaged human osteoarthritic cartilage. A flux in free matrix sodium upon mechanical compression of cartilage was visualised by 23Na -MRI just below the articular surface. This corresponded to a region of reduced tissue stiffness, measured by scanning acoustic microscopy and second harmonic generation microscopy, and where Smad2/3 was phosphorylated upon cyclic compression. Our results describe a novel intrinsic repair mechanism, controlled by matrix stiffness and mediated by the free sodium concentration, in which heparan sulfate-bound growth factors are released from cartilage upon injurious load. They identify aggrecan as a depot for sequestered sodium, explaining why osteoarthritic tissue loses its ability to repair. Treatments that restore matrix sodium to allow appropriate release of growth factors upon load are predicted to enable intrinsic cartilage repair in OA. SIGNIFICANCE STATEMENT: Osteoarthritis is the most prevalent musculoskeletal disease, affecting 250 million people worldwide.1 We identify a novel intrinsic repair response in cartilage, mediated by aggrecan-dependent sodium flux, and dependent upon matrix stiffness, which results in the release of a cocktail of pro-regenerative growth factors after injury. Loss of aggrecan in late-stage osteoarthritis prevents growth factor release and likely contributes to disease progression. Treatments that restore matrix sodium in osteoarthritis may recover the intrinsic repair response to improve disease outcome.

Microstructure and antibacterial efficacy of graphene oxide nanocomposite fibres.

Antibacterial polymer nanocomposite fibre meshes containing graphene oxide (GO) nanosheets were successfully prepared by pressurised gyration. The morphological and chemical composition of the resulting fibre meshes were determined using Scanning Electron Microscopy (SEM), Raman spectroscopy, Raman mapping and Fourier-Transform Infrared Spectroscopy (FT-IR). SEM showed the fibres to have an average diameter increasing from ~1-4 µm as the GO loading increased. FT-IR and Raman spectroscopy confirmed the inclusion of GO nanosheets on the fibre surface. The antibacterial potential of GO nanocomposite fibres were investigated using Escherichia coli K12. Average bacterial reduction ranged from 46 to 85 % with results favouring the strongest bioactivities of the nanocomposite containing 8 wt% of GO. Finally, bacterial toxicity of the nanocomposites was evaluated by reactive oxygen species (ROS) formation. A mechanism for the antibacterial behaviour of the nanocomposite fibres is presented. Stimulated Raman scattering imaging and spectra of the fibres post antibacterial studies showed flakes of GO distributed across the surface of the poly(methyl 2-methylpropenoate) (PMMA) fibres, which contribute to the high killing efficacy of the composites towards E. coli. GO nanosheets embedded in a polymer matrix have demonstrated the ability to retain their antibacterial properties, thus offering themselves as a promising antibacterial agent.

Parental Involvement and Executive Function in Emerging Adults with Type 1 Diabetes.

OBJECTIVE: To examine (a) changes in parental involvement across early emerging adulthood, (b) whether yearly fluctuations in parental involvement were associated with adherence and glycated hemoglobin (HbA1c) over time, and (c) whether higher involvement was more beneficial for those with poorer executive function (EF). METHODS: A total of 228 high school seniors (M age = 17.76) with type 1 diabetes reported on mothers' and fathers' acceptance, knowledge of diabetes activities, disclosure to mothers and fathers regarding diabetes, and adherence at four yearly time points. At baseline, participants completed performance-based measures of EF. HbA1c was collected from assay kits. RESULTS: Growth curve models revealed significant declines in disclosure to fathers and mothers' and fathers' knowledge of diabetes activities; no changes were found in mothers' or fathers' acceptance nor disclosure to mothers. Multilevel models indicated significant between-person effects for nearly all aspects of parental involvement with more acceptance, knowledge, and disclosure associated with better HbA1c and adherence. Within-person effects for disclosure to fathers, and mothers' and fathers' knowledge indicated that in years when emerging adults perceived higher amounts of these types of involvement (compared with their own average), HbA1c was lower. Within-person effects were found for acceptance to mothers, disclosure to mothers and fathers, and mothers' diabetes knowledge for adherence. Disclosure to fathers and mothers' knowledge of diabetes activities were especially beneficial for HbA1c for those with poorer EF performance. CONCLUSIONS: Parental involvement in diabetes management remains important during the high-risk time of emerging adulthood, especially for those with poorer EF.

Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy.

Type II collagen fibril diameters in cartilage are beneath the diffraction limit of optical microscopy, which makes the assessment of collagen organization very challenging. In this work we use polarization sensitive second harmonic generation (P-SHG) imaging to map collagen organization in articular cartilage, addressing in particular its behaviour under strain and changes which occur in osteoarthritis. P-SHG yields two parameters, molecular order and orientation, which provide measures of the degree of organization both at the molecular scale (below the diffraction limit) and above a few hundred nanometres (at the image pixel size). P-SHG clearly demonstrates the zonal collagen architecture and reveals differences in the structure of the fibrils around chondrocytes. P-SHG also reveals sub-micron scale fibril re-organization in cartilage strips exposed to tensile loading, with an increase in local organization in the superficial zone which weakly correlates with tensile modulus. Finally, P-SHG is used to investigate osteoarthritic cartilage from total knee replacement surgery, and reveals widespread heterogeneity across samples both microscale fibril orientations and their sub-micron organization. By addressing collagen fibril structure on scales intermediate between conventional light and electron microscopy, this study provides new insights into collagen micromechanics and mechanisms of degradation.

Subwavelength hyperspectral THz studies of articular cartilage.

Terahertz-spectroscopy probes dynamics and spectral response of collective vibrational modes in condensed phase, which can yield insight into composition and topology. However, due to the long wavelengths employed (λ = 300 µm at 1THz), diffraction limited imaging is typically restricted to spatial resolutions around a millimeter. Here, we demonstrate a new form of subwavelength hyperspectral, polarization-resolved THz imaging which employs an optical pattern projected onto a 6 µm-thin silicon wafer to achieve near-field modulation of a co-incident THz pulse. By placing near-field scatterers, one can measure the interaction of object with the evanescent THz fields. Further, by measuring the temporal evolution of the THz field a sample's permittivity can be extracted with 65 µm spatial resolution due to the presence of evanescent fields. Here, we present the first application of this new approach to articular cartilage. We show that the THz permittivity in this material varies progressively from the superficial zone to the deep layer, and that this correlates with a change in orientation of the collagen fibrils that compose the extracellular matrix (ECM) of the tissue. Our approach enables direct interrogation of the sample's biophysical properties, in this case concerning the structure and permittivity of collagen fibrils and their anisotropic organisation in connective tissue.

The effects of needle damage on annulus fibrosus micromechanics.

Needle puncture of the intervertebral disc can initiate a mechanical and biochemical cascade leading to disc degeneration. Puncture's mechanical effects have been shown near the puncture site, mechanical effects should be observed far, relative to needle size, from the puncture site, given the disc-wide damage induced by the stab. The aim of this work was to quantify these far-field effects, and to observe the local structural damage provoked by the needle. Strips of cow tail annulus fibrosus underwent two consecutive mechanical loadings to 5% tensile strain; fifteen samples were punctured in a radial direction with a randomly assigned needle between the two loadings (needle gauges between 19 and 23). Ten samples (control group) were not punctured. During loading, the tissue strains were imaged using second harmonic generation microscopy in a <600×800µm region about 4.4mm from the puncture site. After mechanical testing, the puncture site was imaged in 3D. Puncture had no significant effect on annulus elastic modulus. Imaging showed a modest change in the shearing between fibre bundles however, the linear strain between bundles, intra-bundle shear and linear strain were not significantly affected. At the puncture site, detached lumps of tissue were present. These results suggest that the mechanical effects observed in intact discs are due to the depressurization of the disc, rather than the local damage to the annulus. Needle profiles could be designed, aiming at separating fibre bundles rather than cutting through them, to avoid leaving dying tissue behind. STATEMENT OF SIGNIFICANCE: Needle puncture of the intervertebral disc can initiate a mechanical and biochemical cascade leading to disc degeneration, but the link between the local damage of the puncture and the disc-wide effects is not well understood. This work aimed at determining the micro-mechanical effects of the puncture far from its site, and to observe the damage induced by the puncture with high resolution imaging. Results show that the puncture had modest effect far from the puncture, but lumps of tissue were left by the needle, detached from the disc; these could cause further damage through friction and inflammation of the surrounding tissues. This suggests that the cascade leading to degeneration is probably driven by a biochemical response rather than disc-wide mechanical effects.

Lipid distribution, composition and uptake in bovine articular cartilage studied using Raman micro-spectrometry and confocal microscopy.

The distribution and composition of endogenous lipids in articular cartilage and transport of exogenous fatty acids have been investigated on a microscopic scale in fresh bovine articular cartilage. To investigate the distribution and composition of the endogenous lipids, hyperspectral Raman maps were taken of chondrocytes and their surrounding matrix in both the deep and superficial zones. These revealed differences in both lipid distribution and composition between the two zones. Extracellular lipid was observed surrounding the cells in the superficial zone but not in the deep zone. Additionally, intracellular lipid droplets were observed that were larger and more numerous in the deep zone (P = 0.01). The extracellular lipid was primarily free saturated fatty acid, whereas the cellular lipid droplets contained triglycerides with unsaturated fatty acid chains. Fatty acid uptake and transport were investigated by incubating cartilage samples in Dulbecco's modified Eagle's medium containing fluorescently labelled palmitate for a range of times and temperatures. After incubation, the palmitate distribution was imaged using confocal microscopy. Palmitate accumulated preferentially in the territorial matrix only in the superficial zone where the concentration was up to 100-fold greater than that in the bulk matrix (P = 0.001). Palmitate uptake by the chondrocytes in both zones showed differential temperature sensitivity (P = 0.05), which would support the idea that cells take up palmitate by both active and passive mechanisms. The study reveals large differences between chondrocytes in the superficial and deep zones in their lipid content, in their extracellular lipid environment and in their access to exogenous fatty acids.

Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

The complex structure of the annulus fibrosus is strongly related to its mechanical properties. Recent work showed that it is possible to observe the relative movement of fibre bundles in loaded cow tail annulus; the aim of this work was to describe and quantify annulus fibrosus micromechanics in degenerated human disc, and compare it with cow tail annulus, an animal model often used in the literature. Second harmonic generation was used to image the collagen matrix in twenty strips of annulus fibrosus harvested from intervertebral disc of seven patients undergoing surgery. Samples were loaded to 6% tensile strain in 1% steps. Elastic modulus was calculated from loading curves, and micromechanical strains were calculated from the images using custom software. The same protocol was applied to twenty strips of annulus harvested from cow tail discs. Significant morphological differences were found between human and cow tail samples, the most striking being the lack of collagen fibre crimp in the former. Fibres were also observed bending and running from one lamella to the other, forming a strong flexible interface. Interdigitation of fibre bundles was also present at this interface. Quantitative results show complex patterns of inter-bundle and inter-lamellar behaviour, with inter-bundle sliding being the main strain mechanism. Elastic modulus was similar between species, and it was not affected by the degree of degeneration. This work gives an insight into the complex structure and mechanical function of the annulus fibrosus, which should be accounted for in disc numerical modelling.

Lamellar and fibre bundle mechanics of the annulus fibrosus in bovine intervertebral disc.

UNLABELLED: The intervertebral disc is a multicomposite structure, with an outer fibrous ring, the annulus fibrosus, retaining a gel-like core, the nucleus pulposus. The disc presents complex mechanical behaviour, and it is of high importance for spine biomechanics. Advances in multiscale modelling and disc repair raised a need for new quantitative data on the finest details of annulus fibrosus mechanics. In this work we explored inter-lamella and inter-bundle behaviour of the outer annulus using micromechanical testing and second harmonic generation microscopy. Twenty-one intervertebral discs were dissected from cow tails; the nucleus and inner annulus were excised to leave a ring of outer annulus, which was tested in circumferential loading while imaging the tissue's collagen fibres network with sub-micron resolution. Custom software was developed to determine local tissue strains through image analysis. Inter-bundle linear and shear strains were 5.5 and 2.8 times higher than intra-bundle strains. Bundles tended to remain parallel while rotating under loading, with large slipping between them. Inter-lamella linear strain was almost 3 times the intra-lamella one, but no slipping was observed at the junction between lamellae. This study confirms that outer annulus straining is mainly due to bundles slipping and rotating. Further development of disc multiscale modelling and repair techniques should take into account this modular behaviour of the lamella, rather than considering it as a homogeneous fibre-reinforced matrix. STATEMENT OF SIGNIFICANCE: The intervertebral disc is an organ tucked between each couple of vertebrae in the spine. It is composed by an outer fibrous layer retaining a gel-like core. This organ undergoes severe and repeated loading during everyday life activities, since it is the compliant component that gives the spine its flexibility. Its properties are affected by pathologies such as disc degeneration, a major cause of back pain. In this article we explored the micromechanical behaviour of the disc's outer layer using second harmonic generation, a technique which allowed us to visualize, with unprecedented detail, how bundles of collagen fibres slide relative to each other when loaded. Our results will help further the development of new multiscale numerical models and repairing techniques.

Depressive symptoms, daily stress, and adherence in late adolescents with type 1 diabetes.

OBJECTIVE: To examine whether depressive symptoms are associated with greater perceived daily stress and moderate the link between stress severity and poorer daily adherence in late adolescents with Type 1 diabetes (T1D). METHOD: 175 late adolescents with T1D completed measures of depressive symptoms and glycemic control during a baseline laboratory assessment. This assessment was followed by a 14-day daily diary during which adolescents rated the severity of general (GS) and diabetes-specific (DSS) stressful events, as well as adherence to their diabetes regimen. RESULTS: Multilevel modeling revealed that adolescents with more depressive symptoms reported more severe daily stress and poorer daily adherence on average, and had poorer glycemic control. On days with more severe DSS, but not GS, adolescents reported poorer adherence. This association was moderated by an interaction between depressive symptoms and the mean level of DSS severity experienced across the 2-week diary. In adolescents with low levels of depressive symptoms, poorer adherence was reported on days with more severe DSS across all levels of mean DSS severity. In adolescents with average or high levels of depressive symptoms, poorer adherence was reported on days with more severe DSS only when mean DSS severity was average or high. CONCLUSIONS: Depressive symptoms are associated with poorer daily adherence and greater stress severity, and interact with mean DSS severity to moderate the link between daily stress and adherence. The results point to the importance of depressive symptoms for understanding associations between stress and adherence during late adolescence.

In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy.

The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.

An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the "negative space" within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.

The structure and micromechanics of elastic tissue.

Elastin is a major component of tissues such as lung and blood vessels, and endows them with the long-range elasticity necessary for their physiological functions. Recent research has revealed the complexity of these elastin structures and drawn attention to the existence of extensive networks of fine elastin fibres in tissues such as articular cartilage and the intervertebral disc. Nonlinear microscopy, allowing the visualization of these structures in living tissues, is informing analysis of their mechanical properties. Elastic fibres are complex in composition and structure containing, in addition to elastin, an array of microfibrillar proteins, principally fibrillin. Raman microspectrometry and X-ray scattering have provided new insights into the mechanisms of elasticity of the individual component proteins at the molecular and fibrillar levels, but more remains to be done in understanding their mechanical interactions in composite matrices. Elastic tissue is one of the most stable components of the extracellular matrix, but impaired mechanical function is associated with ageing and diseases such as atherosclerosis and diabetes. Efforts to understand these associations through studying the effects of processes such as calcium and lipid binding and glycation on the mechanical properties of elastin preparations in vitro have produced a confusing picture, and further efforts are required to determine the molecular basis of such effects.

The mechanical properties of human adipose tissues and their relationships to the structure and composition of the extracellular matrix.

Adipose tissue (AT) expansion in obesity is characterized by cellular growth and continuous extracellular matrix (ECM) remodeling with increased fibrillar collagen deposition. It is hypothesized that the matrix can inhibit cellular expansion and lipid storage. Therefore, it is important to fully characterize the ECM's biomechanical properties and its interactions with cells. In this study, we characterize and compare the mechanical properties of human subcutaneous and omental tissues, which have different physiological functions. AT was obtained from 44 subjects undergoing surgery. Force/extension and stress/relaxation data were obtained. The effects of osmotic challenge were measured to investigate the cellular contribution to tissue mechanics. Tissue structure and its response to tensile strain were determined using nonlinear microscopy. AT showed nonlinear stress/strain characteristics of up to a 30% strain. Comparing paired subcutaneous and omental samples (n = 19), the moduli were lower in subcutaneous: initial 1.6 ± 0.8 (means ± SD) and 2.9 ± 1.5 kPa (P = 0.001), final 11.7 ± 6.4 and 32 ± 15.6 kPa (P < 0.001), respectively. The energy dissipation density was lower in subcutaneous AT (n = 13): 0.1 ± 0.1 and 0.3 ± 0.2 kPa, respectively (P = 0.006). Stress/relaxation followed a two-exponential time course. When the incubation medium was exchanged for deionized water in specimens held at 30% strain, force decreased by 31%, and the final modulus increased significantly. Nonlinear microscopy revealed collagen and elastin networks in close proximity to adipocytes and a larger-scale network of larger fiber bundles. There was considerable microscale heterogeneity in the response to strain in both cells and matrix fibers. These results suggest that subcutaneous AT has greater capacity for expansion and recovery from mechanical deformation than omental AT.

Label-free chemically specific imaging in planta with stimulated Raman scattering microscopy.

The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas. There is currently a lack of analytical tools that provide noninvasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide label-free chemically specific image contrast based on vibrational spectroscopy. Over the past decade, these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis, it does not suffer from photobleaching, and it allows near real-time imaging in 3D with submicrometer spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll), the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored. In this paper, we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally, we demonstrate the potential of SRS for a range of in planta applications by presenting in situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface.

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering.

Stimulated Raman scattering (SRS) has been applied to unstained samples of articular cartilage enabling the investigation of living cells within fresh tissue. Hyperspectral SRS measurements over the CH vibrational region showed variations in protein and lipid content within the cells, pericellular matrix and interterritorial matrix. Changes in the cells and pericellular matrix were investigated as a function of depth into the cartilage. Lipid was detected in the pericellular matrix of superficial zone chondrocytes. The spectral profile of lipid droplets within the chondrocytes indicated that they contained predominantly unsaturated lipids. The mineral content has been imaged by using the PO4³â» vibration at 959 cm⁻¹ and the CO3²â» vibration at 1070 cm⁻¹. Both changes in cells and mineralization are known to be important factors in the progression of osteoarthritis. SRS enables these to be visualized in fresh unstained tissue and consequently should benefit osteoarthiritis research.

A multi-modal multiphoton investigation of microstructure in the deep zone and calcified cartilage.

Multi-modal multiphoton microscopy was used to investigate tissue microstructure in the zone of calcified cartilage, focussing on the collagen fibre organisation at the tidemark and cement line. Thick, unstained and unfixed sagittal sections were prepared from the equine metacarpophalangeal joint. Second harmonic generation (SHG) provided contrast for collagen, two-photon fluorescence (TPF) for endogenous fluorophores, and coherent anti-Stokes Raman scattering (CARS) allowed the cells to be visualised. The structure of radial and calcified cartilage was found to vary with location across the joint, with the palma regions showing a more ordered parallel arrangement of collagen fibres than the cortical ridge and dorsal regions. These patterns may be associated with regional variations in joint loading. In addition, the cell lacunae had a greater diameter in the dorsal region than in the palmar region. At the cement line some collagen fibres were observed crossing between the calcified cartilage and the subchondral bone. At the tidemark the fibres were parallel and continuous between the radial and calcified cartilage. Beneath early superficial lesions the structure of the tidemark and calcified cartilage was disrupted with discontinuities and gaps in the fibrillar organisation. Cartilage microstructure varies in the deep zones between regions of different loading. The variations in collagen structure observed may be significant to the local mechanical properties of the cartilage and therefore may be important to its mechanical interactions with the subchondral bone. The calcified cartilage is altered even below early superficial lesions and therefore is important in the understanding of the aetiology of osteoarthritis.