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

This is the pre-peer reviewed version of the following article: Mansfield, J., Moger, J., Green, E., Moger, C. and Winlove, C. P. (2013), Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering. J. Biophoton., 6: 803–814. doi: 10.1002/jbio.201200213, which has been published in final form at http://dx.doi.org/10.1002/jbio.201200213.© 2013 by WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimStimulated 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 PO₄³⁻ vibration at 959 cm⁻¹ and the CO₃²⁻ 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

Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy

Jessica C. Mansfield ; C. Peter Winlove ; Julian Moger and Steve J. Matcher "Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy", J. Biomed. Opt. 13(4), 044020 (July 15, 2008). Copyright © 2008 Society of Photo-Optical Instrumentation EngineersSecond harmonic generation (SHG) and two-photon fluorescence (TPF) microscopy is used to image the intercellular and pericellular matrix in normal and degenerate equine articular cartilage. The polarization sensitivity of SHG can be used directly to determine fiber orientation in the superficial 10 to 20 microm of tissue, and images of the ratio of intensities taken with two orthogonal polarization states reveal small scale variations in the collagen fiber organization that have not previously been reported. The signal from greater depths is influenced by the birefringence and biattenuance of the overlying tissue. An assessment of these effects is developed, based on the analysis of changes in TPF polarization with depth, and the approach is validated in tendon where composition is independent of depth. The analysis places an upper bound on the biattenuance of tendon of 2.65 x 10(-4). Normal cartilage reveals a consistent pattern of variation in fibril orientation with depth. In lesions, the pattern is severely disrupted and there are changes in the pericellular matrix, even at the periphery where the tissue appears microscopically normal. Quantification of polarization sensitivity changes with depth in cartilage will require detailed numerical models, but in the meantime, multiphoton microscopy provides sensitive indications of matrix changes in cartilage degeneration

In situ chemically specific mapping of agrochemical seed coatings using stimulated Raman scattering microscopy.

This is the author accepted manuscript. The final version is available from Wiley-VCH-Verlag via the DOI in this record.Providing sufficient, healthy food for the increasing global population is putting a great deal of pressure on the agrochemical industry to maximise crop yields without sustaining environmental damage. The growth and yield of every plant with sexual reproduction, depends on germination and emergence of sown seeds, which is affected greatly by seed disease. This can be most effectively controlled by treating seeds with pesticides before they are sown. An effective seed coating treatment requires a high surface coverage and adhesion of active ingredients onto the seed surface and the addition of adhesive agents in coating formulations plays a key role in achieving this. Although adhesive agents are known to enhance seed germination, little is understood about how they affect surface distribution of actives and how formulations can be manipulated to rationally engineer seed coating preparations with optimized coverage and efficacy. We show, for the first time, that stimulated Raman scattering (SRS) microscopy can be used to map the seed surface with microscopic spatial resolution and with chemical specificity to identify formulation components distributed on the seed surface. This represents a major advance in our capability to rationally engineer seed coating formulations with enhanced efficacy.We thank the funding from the Biotechnology and Biological Sciences Research Council (BBSRC, BB/M017915/1 and BB/K013602/1)

Microplastic Ingestion by Zooplankton

Small plastic detritus, termed “microplastics”, are a widespread and ubiquitous contaminant of marine ecosystems across the globe. Ingestion of microplastics by marine biota, including mussels, worms, fish, and seabirds, has been widely reported, but despite their vital ecological role in marine food-webs, the impact of microplastics on zooplankton remains under-researched. Here, we show that microplastics are ingested by, and may impact upon, zooplankton. We used bioimaging techniques to document ingestion, egestion, and adherence of microplastics in a range of zooplankton common to the northeast Atlantic, and employed feeding rate studies to determine the impact of plastic detritus on algal ingestion rates in copepods. Using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy we identified that thirteen zooplankton taxa had the capacity to ingest 1.7–30.6 μm polystyrene beads, with uptake varying by taxa, life-stage and bead-size. Post-ingestion, copepods egested faecal pellets laden with microplastics. We further observed microplastics adhered to the external carapace and appendages of exposed zooplankton. Exposure of the copepod Centropages typicus to natural assemblages of algae with and without microplastics showed that 7.3 μm microplastics (>4000 mL–1) significantly decreased algal feeding. Our findings imply that marine microplastic debris can negatively impact upon zooplankton function and health

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

Analytic philosophy for biomedical research: the imperative of applying yesterday's timeless messages to today's impasses

The mantra that "the best way to predict the future is to invent it" (attributed to the computer scientist Alan Kay) exemplifies some of the expectations from the technical and innovative sides of biomedical research at present. However, for technical advancements to make real impacts both on patient health and genuine scientific understanding, quite a number of lingering challenges facing the entire spectrum from protein biology all the way to randomized controlled trials should start to be overcome. The proposal in this chapter is that philosophy is essential in this process. By reviewing select examples from the history of science and philosophy, disciplines which were indistinguishable until the mid-nineteenth century, I argue that progress toward the many impasses in biomedicine can be achieved by emphasizing theoretical work (in the true sense of the word 'theory') as a vital foundation for experimental biology. Furthermore, a philosophical biology program that could provide a framework for theoretical investigations is outlined

Evolution of a minimal cell.

Na Publicação: D. M. C. Bittencourt

Intramolecular hydrogen transfer reactions of thiyl radicals from glutathione: formation of carbon-centered radical at Glu, Cys and Gly

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3000494Glutathione thiyl radicals (GS•) were generated in H2O and D2O by either exposure of GSH to AAPH#, photoirradiation of GSH in the presence of acetone, or photoirradiation of GSSG. Detailed interpretation of the fragmentation pathways of deuterated GSH and GSH-derivatives during mass spectrometry analysis allowed us to demonstrate that reversible intramolecular H-atom transfer reactions between GS• and C-H bonds at Cys[αC], Cys[βC], and Gly[αC] are possible