A review of some recent developments in polarization-sensitive optical imaging techniques for the study of articular cartilage

This article reviews recent developments in the optical imaging of articular cartilage using polarized-light methods, with an emphasis on tools that could be of use in tissue engineering approaches to treatment. Both second-harmonic generation microscopy and polarization-sensitive optical coherence tomography are described and their potential role in the treatment of cartilage disorders such as osteoarthritis is suggested. Key results are reviewed and future developments are discussed

Novel optical imaging technique to determine the 3-D orientation of collagen fibers in cartilage: variable-incidence angle polarization-sensitive optical coherence tomography

Objective: To investigate a novel optical method to determine the three dimensional (3-D) structure of articular cartilage collagen non-destructively. Methods: Polarization-sensitive optical coherence tomography was used to determine the apparent optical birefringence of articular cartilage for a number of different illumination directions. A quantitative method based on the theory of light propagation in uniaxial crystalline materials was validated on equine flexor tendon. Qualitative maps of fiber polar and azimuthal orientation at sites on the posterior and anterior segments of the equine third metacarpophalangeal (fetlock) joint were produced, and the azimuthal orientations compared with data from a split-line experiment. Results: Polar and azimuthal angles of cut flexor tendon broadly agreed with the nominal values but suggested that the accuracy was limited by our method of determining the apparent birefringence. On intact equine fetlock joints we found a non-zero polar tilt that changed in direction at various points along the apex, moving from the sagittal ridge outwards. The azimuthal orientation changes from being parallel to the sagittal ridge in the posterior region to being inclined to the ridge in the anterior region. This broadly agrees with split-line data for the anterior region but differs in the posterior region, possibly reflecting depth-dependent orientation changes. Conclusion: General quantitative agreement was found between our method and histology in validation experiments. Qualitative results for cartilage suggest a complicated 3-D structure that warrants further study. There is potential to develop this approach into a tool that can provide depth-resolved information on collagen orientation in near real-time, non-destructively and in vivo. (c) 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved

Optical coherence tomography measurements of biological fluid flows with picolitre spatial localization

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.Interest in studying the human and animal microcirculation has burgeoned in recent years. In part this has been driven by recent advances in volumetric microscopy modalities, which allow the study of the 3-D morphology of the microcirculation without the limitations of 2-D intra-vital microscopy. In this paper we highlight the power of optical coherence tomography (OCT) to image the normal and pathological microcirculation with picolitre voxel sizes. Both Doppler and speckle-variance methods are employed to characterize complex rheological flows both in-vitro and in-vivo. GPU accelerated image registration methods are demonstrated in order to mitigate problems of bulk tissue motion in methods based on speckle decorrelation. In-vivo images of the human nailfold microcirculation are shown

A broad host range reporter plasmid for the analysis of divergent promoter regions

Although many vectors exist for Escherichia coli and closely related species, there are few broad host range vectors that can be conjugated into a large variety of Gram-negative bacteria. We have constructed a broad host range vector, pMJ445, that facilitates the analysis of divergent promoters in Gram-negative bacteria. The vector was validated using two intergenic regions derived from gene clusters involved in hydantoin hydrolysis, from the environmental isolates Pseudomonas putida and Agrobacterium tumefaciens. The DNA sequences analysed were capable of activating expression of the reporter enzymes, β-glucuronidase and β-galactosidase, present on pMJ445, indicating the presence of divergent promoters in the sequences selected. In addition, we demonstrated that pMJ445 can be applied to gene regulation studies

Insights into the bacterial diversity in a freshwater-deprived permanently open Eastern Cape estuary, using 16S rRNA pyrosequencing analysis

The aim of this study was to conduct an investigation into the bacterial diversity in the freshwater-deprived Kariega Estuary, situated along the Eastern Cape coastline, using ribosomal RNA gene sequences obtained by pyrosequencing. Shifts in the microbial diversity were correlated to selected physico-chemical variables along the length of the estuary. More than 27 000 sequences were obtained and rarefaction analyses confirmed a comprehensive appraisal of the microbial diversity present in the Kariega Estuary. Distinct patterns in phylotype distribution from the hypersaline upper reaches to the mouth of the estuary were observed; notably, the importance of the detrital food web within the Kariega Estuary was highlighted by the high occurrence of Bacteroidetes and Actinomycetes. Moreover, while the observed chlorophyll-a concentrations were low (< 0.1 ìg..-1), the presence of Pelagibacter and Flavobacteria amongst the microbial community suggests a potentially important contribution of these microbes towards the total primary productivity of the ecosystem. No human pathogenic microbes were detected within waters of the system. We conclude that pyrosequencing provides a versatile and efficient tool for assessing the microbial diversity in the Kariega Estuary and propose that this technology may provide valuable information on the ecosystem functioning and health of aquatic ecosystems

Evaluation of a cheap ultrasonic stage for light source coherence function measurement, optical coherence tomography and dynamic focusing

We evaluate the performance of a cheap ultrasonic stage in setups related to optical coherence tomography. The stage was used in several configurations: (1) optical delay line in an optical coherence tomography (OCT) setup; (2) as a delay line measuring coherence function of a low coherence source (e. g. superluminescent diode) and (3) in a dynamic focusing arrangement. The results are as follows: the stage is suitable for coherence function measurement (coherence length up to 70 mu m) of the light source and dynamic focusing. We found it unsuitable for OCT due to an unstable velocity profile. Despite this, the velocity profile has a repeatable shape (4% over 1000 A-scans) and slight modifications to the stage promise wider applications

Characterization of the hydantoin-hydrolysing system of Pseudomonas putida RU-KM3s

The biocatalytic conversion of 5-monosubstituted hydantoin derivatives to optically pure amino acids involves two reaction steps: the hydrolysis of hydantoin to N-carbamylamino acid by an hydantoinase or dihydropyrimidinase enzyme, followed by conversion of the Ncarbamylamino acid to the corresponding amino acid by an N-carbamoylase enzyme. This biocatalytic process has been successfully applied in several industrial processes for the production of enantiomerically pure amino acids used in the synthesis of pharmaceuticals, insecticides, hormones, and food additives. P. putida RU-KM3S was selected for study based on inherent high levels of hydantoinase and N-carbamoylase activity. Subsequent biocatalytic analysis of the enzyme activity within this strain revealed unique properties thus prompting further characterization. The main focus of this research was the isolation of the genes encoding the hydantoin-hydrolysing pathway in RU-KM3S. A genomic library was constructed and screened for heterologous expression of the hydantoin-hydrolysing enzymes. However, this approach was unsuccessful prompting the use of transposon mutagenesis in order to circumvent the drawbacks associated with complementation studies. The enzymes responsible for hydantoin-hydrolysis were identified by insertional inactivation as a dihydropyrimidinase and b-ureidopropionase encoded by dhp and bup respectively. A third open reading frame, encoding a putative transport protein, was identified between the dhp and bup genes and appeared to share a promoter with bup. Analysis of the amino acid sequence deduced from bup and dhp substantiated the distinctive properties and potential industrial application of the L-enantioselective b-ureidopropionase and provided targets for potential optimisation of the substrate-selectivity and activity of the dihydropyrimidinase by site directed mutagenesis. Several transposon-generated mutants with an altered phenotype for growth on minimal medium with hydantoin as the sole source of nitrogen were also isolated. Analysis of the insertion events in these mutants revealed disruptions of genes encoding key elements of the Ntr global regulatory pathway. However, inactivation of these genes had no effect on the dihydropyrimidinase and b-ureidopropionase activity levels. An additional mutant in which the gene coding for the dihydrolipoamide succinyltransferase, which is involved in the TCA cycle, was isolated with reduced levels of both dihydropyrimidinase and b-ureidopropionase activities. These results indicated that the hydantoin-hydrolysis pathway in RU-KM3S is regulated by carbon rather than nitrogen catabolite repression. This was confirmed by the reduction of hydantoin-hydrolysis in cells grown in excess carbon as opposed to nitrogen. Identification of a putative CRP-binding site within the promoter region of these enzymes further supported the regulatory role of carbon catabolite repression (CCR). As CCR in Pseudomonads is poorly understood, elucidation of the mechanism by which the hydantoinhydrolysing pathway in RU-KM3S is regulated would provide valuable insight into this complex process

Ghosting artifact reduction of polarization sensitive optical coherence tomography images through wavelet-FFT filtering

Undesirable cross-coupling between polarisation-maintaining (PM) fibers can result in detrimental ghost artefacts within polarisation sensitive optical coherence tomography (PS-OCT) images. Such artefacts combine with coherence noise stripes (originating from Fresnel reflections of optical components), complex-conjugate derived mirror-images and further irregular autocorrelation terms originating from the sample. Together, these artefacts can severely degrade the detected images, making quantitative measurements of the tissue birefringence challenging to perform. In this work, we utilize the recently presented wavelet-FFT filter1 to efficiently suppress these imaging artefacts entirely through post-processing. While the original algorithm was designed to suppress one-dimensional stripe artefacts, we extend this methodology to also facilitate removal of artefacts following a duplicate or inverse (mirror) profile to that of the skin surface. This process does not require any hardware modification of the system and can be applied retroactively to previously acquired OCT images. The performance of this methodology is evaluated by processing artefact-corrupted PS-OCT images of skin consisting of simultaneously detected horizontal and vertical polarized light. The resulting images are used to calculate a phase retardance map within the skin, the profile of which is indicative of localized birefringence. Artefacts in the resulting processed PSOCT images were notably attenuated compared to the unprocessed raw-data, with minimal degradation to the underlying phase retardation information. This should improve the reliability of curve-fitting for measurements of depth-resolved birefringence

Morphological parametric mapping of 21 skin sites throughout the body using optical coherence tomography

Background Changes in body posture cause changes in morphological properties at different skin sites. Although previous studies have reported the thickness of the skin, the details of the postures are not generally given. This paper presents the effect of a change in posture on parameters such as thickness and surface roughness in 21 load-bearing and non-load-bearing sites. Materials and methods A total of 12 volunteers (8 males and 4 females) were selected in an age group of 18–35 years and of Fitzpatrick skin type I-III. Images were captured using a clinically-approved VivoSight® optical coherence tomography system and analysed using an algorithm provided by Michelson Diagnostics. Results Overextension (extending joints to full capacity) resulted in changes to thickness, roughness and undulation of the skin around the body. Discussion and conclusion The load-bearing regions have thicker skin compared to non-load-bearing sites. This is the first time that undulation topography of the stratum corneum–stratum lucidum and the dermal–epidermal junction layers have been measured and reported using statistical values such as Ra. The data presented could help to define new skin layer models and to determine the variability of the skin around the body and between participants

A new mode of contrast in biological second harmonic generation microscopy

Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can produce large changes in the wavelength-dependency of epi-generated SHG. However, in the case of forward SHG, although the same changes impact upon absolute intensity at a given wavelength, they have very little effect on wavelength-dependency beyond the expected monotonic fall. We also propose that this difference between forward and epi-generated SHG provides an explanation for many of the wavelength-dependency discrepancies in the published literature