We present a three-dimensional computer reconstruction of a plaice (Pleuronectes platessa) otolith from data acquired by the Diamond Light synchrotron, beamline I12, X-ray source, a high energy (53 – 150 keV) source particularly well suited to the study of dense objects. Our data allowed non-destructive rendering of otolith structure, and for the first time allows examination of otolith annuli (internal ring structures) to be analysed in X-ray tomographic images

Bailey

Brophy

Cadrin

Campana

Doubleday

Hamrin

Jolivet

Limburg

Paganin

Pontual

Popper

Sturrock

Mapp, James

Fisher, Mark

Atwood, Robert

Bell, Duncan

Greco, Mark

Songer, Sally

Hunter, Ewan

University of East Anglia digital repository

Journal of Fish Biology Three-dimensional rendering of otolith growth using phase contrast synchrotrontomography--Manuscript Draft-- Manuscript Number: MS 15-592R1Full Title: Three-dimensional rendering of otolith growth using phase contrast synchrotrontomographyShort Title: 3-D rendering of otolith growthArticle Type: Brief CommunicationKeywords: 3-D Reconstruction;  Fish Ageing;  Otolith Growth;  Phase Contrast;  SynchrotronTomographyCorresponding Author: Mark Henry Fisher, B.Sc., M.Sc., Ph.D.University of East AngliaNorwich, UNITED KINGDOMCorresponding Author SecondaryInformation:Corresponding Author's Institution: University of East AngliaCorresponding Author's SecondaryInstitution:First Author: James Mapp, M.Sc. Ph.D.First Author Secondary Information:Order of Authors: James Mapp, M.Sc. Ph.D.Mark Henry Fisher, B.Sc., M.Sc., Ph.D.Robert Atwood, Ph.D.Duncan Bell, M.D., M.Sc.Mark Greco, Ph.D.Sally Songer, B.Sc.Ewan Hunter, Ph.D.Order of Authors Secondary Information:Abstract: We present a three-dimensional computer reconstruction of a plaice (Pleuronectesplatessa) otolith from data acquired by the Diamond Light synchrotron, beamline I12,X-ray source, a high energy (53 - 150 keV) source particularly well suited to the studyof dense objects. Our data allowed non-destructive rendering of otolith structure, andfor the first time allows examination of otolith annuli (internal ring structures) to beanalysed in X-ray tomographic images.Powered by Editorial Manager® and ProduXion Manager® from Aries Systems CorporationEthics questionnaire for JFB  This questionnaire relates to the Editorial published in JFB 68, 1-2, which you have been asked to read. Please note that submitted manuscripts will only be considered if the experimental methods employed are ethically justified. PLEASE SUBMIT THE COMPLETED QUESTIONNAIRE WITH YOUR MANUSCRIPT ONLINE THROUGH EDITORIAL MANAGER.   Corresponding author’s name: _Mark Henry Fisher_________________________________   Question 1: If the fishes have been collected as part of faunal surveys, have the fishes, where feasible, been killed rapidly or returned to the wild after being held in aquaria?   Yes x No     Question 2: What method was used if they were killed? The study used a single specimen (plaice) caught by a CEFAS survey vessel.      Question 3: If you have undertaken experimental work, has the care and use of experimental animals complied with local and or national animal welfare laws, guidelines and policies?   Yes  No    If ‘Yes’, state these and provide suitable evidence (e.g. for the U.K. a Home Office PPL number is sufficient) that protocols have undergone an ethical review process by an institutional animal care and use (or similar) committee, a local ethics committee, or by appropriately qualified scientific and lay colleagues.  N/A    Please read the exceptions below (Questions 4 to 7). If any of these exceptions apply to your study, complete the appropriate section. Otherwise leave blank.  If ‘No’, because these laws do not exist in your country, please state this.     Please read the exceptions below (Questions 4 to 7). If any of these exceptions apply to your study, complete the appropriate section. Otherwise leave blank.  Question 4: Did you use experimental conditions that severely distressed the animals?   Yes  No    If ‘Yes’, state the conditions and how they can be justified. N/A    Ethics Questionaire Question 5: Did you use humane endpoints that minimized adverse effects?   Yes  No    Question 6: Have you performed surgical procedures?   Yes  No    If ‘Yes’, have you suitably described these in your manuscript?       Question 7: If the procedures caused more than slight pain or distress, did you use appropriate sedation, analgesia and anaesthesia, with appropriate post-operative care?   Yes  No    If ‘Yes’, outline these.      If ‘No’, did any of your procedures involve sentient, un-anaesthetized animals paralysed by chemical agents such as muscle relaxants?   Yes   No     If ‘Yes’, provide details. Normally these procedures will be considered unacceptable by JFB.      If ‘No’, did any of the procedures, particularly those that involve lethal endpoints, cause adverse effects or lasting harm to a sentient animal?   Yes  No    If ‘Yes’, provide details. Normally these procedures will be considered unacceptable by JFB unless any harm caused can be justified against the benefit gained.      3-D rendering of otolith growth   1  Three-dimensional rendering of otolith growth using phase contrast synchrotron tomography 1  2 J. J. I. Mapp, M. H. Fisher*, School of Computing Sciences, University of East Anglia, 3 Norwich Research Park, Norwich, NR4 7TJ, UK. 4 R. C. Atwood, Diamond Light Source Ltd., Diamond House, Harwell Science and Innovation 5 Campus, Didcot, Oxfordshire, OX11 0DE, UK. 6 G. D. Bell, Faculty of Health and Science, University Campus Suffolk, Waterfront Building, 7 Neptune Quay, Ipswich, IP4 1QJ, UK. 8 M. K. Greco, Faculty of Science, School of Dentistry and Health Sciences, Charles Sturt 9 University, Panorama Avenue, Bathurst, New South Wales, 2795, Australia. 10 S. Songer, E. Hunter, Centre for Environment Fisheries and Aquaculture Science, Pakefield 11 Rd., Lowestoft, Suffolk, NR33 0HT, UK. 12 13 Manuscript3-D rendering of otolith growth   2   14 Abstract 15 We present a three-dimensional computer reconstruction of a plaice (Pleuronectes platessa) 16 otolith from data acquired by the Diamond Light synchrotron, beamline I12, X-ray source, a 17 high energy (53 – 150 keV) source particularly well suited to the study of dense objects. Our 18 data allowed non-destructive rendering of otolith structure, and for the first time allows 19 examination of otolith annuli (internal ring structures) to be analysed in X-ray tomographic 20 images.  21  22 Keywords 23 3-D Reconstruction; Fish Ageing; Otolith Growth; Phase Contrast; Synchrotron Tomography. 24 25 3-D rendering of otolith growth   3  Fish otoliths have long played an important role in sustainable fisheries management 26 (Campana 2005, Popper et al. 2005). The stock assessment models currently used rely on 27 species specific age profiles obtained from the seasonal patterns of growth marks that their 28 otoliths exhibit (Brophy 2014). Since the shape of otoliths is known to vary both within and 29 between species due to the combined influences of genetic and environmental factors otolith 30 shape is becoming increasingly important in determining the structure of mixed assemblages 31 of fish stocks (Cadrin et al. 2014). Evidence of otolith microstructure, recovered by electron 32 microscopy (Campana & Neilson 1985) and more recent chemical analysis of material at 33 specific points in the accretion time-series and investigations using Raman 34 microspectrometry have highlighted the physico-chemical characteristics of fish otoliths 35 (Jolivet et al. 2008, Sturrock et al. 2012). But although these methods provide increasingly 36 detailed information, all are invasive, ultimately resulting in degradation or even destruction 37 of the tissue once the sampling is performed. 38  39 With X-ray tomography comes the possibility of recovering a full three-dimensional (3-D) 40 otolith model of shape, density and composition, hitherto only approximated by stacking 41 ablated serial sections (Bailey et al. 1995). Preliminary attempts to achieve this using 42 conventional absorption X-ray micro-CT were reported by Hamrin et al. (Hamrin et al. 1999), 43 but only the outer shape of the otolith was recovered ( 44 Arneri et al. ). Although the ambition of the implementation of phase-contrast micro-CT was 45 to reveal the internal otolith structure, this ultimately proved unsuccessful. Working with 46 SkyScan (http://bruker-microct.com) we recently repeated this experiment using a SkyScan 47 1172 desktop microCT with 5 micron spot size source operating at 60 kV. The detector used 48 a 12-bit CCD  camera fitted with Gallium Oxide scintillator, coupled by fibre optic 49 connection.  Our results show that a polychromatic X-ray source was unable to resolve 50 3-D rendering of otolith growth   4  sufficient X-ray absorption contrast to differentiate seasonal growth marks from the bulk 51 composition within the otolith. These results are consistent with those reported previously by 52 Hamrin et al. in 1999. 53  54 To investigate if higher energy X-rays could be used to recover internal growth features we 55 initiated an experiment at Diamond Light (http://www.diamond.ac.uk/) to examine an otolith 56 of the flatfish, plaice (Pleuronectes platessa).  For this experiment we obtained X-ray images 57 from a section of the otolith near the rostrum (highlighted in microscope image Fig. 1) with 58 the aim of demonstrating proof of principle. The field of view is limited in this preliminary 59 study due as the specimen was presented using general purpose mount. The images were 60 processed using the method presented by Paganin et.al. (Paganin et al. 2002) that allows 61 simultaneous phase and amplitude to be extracted from a single defocused image, then three-62 dimensional images were reconstructed using filtered back-projection (Kak & Slaney 1988). 63 A reconstruction of the otolith is shown in Fig. 2.  64  65 Internal growth marks (rings or annuli) are visible in the individual slices (Fig. 3).  The slice 66 exhibits a number of primary rings, suggesting a fish age of 5 years. Software rendering 67 programs such as DISECT (http://www.disectsystems.com/) and TOMOMASK 68 (http://www.tomomask.com/) offer many possibilities for analyses of the reconstructed 69 otolith volume. For example, while physical sections are limited to flat planes the virtual 3-D 70 reconstructed volume can be cut along any curved surface or manifold. We illustrate this in 71 Fig. 4 by reconstructing a surface orthogonal to the x-y plane along a spline curve fitted to the 72 locus of maximum seasonal growth in a selection of slices (an example curve is plotted in 73 Fig. 3). The reconstruction in Fig. 4 illustrates that otolith growth is complex and non-linear. 74 This in turn highlights possible errors resulting from assessing age and growth of individuals 75 3-D rendering of otolith growth   5  from plane sections (current operational practice). An animation of the 3-D reconstruction 76 and a virtual fly-through otolith can be found at 77 https://www.youtube.com/channel/UCbvdIX_NY1EhJeJcYODG6tg. Although some imaging 78 artefacts are evident in the reconstruction (e.g. the corona around the air/otolith interface), 79 this proof of principle study provides a glimpse of the possibilities for otolith imaging using 80 phase contrast synchrotron radiation and also demonstrates the potential for further 3-D 81 rendered tomographic reconstructions using the Diamond Light I12 beamline.  82  83 Previous synchrotron studies of otoliths have used Synchrotron Rapid Scanning X-ray 84 Fluorescence (SRS-XRF) to investigate trace elements (e.g. (Doubleday et al. 2014, Limburg 85 et al. 2007)). This technique is inherently limited to an examination of the otolith surface and 86 so may require thin sections of material to be prepared, resulting in damage to the specimen. 87 The preliminary data from this study are encouraging because they illustrate the potential for 88 more accurate measurement of total seasonal accreted volume (as opposed to growth 89 estimates from 2-D sections). 3-D analysis is also potentially more robust to anomalous 90 secondary growth signatures that do not correspond to seasonal deposits. Such artefacts 91 continue to challenge even the most experienced readers of 2-D otolith sections thereby 92 contributing to uncertainties in age estimates, and consequently stock assessments 93 (de Pontual et al. 2006). Currently, synchrotron studies are very expensive but as the 94 technology becomes more accessible virtual otolith studies using computer graphics could 95 provide a historical perspective for each individual within their environmental context. 96  97 We hope to build on this preliminary study through funded access to the beamline to allow us 98 to render whole otolith specimens, investigate species specific growth traits, and factors that 99 affect the direction of maximal accretion, which seems to change with age (Fig. 3). This may 100 3-D rendering of otolith growth   6  help in understanding why certain species are particularly problematic for human otolith 101 readers and provide a valuable insight into how and why the accreted biomineralisation is 102 related to anatomy, physiology and lifecycle. Preliminary observations of the specimen 103 suggest the study was totally non-invasive and that the otolith in this study was undamaged 104 by the experimental process. This feature of tomographic analysis could be particularly 105 valuable, for example where the otoliths of individual fish have been recovered accompanied 106 by archival data storage tags which may have recorded ambient experience of the same fish 107 for periods over seasons or sometimes years (Sturrock et al. 2012). Such otoliths with 108 accompanying “ground-truth” data represent a rich resource of information concerning 109 individual lifetime movements.  110  111 We would like to thank Mr. N. Corps of Bruker-MicroCT for his help in repeating the 112 MicroCT study and Diamond Light Ltd. for donating the I12 beam time used in this study.  113  114 Bibliography 115 Arneri, E., Bergstrom, N., Cardinale, M., Claes, E., Clevestam, P., de Clerck, N., Doering-116 Arjes, P., Donato, F., Giannetti, G., Hall, R., Jakupsstovu, H., Kastowsky, M., Magnussen, 117 E., Mosegaard, H., Mouritsen, R., Patwardhan, A., Paulsen, H., Sasov, A., Sayles, R., Schatz, 118 M., Schmidt, R., van Dyck, D., van Heel, M. & Wickström, H. (2002), Fish ageing by otolith 119 shape analysis : Final report to the European Commission, [s.n.]. FAIR CT97 3402. 120 http://www.ices.dk/community/Documents/PGCCDBS/fabosa_rapp02_hel.pdf 121 Bailey, K. M., Brown, A. L., Nishimura, A. & Reilly, M. T. (1995), ‘Three-dimensional 122 imaging of walleye pollock otoliths: Reconstruction from serial sections and fluorescent laser 123 cytometry’, Journal of Fish Biology 47, 296–302. 124 3-D rendering of otolith growth   7  Brophy, D. (2014), Chapter Eight - Analysis of growth marks in calcified structures: Insights 125 into stock structure and migration pathways, in S. X. Cadrin, L. A. Kerr & S. Mariani, eds, 126 ‘Stock Identification Methods (Second Edition)’, second edn, Academic Press, San Diego, 127 pp. 141 – 170. http://www.sciencedirect.com/science/article/pii/B9780123970039000084 128 Cadrin, S. X., Karr, L. A. & Mariani, S. (2014), Chapter One - Stock identification methods: 129 An overview, in S. X. C. A. K. Mariani, ed., ‘Stock Identification Methods (Second Edition)’, 130 second edn, Academic Press, San Diego, pp. 1 – 5. 131 http://www.sciencedirect.com/science/article/pii/B9780123970039000011 132 Campana, S. (2005), ‘Otolith science entering the 21st century’, Marine and Freshwater 133 Research 56, 485–495. 134 Campana, S. & Neilson, J. (1985), ‘Microstructure of fish otoliths’, Canadian Journal of 135 Fisheries and Aquatic Sciences 42, 1014–1032. 136 de Pontual, H., Groison, A. L., Piñeiro, C. & Bertignac, M. (2006), ‘Evidence of 137 underestimation of European hake growth in the Bay of Biscay, and its relationship with bias 138 in the agreed method of age estimation’, ICES Journal of Marine Science: Journal du 139 Conseil 63(9), 1674–1681. 140 Doubleday, Z. A., Harris, H. H., Izzo, C. & Gillanders, B. M. (2014), ‘Strontium randomly 141 substituting for calcium in fish otolith aragonite’, Analytical Chemistry 86(1), 865–869. 142 PMID: 24299165. http://dx.doi.org/10.1021/ac4034278 143 Hamrin, S. F., Arneri, E., Doering-Arjes, P., Mosegaard, H., Patwardhan, A., Sasov, A., 144 Schatz, M., Dyck, D. V., Wickström, H. & Van Heel, M. (1999), ‘A new method for three-145 dimensional otolith analysis’, Journal of Fish Biology 54(1), 223–225. 146 http://dx.doi.org/10.1111/j.1095-8649.1999.tb00627.x 147 Jolivet, A., Bardeau, J.-F., Fablet, R., Paulet, Y.-M. & de Pontual, H. (2008), ‘Understanding 148 otolith biomineralization processes: new insights into microscale spatial distribution of 149 3-D rendering of otolith growth   8  organic and mineral fractions from raman microspectrometry’, Analytical and Bioanalytical 150 Chemistry 392(3), 551–560. http://dx.doi.org/10.1007/s00216-008-2273-8 151 Kak, A. & Slaney, M. (1988), Principles of Tomographic Imaging, IEEE Press Inc., New 152 York. 153 Limburg, K. E., Huang, R. & Bilderback, D. H. (2007), ‘Fish otolith trace element maps: new 154 approaches with synchrotron microbeam X-ray fluorescence’, X-Ray Spectrometry 155 36(5), 336–342. http://dx.doi.org/10.1002/xrs.980 156 Paganin, D., Mayo, S. C., Gureyev, T. E., Miller, P. R. & Wilkins, S. W. (2002), 157 ‘Simultaneous phase and amplitude extraction from a single defocused image of a 158 homogeneous object’, Journal of Microscopy 206(1), 33–40. 159 http://dx.doi.org/10.1046/j.1365-2818.2002.01010.x 160 Popper, A. N., Ramcharitar, J. & Campana, S. E. (2005), ‘Why otoliths? Insights from inner 161 ear physiology and fisheries biology’, Marine and Freshwater Research 56, 497–504. 162 Sturrock, A. M., Trueman, C. N., Darnaude, A. M. & Hunter, E. (2012), ‘Can otolith 163 elemental chemistry retrospectively track migrations in fully marine fishes?’, Journal of Fish 164 Biology 81(2), 766–795. http://dx.doi.org/10.1111/j.1095-8649.2012.03372.x 165  166 Fig. 1: Microscope image of plaice otolith captured after exposure to synchrotron radiation. 1 (Note: Area highlighted indicates the approximate location of the volume reconstructed by X-2 ray tomography). 3 Fig. 2: 3-D tomographic rendering of a section of plaice otolith highlighted in Fig. 1 4 recovered by the high energy Joint Engineering, Environmental and Processing (JEEP) I12 5 synchrotron beamline at the UK’s national synchrotron science facility, Diamond Light 6 (http://www.diamond.ac.uk/).  The otolith was imaged using monochromatic X-rays of 7 wavelength λ=.0234 nm (53 keV) and propagation phase contrast (Davis et al. 1995, Snigirev 8 et al. 1995) inherent in such monochromatic X-ray images, was exploited to observe small 9 variations in the sample. The camera was placed 1000mm beyond the sample to obtain this 10 effect. This technique has been previously used with success for fossils and other specimens 11 exhibiting weak variation in contrast (Tafforeau et al. 2006).  The detector used was a 12 CdWO4 (Cadmium Tungstate)  scintillator viewed through bespoke radiation-hard 13 microscope optics (SILL, Germany) by a PCO.EDGE (PCO, Germany) camera having 14 2560x2150 pixels, the resulting image pixel size being 5um square. 15 Fig. 3: Spline curve fitted to a set of maximal annular growth features identified in one of the 16 image slices. Note: The direction of maximal growth varies throughout the life of the fish. 17 Fig. 4: Virtual section through the otolith taken along the curved surface orthogonal to the 18 spline curve shown in Fig. 3. 19 Figure CaptionsFigure Click here to download Figure Fig_1.tif Figure Click here to download Figure Fig_2.tif Figure Click here to download Figure Fig_3.tif Figure Click here to download Figure Fig_4.tif 

Three-dimensional rendering of otolith growth using phase contrast synchrotron tomography

Three-dimensional rendering of otolith growth using phase contrast synchrotron tomography

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