Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

Background The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals. Results Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish. Conclusions The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration

Design, Construction and Installation of the ATLAS Hadronic Barrel Scintillator-Tile Calorimeter

The scintillator tile hadronic calorimeter is a sampling calorimeter using steel as the absorber structure and scintillator as the active medium. The scintillator is located in "pockets" in the steel structure and the wavelength-shifting fibers are contained in channels running radially within the absorber to photomultiplier tubes which are located in the outer support girders of the calorimeter structure. In addition, to its role as a detector for high energy particles, the tile calorimeter provides the direct support of the liquid argon electromagnetic calorimeter in the barrel region, and the liquid argon electromagnetic and hadronic calorimeters in the endcap region. Through these, it indirectly supports the inner tracking system and beam pipe. The steel absorber, and in particular the support girders, provide the flux return for the solenoidal field from the central solenoid. Finally, the end surfaces of the barrel calorimeter are used to mount services, power supplies and readout crates for the inner tracking systems and the liquid argon barrel electromagnetic calorimeter

The Optical Instrumentation of the ATLAS Tile Calorimeter

The purpose of this Note is to describe the optical assembly procedure called here Optical Instrumentation and the quality tests conducted on the assembled units. Altogether, 65 Barrel (or LB) modules were constructed - including one spare - together with 129 Extended Barrel (EB) modules (including one spare). The LB modules were mechanically assembled at JINR (Dubna, Russia) and transported to CERN, where the optical instrumentation was performed with personnel contributed by several Institutes. The modules composing one of the two Extended Barrels (known as EBA) were mechanically assembled in the USA, and instrumented in two US locations (ANL, U. of Michigan), while the modules of the other Extended barrel (EBC) were assembled in Spain and instrumented at IFAE (Barcelona). Each of the EB modules includes a subassembly known as ITC that contributes to the hermeticity of the calorimeter; all ITCs were assembled at UTA (Texas), and mounted onto the module mechanical structures at the EB mechanical assembly locations.The Tile Calorimeter, covering the central region of the ATLAS experiment up to pseudorapidities of ±1.7, is a sampling device built with scintillating tiles that alternate with iron plates. The light is collected in wave-length shifting (WLS) fibers and is read out with photomultipliers. In the characteristic geometry of this calorimeter the tiles lie in planes perpendicular to the beams, resulting in a very simple and modular mechanical and optical layout. This paper focuses on the procedures applied in the optical instrumentation of the calorimeter, which involved the assembly of about 460,000 scintillator tiles and 550,000 WLS fibers. The outcome is a hadronic calorimeter that meets the ATLAS performance requirements, as shown in this paper

The Production and Qualification of Scintillator Tiles for the ATLAS Hadronic Calorimeter

The production of the scintillator tiles for the ATLAS Tile Calorimeter is presented. In addition to the manufacture and production, the properties of the tiles will be presented including light yield, uniformity and stability

Mechanical construction and installation of the ATLAS tile calorimeter

This paper summarises the mechanical construction and installation of the Tile Calorimeter for the ATLAS experiment at the Large Hadron Collider in CERN, Switzerland. The Tile Calorimeter is a sampling calorimeter using scintillator as the sensitive detector and steel as the absorber and covers the central region of the ATLAS experiment up to pseudorapidities +/- 1.7. The mechanical construction of the Tile Calorimeter occurred over a period of about 10 years beginning in 1995 with the completion of the Technical Design Report and ending in 2006 with the installation of the final module in the ATLAS cavern. During this period approximately 2600 metric tons of steel were transformed into a laminated structure to form the absorber of the sampling calorimeter. Following instrumentation and testing, which is described elsewhere, the modules were installed in the ATLAS cavern with a remarkable accuracy for a structure of this size and weight

Integrated monitoring of mola mola behaviour in space and time

Over the last decade, ocean sunfish movements have been monitored worldwide using various satellite tracking methods. This study reports the near-real time monitoring of finescale (< 10 m) behaviour of sunfish. The study was conducted in southern Portugal in May 2014 and involved satellite tags and underwater and surface robotic vehicles to measure both the movements and the contextual environment of the fish. A total of four individuals were tracked using custom-made GPS satellite tags providing geolocation estimates of fine-scale resolution. These accurate positions further informed sunfish areas of restricted search (ARS), which were directly correlated to steep thermal frontal zones. Simultaneously, and for two different occasions, an Autonomous Underwater Vehicle (AUV) videorecorded the path of the tracked fish and detected buoyant particles in the water column. Importantly, the densities of these particles were also directly correlated to steep thermal gradients. Thus, both sunfish foraging behaviour (ARS) and possibly prey densities, were found to be influenced by analogous environmental conditions. In addition, the dynamic structure of the water transited by the tracked individuals was described by a Lagrangian modelling approach. The model informed the distribution of zooplankton in the region, both horizontally and in the water column, and the resultant simulated densities positively correlated with sunfish ARS behaviour estimator (r(s) = 0.184, p < 0.001). The model also revealed that tracked fish opportunistically displace with respect to subsurface current flow. Thus, we show how physical forcing and current structure provide a rationale for a predator's finescale behaviour observed over a two weeks in May 2014

Comparing the effectiveness of hyperspectral imaging and Raman spectroscopy:A case study on Armenian manuscripts

There is great practical and scholarly interest in the identification of pigments in works of art. This paper compares the effectiveness of the widely used Raman Spectroscopy (RS), with hyperspectral imaging (HSI), a reflectance imaging technique, to evaluate the reliability of HSI for the identification of pigments in historic works of art and to ascertain if there are any benefits from using HSI or a combination of both. We undertook a case study based on six Armenian illuminated manuscripts (eleventh–eighteenth centuries CE) in the Bodleian Library, University of Oxford. RS, and HSI (380–1000 nm) were both used to analyse the same 10 folios, with the data then used to test the accuracy and efficiency of HSI against the known results from RS using reflectance spectra reference databases compiled by us for the project. HSI over the wavelength range 380–1000 nm agreed with RS at best 93% of the time, and performance was enhanced using the SFF algorithm and by using a database with many similarities to the articles under analysis. HSI is significantly quicker at scanning large areas, and can be used alongside RS to identify and map large areas of pigment more efficiently than RS alone. HSI therefore has potential for improving the speed of pigment identification across manuscript folios and artwork but must be used in conjunction with a technique such as RS

Multi-analytical characterisation of D’Aprés Cormon by José Veloso Salgado

This case study reveals the analytical characterisation carried out on ‘D’Aprés Cormon’ (1891) by Veloso Salgado, a copy of ‘Caïn’ (1880) by Fernando Cormon, belonging nowadays to Musée d’Orsay. Infrared Reflectography made in situ revealed underdrawing grid that was used to transpose the original painting to a smaller scale, pictorial style, execution method and retouched/restored areas. In situ EDXRF analysis together with Raman microscopy allowed the identification of gypsum, lead white, titanium white, yellow ochre, vermilion, ultramarine and lamp black. These results provide valuable information about the Salgado’s palette and his production technique

Analytical characterization of academic nude paintings by José Veloso Salgado

This case study illustrates the analytical characterization of 12 academic nude paintings by Veloso Salgado, made between 1883 and 1893. The study was done with in situ methods (infrared reflectography and EDXRF) and benchtop equipment (Raman and FTIR microscopes and SEM-EDS). Infrared reflectography revealed underdrawings, painting style, execution method and retouched/restored areas. Analytical techniques allowed the identification of gypsum, lead white, barium white, zinc white, yellow ochre, chrome yellow, zinc yellow, vermilion, synthetic ultramarine blue, cerulean blue, Prussian blue, viridian, green earth, Paris green, red ochre, raw umber and lamp black. These results provide valuable information on Salgado’s palette and his artistic production technique and allow the distinction between palettes used in Lisbon and Paris and consequently, technical interchanges between both Academies. Finally, this study intends to start a systematic analytical study of the Faculty of Fine Arts’ collection, allowing the characterization of many other authors and their influences for the development of painting methodology in this Academy