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

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