Combined use of hyperspectral imaging and chemometrics to investigate the development of zebrafish embryos

Abstract

Zebrafish (Danio rerio) is a widely used model organism in biological research due to its high genetic similarity to humans. The optical transparency of the embryos, easy accessibility and fast development are the characteristics that make zebrafish particularly suitable for developmental and biomedical research. This project will focus on the investigation of the lipid distribution during zebrafish embryonic development, specifically at the middle-epiboly stage. To achieve this goal, embryos were stained using two lipidic-selective fluorophores, Nile Red and Lipi-Green, and analysed using confocal fluorescence hyperspectral imaging. Initially, embryos were stained with a single fluorophore at a time to evaluate its specificity and distribution. Subsequently, both fluorophores were applied simultaneously to enable a combined analysis of lipid components across the different embryonic regions. Due to the large and complex nature of hyperspectral datasets, chemometric analysis based on Multivariate Curve Resolution – Alternating Least Squares (MCR-ALS) was applied to extract meaningful information, obtaining the pure spectral signatures and the spatial distribution of the different lipid components. The results showed that Nile Red stained the three main embryonic regions – the yolk, the yolk syncytial layer (YSL) and the embryonic cells –, exhibiting two distinct emission shifts associated with differences in lipid polarity of the components. In contrast, Lipi-Green labelled a single lipid component, present in specific parts of the three regions. Overall, this study demonstrates that the combination of fluorescence hyperspectral imaging and chemometric analysis is a powerful approach for characterizing chemical and morphological changes during zebrafish embryonic development.