SPIMTOP

Combined Selective Plane Illumination Microscopy and Optical Projection Tomography for in vivo quantitative imaging of the developing zebrafish vasculature

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 Obiettivo del progetto (Objective)

'Recently conceived three-dimensional microscopy techniques are revolutionizing developmental biology and embryology studies, being able to visualize entire organisms in-vivo. Despite increasingly proposed imaging solutions, methods suitable to quantify dynamic and mechanical parameters over a large region of a biological specimen are still lacking. This proposal aims to construct an optical system and a computational framework able to quantitatively study the developing embryonic zebrafish vasculature, looking at the interaction between gene expression and hemodynamic parameters, at high spatial resolution, over a large volume of the organism. The instrument will exploit an advanced version of Optical Projection Tomography, in order to provide a detailed three-dimensional map of the zebrafish cardiovascular system annotated with blood flow and shear stress parameters. Co-registered Selective Plane Illumination Microscopy images will enable the users to observe the expression of fluorescent markers at sub-cellular resolution and to correlate it to the hemodynamic/mechanical parameters. Low photo-bleaching and high acquisition speeds make this multimodal technology ideally suited for extended time-lapse experiments in live embryos. The system will be employed to characterize the development of the embryonic cardiovascular system in normal and mutant zebrafish embryos. The researcher, proponent of this project, will be integrated in a highly multidisciplinary team and will be trained on microscopy and biological aspects of the research, being able to acquire new skills relevant for the development of his optical tomography laboratory and his career.'

Introduzione (Teaser)

Merging two optical systems means that researchers can visualise the development of entire living organisms. Researchers worked to realise such a system.

Descrizione progetto (Article)

Selective plane illumination microscopy (SPIM), based on fluorescence of proteins in a sample, can image developmental processes in vivo with rapid high-resolution optical sectioning. Unable to image structures beyond the labelled tissue, SPIM can be complemented with an appropriate technique such as optical projection tomography (OPT).

A multimodality system that exploits the features of SPIM and OPT has not previously been fully investigated. The EU-funded project SPIMTOP (Combined selective plane illumination microscopy and optical projection tomography for in vivo quantitative imaging of the developing zebrafish vasculature) has combined the two systems.

Researchers developed a data analysis method to yield a detailed dynamic map of the developing vascular system of the zebrafish. Combining the two systems revealed a limitation as OPT requires a long depth of field and SPIM benefits from a shallow range. Overcoming this technical hitch without modifications to hardware, SPIMTOP took a stack of transmission images and created a dataset for tomographic reconstruction.

The new method can be readily adopted for a large number of systems including commercial light sheet microscopes. The researchers also developed a real-time routine. This records only relevant data for the reconstruction, is compatible with in vivo imaging and time-lapsed observation of development and also integrates well with rapid SPIM recording.

Developmental biology is a rapidly growing area especially with the input of gene expression to morphological data. SPIMTOP technology will broaden the horizons for quantitatively imaging development of translucent organisms such as the zebrafish. Project innovations promise to lend a competitive edge to EU research.