Image analysis in light sheet fluorescence microscopy images of transgenic zebrafish vascular development

The zebrafish has become an established model to study vascular development and disease in vivo. However, despite it now being possible to acquire high-resolution data with state-of-the-art fluorescence microscopy, such as lightsheet microscopy, most data interpretation in pre-clinical neurovascular research relies on visual subjective judgement, rather than objective quantification. Therefore, we describe the development of an image analysis workflow towards the quantification and description of zebrafish neurovascular development. In this paper we focus on data acquisition by lightsheet fluorescence microscopy, data properties, image pre-processing, and vasculature segmentation, and propose future work to derive quantifications of zebrafish neurovasculature development

Enhancement and Segmentation Workflow for the Developing Zebrafish Vasculature †

Zebrafish have become an established in vivo vertebrate model to study cardiovascular development and disease. However, most published studies of the zebrafish vascular architecture rely on subjective visual assessment, rather than objective quantification. In this paper, we used state-of-the-art light sheet fluorescence microscopy to visualize the vasculature in transgenic fluorescent reporter zebrafish. Analysis of image quality, vascular enhancement methods, and segmentation approaches were performed in the framework of the open-source software Fiji to allow dissemination and reproducibility. Here, we build on a previously developed image processing pipeline; evaluate its applicability to a wider range of data; apply and evaluate an alternative vascular enhancement method; and, finally, suggest a work-flow for successful segmentation of the embryonic zebrafish vasculature

3D Quantification and Description of the Developing Zebrafish Cranial Vasculature

Background: Zebrafish are an excellent model to study cardiovascular development and disease. Transgenic reporter lines and state-of-the-art microscopy allow 3D visualization of the vasculature in vivo. Previous studies relied on subjective visual interpretation of vascular topology without objective quantification. Thus, there is the need to develop analysis approaches that model and quantify the zebrafish vasculature to understand the effect of development, genetic manipulation or drug treatment. Aim: To establish an image analysis pipeline to extract quantitative 3D parameters describing the shape and topology of the zebrafish vasculature, and examine how these are impacted during development, disease, and by chemicals. Methods: Experiments were performed in zebrafish embryos, conforming with UK Home Office regulations. Image acquisition of transgenic zebrafish was performed using a Z.1 Zeiss light-sheet fluorescence microscope. Pre-processing, enhancement, registration, segmentation, and quantification methods were developed and optimised using open-source software, Fiji (Fiji 1.51p; National Institutes of Health, Bethesda, USA). Results: Motion correction was successfully applied using Scale Invariant Feature Transform (SIFT), and vascular enhancement based on vessel tubularity (Sato filter) exceeded general filter outcomes. Following evaluation and optimisation of a variety of segmentation methods, intensity-based segmentation (Otsu thresholding) was found to deliver the most reliable segmentation, allowing 3D vascular volume measurement. Following successful segmentation of the cerebral vasculature, a workflow to quantify left-right intra-sample symmetry was developed, finding no difference from 2-to-5dpf. Next, the first vascular inter-sample registration using a manual landmark-based approach was developed and it was found that conjugate direction search allowed automatic inter-sample registration. This enabled extraction of age-specific regions of similarity and variability between different individual embryos from 2-to-5dpf. A workflow was developed to quantify vascular network length, branching points, diameter, and complexity, showing reductions in zebrafish without blood flow. Also, I discovered and characterised a previously undescribed endothelial cell membrane behaviour termed kugeln. Conclusion: A workflow that successfully extracts the zebrafish vasculature and enables detailed quantification of a wide variety of vascular parameters was developed