Abstract Advances in fluorescence microscopy and tissue-clearing technology have revolutionized three-dimensional (3D) imaging of fluorescently labeled tissues, organs, and embryos. However, the complexity and high cost of existing software and computer solutions for such imaging limit its widespread adoption by researchers with limited resources. We here introduce Acto3D as a user- and budget-friendly, open-source computer software application designed to streamline the generation and observation of high-resolution 3D images of targets labeled with multiple fluorescent probes. Acto3D features an intuitive interface that simplifies the importation, visualization, and analysis of data sets, has an associated tool for annotation of vascular lumens, and incorporates multiple fluorescence channels for comprehensive imaging. Underpinned by an integrated graphics processing unit, Acto3D allows accurate image reconstruction and efficient data processing without the need for expensive high-performance computers. We validated the software by imaging mouse embryonic structures. Acto3D thus constitutes a cost-effective and efficient platform to support biological research.

Acto3D: user- and budget-friendly software for multichannel high-resolution three-dimensional imaging