Summary This application seeks support to develop the next generation of the Advanced Normalization Tools (ANTs) ecosystem, an open-source library spanning multiple software packages and programming languages that houses top-performing algorithms used worldwide by a large community of researchers. The core software library, ANTs, is built upon and contributes to the well-known NIH-funded Insight Toolkit (ITK), which has historically been a significant resource of open-source algorithmic and software innovation. Over the course of its decade-long development, ANTs has enabled hundreds of academic and industrial scientists to meet modern quantitative imaging needs with particular focus on issues in biomedical imaging-a broad range of applications and published research literature sample the study of organisms from small animals to humans as well as target organ systems such as respiratory, cardiovascular, and nervous. Today, ANTs is a freely available, cross-platform toolkit for multiple modality image processing which continues to set the standard in the field. However, despite its success in meeting the real-world needs of a broad community of investigators, the rapidly growing use of imaging, and the associated need to solve ever more complex image registration problems, require a dynamic update of ANTs in order to meet current and emerging needs of the biomedical research community. This major update, representing the first ever application for dedicated funding support of ANTs development, will comprise of the four specific aims of this project: 1) to significantly broaden the range of multiple modality registration tasks able to benefit from AN Ts-based solutions; 2) to modernize and expand the tool's support of very large datasets and scalable computing platforms; 3) to provide reference registration protocols and validate their reliability in investigator-driven research studies; and 4) to enhance support and outreach to the user community. These developments will ensure the long-term viability of ANTs in the face of significant advancements in computing and imaging technologies, as well as enable an even broader research audience to leverage ANTs in their scientific output.