ABSTRACT Cellular behavior is regulated by diverse signaling mechanisms that often depend on molecular dynamics precisely organized in space and time. The transient positioning and kinetics of molecular events is lost in bulk biochemical analysis and in single cell proteomics. Understanding such factors requires visualization and quantitative analysis of molecular events in living cells and tissues, now made possible by combining molecular probe design, high-resolution live cell microscopy and computational image analysis. However, combining these diverse technology into a working whole for the quantitative analysis of live cell signaling networks remains challenging. The proposed Center for Cell Signaling Analysis aims to democratize advanced methods that put these tools in the hands of scientists who have not devoted their careers to imaging. We propose to develop and disseminate a user-friendly and integrated pipeline that combines 1) biosensors, optogenetics and chemogenetics 2) modular, high-speed, and high-resolution light-sheet microscopes, and 3) image analysis and computational modeling to derive signaling network architecture, including the causality and kinetics of connections. The Center will concentrate its integration effort between 3 Technology Development Projects: i) Optogenetics, chemogenetics, and biosensors based on alternate approaches with complementary capabilities and reduced perturbation of signaling. ii) Multiple modular, cost-effective, and high-resolution 3D light-sheet microscopes that can be assembled rapidly by non-experts and deliver ~220 and ~450 nm lateral and axial resolution. These will be designed to maximally leverage computer vision workflows, biosensors, and optogenetics/chemogenetics. iii) A library of open-source Fiji-based plugins for the analysis of subcellular signal transduction in 2D and 3D live cell time-lapse data using advanced methods in statistical time series analysis. Through our collaborative Driving Biological Projects (DBPs), we will iteratively refine and improve our probes, imaging platforms and image analysis methods. We have identified 8 DBPs that address topics in immune cell migration, immunological synapse function, mechanosensation, sarcomere formation, cancer dissemination and drug resistance, synaptic plasticity, and ventral furrow ingression. We propose a strong dissemination component that maximally leverages existing infrastructure, including imaging facilities with >3,000 users located throughout the United States, the widely adopted Fiji software ecosystem, Addgene, GitHub, and Applied Scientific Instrumentation. We will provide extensive training (in person, remote, and topic-driven courses) and centrally organize the Center outputs on a comprehensive and continuously updated website.