Project Summary/Abstract The ability of experimentalists to investigate biological systems has often been limited to pre-programmed open-loop protocols whereby systems are probed based on assumptions and predetermined models. In contrast, real-time feedback control allows systems to be dynamically probed with parameter perturbations calculated as functions of instantaneous closed-loop system measurements, enabling researchers to address questions not amenable to open-loop approaches. While there are many biological problems that can be made more tractable by such feedback control – such as dynamic probing of ion-channel function in neurons or manipulation of sleep network dynamics using auditory and electrical stimuli governed by real- time EEG feedback – technical complexities often hinder its adoption. Because there has been a many-decades advance in computing technologies, it is counter-intuitive that real-time experiment control is not possible with standard computer operating systems (without expensive add-on components). They are simply not designed for such strictly timed tasks. Furthermore, commercial real-time systems are costly and require end-user customization for the lab, such that they are typically not portable to the broader scientific community. To circumvent these limitations, we developed a fast and highly versatile real-time biological experimentation system – the Real-Time eXperiment Interface (RTXI). RTXI is free and open source, compatible with an extensive range of experimentation hardware, and delivers reproducible, hard real-time performance via a user-tailorable interface on standard laboratory computers. Importantly, RTXI offers extensive versatility and high-performance to “power users,” while simultaneously providing a rich – and ever growing – library of experiment-control modules that require little effort for those who are not computationally savvy. RTXI has grown to the point where it is now an invaluable part of the scientific programs of many leading research groups. In addition to updating and maintaining RTXI for those, and future, users, there remain important avenues for development that would substantially expand RTXI’s functionality, reproducibility enhancement, and utility for an even broader group of biological researchers. Thus, we propose: 1. To keep RTXI pushing scientific innovation by overhauling its base code and core modules. 2. To enable several new real-time experiment paradigms. 3. To ease and enrich experimentalists’ user experience and reproducibility. Critically, the work proposed here will ensure that RTXI remains a valuable research tool for its varied group of outstanding biological scientist end users. Furthermore, we will expand RTXI’s utility significantly by building and supporting additional classes of experiments and the groundbreaking science they will enable.