Automated <scp>FRAP</scp> microscopy for high‐throughput analysis of protein dynamics in chromatin organization and transcription

Fluorescence recovery after photobleaching (FRAP) is a quantitative technique to study the dynamics of fluorescently tagged proteins in living cells. Current FRAP workflows are limited in throughput because of the requirement for human interaction. Here, we present RoboMic, a fully automated confocal microscopy platform for high-throughput imaging assays such as FRAP. We demonstrate its capabilities using two complementary approaches: sequential FRAP (sFRAP) and a novel parallel FRAP (pFRAP). The latter enables simultaneous photobleaching and monitoring of multiple cells within one imaging cycle, increasing throughput by approximately five- to 10-fold while maintaining spatiotemporal resolution. The protocol consists of microscope control software for automated, AI-based selection and segmentation of cell nuclei, sub-nuclear ROI definition, photobleaching, and time-lapse imaging. As proof of concept, we examined the nuclear dynamics of the androgen receptor and the cohesin complex under diverse conditions, demonstrating that RoboMic generates robust and reproducible data. In a single session, the platform yields hundreds of FRAP measurements, thereby increasing statistical power and scalability for large-scale studies of protein mobility. While we focus here on FRAP, RoboMic can be readily applied to a wide range of quantitative functional imaging assays.