Progress and new challenges in image-based profiling.

For over two decades, image-based profiling has revolutionized cellular phenotype analysis. Image-based profiling processes rich, high-throughput, microscopy data into unbiased measurements that reveal phenotypic patterns powerful for drug discovery, functional genomics, and cell state classification. Here, we review the evolving computational landscape of image-based profiling, detailing current procedures, discussing limitations, and highlighting future development directions. Deep learning has fundamentally reshaped image-based profiling, improving feature extraction, scalability, and multimodal data integration. Methodological advancements such as single-cell analysis and batch effect correction, drawing inspiration from single-cell transcriptomics, have enhanced analytical precision. The growth of open-source software ecosystems and the development of community-driven standards have further democratized access to image-based profiling, fostering reproducibility and collaboration across research groups. Despite these advancements, the field still faces significant challenges requiring innovative solutions. By focusing on the technical evolution of image-based profiling rather than the wide-ranging biological applications, our aim with this review is to provide researchers with a roadmap for navigating the progress and new challenges in this rapidly advancing domain.