Progress and new challenges in image-based profiling.

For over two decades, image-based profiling has revolutionized cell phenotype analysis. Image-based profiling processes rich, high-throughput, microscopy data into thousands of 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 the bioinformatics processes involved from feature extraction to normalization and batch correction. We discuss how deep learning has fundamentally reshaped the field. We examine key methodological advancements, such as single-cell analysis, the development of robust similarity metrics, and the expansion into new modalities like optical pooled screening, temporal imaging, and 3D organoid profiling. We also highlight the growth of public benchmarks and open-source software ecosystems as a key driver for fostering reproducibility and collaboration. Despite these advances, the field still faces substantial challenges, particularly in developing methods for emerging temporal and 3D data modalities, establishing robust quality control standards and workflows, and interpreting the processed features. 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.