Imaging-Based Spatial Transcriptomics: Data Interpretation Methods and Biomedical Applications.

Imaging-based spatial transcriptomics has advanced from low-plex single-molecule fluorescence in situ hybridization to a diverse set of highly multiplexed platforms, with recent multimodal and pathology-compatible capabilities. Despite major differences in chemistry, coding, and imaging strategies across different platforms, their biological interpretation often converges on a few notable computational biology problems. This review examines imaging-based spatial transcriptomics through the lens of data interpretation and applications, focusing on the analytical framework that converts raw fluorescence signals or accompanying in situ sequencing data into molecule-, cell-, and tissue-level representations. We discuss the key challenges in preprocessing, registration, restoration, feature detection, barcode decoding, molecule calling, cell segmentation, transcript assignment, probabilistic cell typing, spatial-domain inference, and atlas integration. We also highlight how optical crowding, tissue thickness, panel bias, and multimodal complexity increase computational difficulty. Finally, we summarize applications of imaging-based spatial transcriptomics techniques, ranging from subcellular RNA localization to atlas-scale and pathology-aware spatial analysis.