Correlative multimodal imaging for microscale spatial mapping of collagen-gene activity interactions in human tissues

Abstract Understanding how gene activity relates to other biological structures is critical to investigate tissue remodeling processes, disease, and regeneration. RNAscope in situ hybridization assay provides single-molecule detection of targeted transcripts, while label-free multiphoton microscopy enables high-resolution, quantitative imaging of extracellular matrix collagen. These modalities have not previously been combined to extract spatially resolved correlations between molecular and structural features within the same tissue section. Here, we introduce correlative multimodal imaging that integrates RNAscope with Second Harmonic Generation microscopy to align transcript localization with quantitative metrics of collagen architecture at microscale resolution. We applied this approach to human skeletal muscle biopsies of healthy and diseased patients, affected by Duchenne Muscular Dystrophy. Applying our workflow, we observed that, in this proof-of-concept, regions enriched in specific dystrophin transcripts (targeting exons 37–42 and 63–75) are associated with localized increases in collagen fiber length and density, suggesting a potential spatial correlation between dystrophin transcript distribution and collagen organization. This workflow enables microscale integration of molecular and structural data. Moreover, it can be readily extended to diverse tissues, targets, and disease contexts, providing a versatile platform for a deeper spatial biomarker discovery, fibrosis and regeneration studies, microscale evaluation of morphological effects on tissue of transcript-based therapies.