Despite fundamental advances in spatial omics, investigating cellular and molecular functions within their native environment remains a challenge in multiscale systems biology, especially in response to organ-level events. Here, we introduce Spatial ProBe (micro-ProBe), a multimodal imaging approach for spatial profiling of biological micro-environments. Spatial ProBe enables preprocessing, registration and correlative analysis of 2D and 3D imaging modalities, supported by an intuitive user interface. As an application, we investigated bone mechanobiology and characterised the cellular and molecular responses to mechanical loading during adaptation and regeneration, which continuously regulate the local microarchitecture. By integrating time-lapsed micro-computed tomography and end-point spatial transcriptomics, we profiled the local mechanical in vivo environment of thousands of musculoskeletal cells, revealing the spatiotemporal interplay between local mechanics and gene expression driving tissue development. Spatial ProBe marks a crucial advance in the characterisation of multiscale tissues and signalling, facilitating the exploration of targeted molecular therapies for pathological conditions.