Nanoneedles enable spatiotemporal lipidomics of living tissues.

Spatial biology provides high-content diagnostic information by mapping the molecular composition of tissues. However, traditional spatial biology approaches typically require non-living samples, limiting temporal analysis. Here, to address this limitation, we present a workflow using porous silicon nanoneedles to repeatedly collect biomolecules from live brain tissues and map lipid distribution through desorption electrospray ionization mass spectrometry imaging. This method preserves the integrity of the original tissue while replicating its spatial molecular profile on the nanoneedle substrate, accurately reflecting lipid distribution and tissue morphology. Machine learning analysis of 23 human glioma biopsies demonstrated that nanoneedle sampling enables the precise classification of disease states. Furthermore, a spatiotemporal analysis of mouse gliomas treated with temozolomide revealed time- and treatment-dependent variations in lipid composition. Our approach enables non-destructive spatiotemporal lipidomics, advancing molecular diagnostics for precision medicine.