Insight, innovation, integrationCell and tissue fate is intimately linked to the biochemistry and physical properties of their microenvironments (including mesenchyme, stroma and stem cell niches), yet no tools were available to probe how cell-generated tensile forces physically alter the properties of 3D extracellular matrix. FRET-labeled fibronectin is exploited as a mechanical strain sensor to monitor time-dependent changes of either tissue-mimetic 3D, ECM scaffolds reseeded with cells or of the newly assembled matrix fibrils. Thisin situmethod is versatile, since cells, provided exogenously with FRET-fibronectin, typically co-assembled it into their ECM fibers. Probing how environmental conditions regulate cell-mediated stretching of ECM fibers is physiologically significant because stretching alters fiber rigidity and the presentation of molecular and cellular recognition sites (mechanotransduction).