Contact‐Free Mechanical Mapping of 3D Microprinted Hydrogels Using FLIM

Mechanical characterization of 3D printed hydrogel microstructures typically relies on invasive, contact‐based methods that are challenging to perform in aqueous environments. Here, we present a fluorescence lifetime imaging microscopy (FLIM)‐based approach that exploits the intrinsic autofluorescence of two‐photon 3D laser‐printed hydrogel microstructures to enable label‐ and contact‐free mechanical mapping. Poly(ethylene glycol) (PEG)‐based micropillar arrays are fabricated with systematically varied laser doses to modulate the degree of network crosslinking, thereby tuning the material stiffness. FLIM measurements reveal a monotonic increase in the intensity‐averaged autofluorescence lifetime with increasing laser dose, reflecting a concomitant increase in stiffness. To quantify the mechanical properties of the 3D printed microstructures in aqueous media, we develop a buoyancy‐corrected nanoindentation protocol. The resulting reduced moduli exhibit the same trend and show strong agreement with the fluorescence lifetime measurements. In addition, the autofluorescence signal of the micropillars proved highly sensitive to changes in the hydration state. Thus, these results demonstrate that FLIM is a powerful, noninvasive tool for high‐resolution mechanical mapping of soft materials, particularly in conditions where other more conventional methodologies are challenging.