In this work, we present a contact-free method to map mechanical properties of microscale 3D printed hydrogel microstructures using fluorescence-lifetime imaging microscopy (FLIM). Specifically, polyethylene glycol (PEG)-based cylindrical pillars were printed using two-photon 3D laser printing, and their autofluorescence lifetime imaging data are correlated to their mechanical properties, obtained by nanoindentation. The autofluorescence lifetime signal of the printed PEG-based hydrogel varies with the printing laser dose, from approximately 1.0 ns at the lowest dose to approximately 3.0 ns at the highest dose, while the actual stiffness of the structures spans a narrow range between 3 and 5 MPa. In addition, the effect of hydration on the 3D-printed microstructures was studied, revealing that dehydration and subsequent rehydration irreversibly alter both the autofluorescence signal and the mechanical properties.

This dataset contains four types of raw data: (1) fluorescence lifetime imaging microscopy (FLIM) data; (2) Raman spectroscopy data; (3) nanoindentation data in the form of load–displacement curves, enabling the determination of local mechanical properties such as the reduced elastic modulus; and (4) microscopy images.