Autofluorescence lifetime of gelatin-methacrylate hydrogels is sensitive to changes in crosslinking and post-gelation...

There are limited tools to non-destructively evaluate the physicochemical properties of biomaterials in in vivo and in vitro settings. Here, we evaluate the autofluorescence of gelatin-methacrylate (GelMA) hydrogels through fluorescence lifetime imaging (FLIM) to assess its sensitivity to hydrogel photocrosslinking and post-gelation pH. GelMA is increasingly used as a biomaterial because it is highly tunable and has great biocompatibility.

Using two-photon excitation, we identified optimal acquisition and analysis approaches to detect changes in the autofluorescence lifetime of gelatin. Through a combination of phasor analysis and multi-exponential modeling, we determined that a tri-exponential decay fit accurately captured multiple intrinsic fluorescent species in gelatin.

The mean lifetime ( and the relative contribution ratios of the lifetime species (A₁/A₂, A₁/A₃, and A₂/A₃) were sensitive to both crosslinking and microenvironmental pH. As hydrogel crosslinking increased following an increase in photoinitiator concentration from 0% to 0.50%, lifetime parameters, A₁/A₃ and A₂/A₃ were altered. Moreover, as hydrogels were submerged in buffers with pH values increasing from 5.8 to 9.0, lifetime parameters , A₁/A₂ and A₁/A₃ changed.

These results demonstrate that FLIM has potential in distinguishing between distinct alterations to the molecular microenvironment of biomaterials without the need for exogenous fluorophores. In addition, we demonstrate that lifetime changes associated with crosslinking are related to gelatin autofluorescence rather than any crosslink autofluorescence.