This paper reports a multifunctional platform based on a nanocomposite hydrogel combining poly(ethylene glycol), with rhodamine B‐containing silica nanoparticles (RhB@SiO2), as temperature sensors, and gold nanorods (AuNRs) as plasmonic heaters. This composite material acts as a light‐addressable cellular matrix able to induce 3D temperature gradients locally and dynamically using the localized surface plasmon resonance (LSPR) of AuNRs under near‐infrared (NIR) laser illumination. At the same time, the temperature changes are probed locally by monitoring changes of the RhB@SiO2 NPs fluorescence. As a result of plasmonic heating, and, depending on the preparation protocol, the light‐addressable hydrogel also deforms controllably and reversibly, allowing mechanical and thermal cellular stimulation in a 3D matrix. The hydrogel deformation is quantified by means of inline holographic microscopy. This approach makes it possible to accurately and locally control and simultaneously measure temperature gradients and deformation in soft, 3D deformable materials and will enable novel platforms for studying cellular thermo‐ and mechanobiology.

Shear rheology and atomic force microscopy (AFM) are used to characterize the stiffness of hydrogels in tissue engineering applications, with several studies reporting differences of several orders of magnitude in the elastic moduli determined by these two methods. This work compares the elastic properties of soft fibrin and polyethylene glycol (PEG) hydrogels used for stem cell applications, determined by AFM indentation with different probe sizes (from nano‐ to micrometer) to shear rheometry data. For all hydrogels, AFM nanoscale probing consistently yields higher elastic modulus (E) values and variability than micrometer‐probe indentation, while the shear modulus (G) values determined are the lowest. Colloidal probe AFM results are closer to rheology data for the stiffest samples, where E/G ratios converge to the theoretical Trouton ratio of 3. The results suggest that high polymer concentration hydrogels are better described by the affine elastic network theory, whereas low polymer concentration hydrogels deviate significantly from the Trouton ratio. Thus, for soft hydrogels relevant for stem cell culture, the assumption E = 3G is often invalid and care should be taken when comparing data from studies where different characterization methods are used in order to discern the impact of material properties on cell behavior.

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