Stretching individual living cells with light is a standard method to assess their mechanical properties. Yet, heat introduced by the laser light of optical stretchers may unwittingly change the mechanical properties of cells therein. To estimate the temperature induced by an optical trap, we introduce cell-sized, elastic poly(N-isopropylacrylamide) (PNIPAAm) microgels that relate temperature changes to hydrogel swelling. For their usage as a standardized calibration tool, we analyze the effect of free-radical chain-growth gelation (FCG) and polymer-analogous photogelation (PAG) on hydrogel network heterogeneity, micromechanics, and temperature response by Brillouin microscopy and optical diffraction tomography. Using a combination of tailor-made PNIPAAm macromers, PAG, and microfluidic processing, we obtain microgels with homogeneous network architecture. With that, we expand the capability of standardized microgels in calibrating and validating cell mechanics analysis, not only considering cell and microgel elasticity but also providing stimuli-responsiveness to consider dynamic changes that cells may undergo during characterization.
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