Significance: Research in tissue engineering and in vitro organ formation has recently intensified. To assess tissue morphology, the method of choice today is restricted primarily to histology. Thus novel tools are required to enable noninvasive, and preferably label-free, three-dimensional imaging that is more compatible with futuristic organ-on-a-chip models.

Aim: We investigate the potential for using multiphoton microscopy (MPM) as a label-free in vitro approach to monitor calcium-induced epidermal differentiation.

Approach: In vitro epidermis was cultured at the air–liquid interface in varying calcium concentrations. Morphology and tissue architecture were investigated using MPM based on visualizing cellular autofluorescence.

Results: Distinct morphologies corresponding to epidermal differentiation were observed. In addition, Ca^2  +-induced effects could be distinguished based on the architectural differences in stratification in the tissue cultures.

Conclusions: Our study shows that MPM based on cellular autofluorescence enables visualization of Ca^2  +-induced differentiation in epidermal skin models in vitro. The technique has potential to be further adapted as a noninvasive, label-free, and real-time tool to monitor tissue regeneration and organ formation in vitro.