Multiphoton fluorescence lifetime imaging microscopy (MPM-FLIM) is extensively proposed as a non-invasive optical method to study tissue metabolism. The approach is based on recording changes in the fluorescence lifetime attributed to metabolic co-enzymes, of which nicotinamide adenine dinucleotide (NADH) is of major importance. However, intrinsic tissue fluorescence is complex. Particularly when utilizing two-photon excitation, as conventionally employed in MPM. This increases the possibility for spectral crosstalk and incorrect assignment of the origin of the FLIM signal. Here we demonstrate that in keratinocytes, proteins such as keratin may interfere with the signal usually assigned to NADH in MPM-FLIM by contributing to the lifetime component at 1.5 ns. This is supported by a change in fluorescence lifetime distribution in KRT5- and KRT14-silenced cells. Altogether, our results suggest that the MPM-FLIM data originating from cellular autofluorescence is far more complex than previously suggested and that the contribution from other tissue constituents should not be neglected—changing the paradigm for data interpretation in this context.

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 2þ-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 2þ-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.

Keratins are structural proteins that are abundant in human skin, nails, and hair, where they provide mechanical strength. In the present study, we investigate the molecular mobilities and structures of three keratin-rich materials with clearly different mechanical properties: nails, stratum corneum (upper layer of epidermis), and keratinocytes (from lower layer of epidermis). We use solid-state NMR on natural-abundance 13 C to characterize small changes in molecular dynamics in these biological materials with close to atomistic resolution. One strong advantage of this method is that it detects small fractions of mobile components in a molecularly complex material while it simultaneously gives information on the rigid components in the very same sample. The molecular mobility can be linked to mechanical material properties in different conditions, including hydration or exposure to osmolytes or organic solvents. Importantly, the study revealed that the response to both hydration and addition of urea is clearly different for the nail keratin compared to the stratum corneum keratin. The comparative examination of these materials may provide a better understanding of skin diseases originating from keratin malfunction and contributes to the design and development of new materials.

Development of organs-on-a-chip calls for non-invasive methods for characterization of tissues in real-time. Multiphoton microscopy with FLIM in combination with stage-adopted cell culturing will potentially solve this problem, enabling monitoring of metabolic changes “online”.