Detection of Calcium Gradients in Live Ex Vivo Human Epidermis and 3D Human Epidermal Equivalents using Phasor Analysis of Two-Photon Excitation Fluorescence Lifetime Imaging (FLIM) and the Genetically Encoded ER Calcium Sensor D1Er

Abstract

Ionic gradients regulate the function and the structure of many tissues and organs. However, traditional techniques used to measure ionic concentrations in tissue (such as Proton induced X-ray Emission or ion capture cytochemistry) are often highly invasive (requiring fixation and dehydration of the specimen) and offer poor spatial resolution. In skin, Ca2+ gradients have been shown to regulate epidermal barrier function, homeostasis and repair after perturbation, but a clear understanding of the mechanisms regulating such gradients is still lacking. We applied the phasor analysis approach to two-photon excitation FLIM measurements of Calcium Green 5N to quantify and localize Ca2+ pools in human epidermis before and after barrier perturbation. The high spatial resolution offered by this method and the data analysis performed allowed us to measure intracellular average calcium concentrations directly in tissue and detect a previously unknown heterogeneous distribution of Ca2+ concentrations in the proliferative epidermal basal layer following barrier disruption. More importantly, this approach revealed that the extracellular space in epidermis is much narrower than previously thought. In fact, most epidermal Ca2+ localizes to intracellular stores, not extracellular space. To test the hypothesis that epidermal barrier repair after experimental barrier abrogation is driven by Ca2+ loss from these intracellular stores, not from the extracellular space, we developed a flexible in vitro 3D skin equivalent model that allows intra-organelle monitoring of calcium levels by genetically encoded sensors. These new findings suggest that long-held hypotheses addressing Ca2+ control of differentiation and barrier formation require revision.