Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis

摘要

Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca²⁺ concentrations and their regulation are therefore especially important to assess when observing epidermal barrier homeostasis and repair, neonatal barrier establishment, in differentiation, signaling, cell adhesion, and in various pathological states. Yet, tissue- and cellular Ca²⁺ concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca²⁺ distribution in skin were based on Ca²⁺ precipitation followed by electron microscopy, or proton-induced X-ray emission. Neither cellular and/or subcellular localization could be determined through these approaches. In cells in vitro, fluorescent dyes have been used extensively for ratiometric measurements of static and dynamic Ca²⁺ concentrations, also assessing organelle Ca²⁺ concentrations. For lack of better methods, these findings together build the basis for the current view of the role of Ca²⁺ in epidermis, their limitations notwithstanding. Here we report a method using Calcium Green 5N as the calcium sensor and the phasor-plot approach to separate raw lifetime components. Thus, fluorescence lifetime imaging (FLIM) enables us to quantitatively assess and visualize dynamic changes of Ca²⁺ at light-microscopic resolution in ex vivo biopsies of unfixed epidermis, in close to in vivo conditions. Comparing undisturbed epidermis with epidermis following a barrier insult revealed major shifts, and more importantly, a mobilization of high amounts of Ca²⁺ shortly following barrier disruption, from intracellular stores. These results partially contradict the conventional view, where barrier insults abrogate a Ca²⁺ gradient towards the stratum granulosum. Ca²⁺ FLIM overcomes prior limitations in the observation of epidermal Ca²⁺ dynamics, and will allow further insights into basic epidermal physiology.