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

@entity141 @entity460
@entity460 controls an array of key events in keratinocytes and epidermis: localized changes in @entity141 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 @entity141 concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the @entity141 distribution in skin were based on @entity141 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 @entity141 concentrations, also assessing organelle @entity141 concentrations. For lack of better methods, these findings together build the basis for the current view of the role of @entity141 in epidermis, their limitations notwithstanding. Here we report a method using @entity460 Green 5N as the @entity460 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 @entity141 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 @entity141 shortly following barrier disruption, from intracellular stores. These results partially contradict the conventional view, where barrier insults abrogate a @entity141 gradient towards the stratum granulosum. @entity141 FLIM overcomes prior limitations in the observation of epidermal @entity141 dynamics, and will allow further insights into basic epidermal physiology.
