Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of .uorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca2+gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca2+stores than those used in previous studies, and revealed that the bulk of free Ca2+measured in the epidermis comes from intracellular Ca2+stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca2+making a relatively small contribution to the epidermal Ca2+gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca2+, cease proliferation, and differentiate passively in response to changes in extracellular Ca2+. The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca2+gradient, and it should be broadly applicable to the study of other tissues with ionic gradients. © 2010 by the Biophysical Society.