The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging

Abstract

Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence 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 Ca(2+) 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 Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) 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 Ca(2+), cease proliferation, and differentiate passively in response to changes in extracellular Ca(2+). 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 Ca(2+) gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.

Figure 1

Calcium calibration in tissue. (A) Phasor representation of FLIM acquired from buffered solutions of known calcium concentrations. (B) CG5N phasor distribution from three different human skin samples. The calibration curve in the tissue (black line) and the comparison with the calibration curve in solution (purple line) are shown. (C) CG5N lifetime distribution at different depths of adult truncal skin. (D) CG5N lifetime distribution of adult truncal skin treated with 260 μM EGTA overnight. The same lifetime distribution is observed at all depths in the EGTA-treated samples. The broadness of the distribution is due to the fact that the EGTA does not homogeneously chelate the sample at all depths.

Figure 2

Autofluorescence lifetime of adult skin samples. (A) Lifetime distribution of adult skin autofluorescence. (B) Lifetime distribution of adult skin incubated with CG5N for 2 h measured at 0 μm and 40 μm. The figure shows how the phasor analysis allows immediate distinction of autofluorescence contribution in deeper tissue layers from the CG5N signal.

Figure 3

Human skin morphology. Optical coronal sections of adult skin sample stained with CG5N allow us to identify different strata of the epidermis in living samples. (A) 0 μm, stratum corneum. (B) 40 μm, stratum granulosum. (C) 60 μm, stratum spinosum. (D) 80 μm, stratum basale. (E) 120 μm, dermis.

Figure 4

The distributions of the fraction of CG5N bound to calcium (Fb) at different depths in the epidermis show statistically significant differences in calcium concentration at different epidermal strata. (A) Fb distribution at different epidermal layers; B) average Fb as a function of depth in samples 1 (solid diamonds) and 2 (open squares). (C) Average Fb from unperturbed skin samples 1–3 (black bars) and from EGTA-treated skin (gray bars).

Figure 5

Calculation of calcium concentration in unperturbed adult skin distribution using the phasor approach. A specific calcium range is selected by moving a circular cursor along the calibration curve as shown in A–D. The pixels with a calcium concentration in the selected range are highlighted in red in the fluorescence images. This figure shows that low calcium concentrations are only found in the SC (E) of adult unperturbed skin. In the SB, we observe both regions with intermediate calcium concentration (N and O) and regions with high calcium (P). In both the SG and dermis, we find a homogeneous high calcium concentration (L and T, respectively).

Figure 6

Calcium distributions in different layers of adult skin sample. The sensitivity range of CG5N to calcium was divided into four calcium intervals and the fraction of pixels with a calcium concentration in each interval was plotted.