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Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca(2+) 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 Ca(2+) concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca(2+) distribution in skin were based on Ca(2+) 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 Ca(2+) concentrations, also assessing organelle Ca(2+) concentrations. For lack of better methods, these findings together build the basis for the current view of the role of Ca(2+) 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 Ca(2+) 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 Ca(2+) shortly following barrier disruption, from intracellular stores. These results partially contradict the conventional view, where barrier insults abrogate a Ca(2+) gradient towards the stratum granulosum. Ca(2+) FLIM overcomes prior limitations in the observation of epidermal Ca(2+) dynamics, and will allow further insights into basic epidermal physiology.

Measurements of ionic concentrations in skin have traditionally been performed with an array of methods which either did not reveal detailed localization information, or only provided qualitative, not quantitative information. FLIM combines a number of advantages into a method ideally suited to visualize concentrations of ions such as FT in intact, unperturbed epidermis and stratum corneum (SC). Fluorescence lifetime is dye concentration- independent, the method requires only low light intensities and is therefore not prone to photobleaching or phototoxic artifacts, and because multiphoton lasers of IR wavelength are used, light penetrates deep into intact tissue. The standard method to measure SC pH is the flat pH electrode, which provides reliable information only about surface pH changes, without further vertical or subcellular spatial resolution; i.e., specific microdomains such as the corneocyte interstices are not resolved, and the deeper SC is inaccessible without resorting to inherently disruptive stripping methods. Furthermore, the concept of a gradient of pH through the SC stems from such stripping experiments, but other confirmation for this concept is lacking. Our investigations into the SC pH distribution so far have revealed the crucial role of the Sodium/Hydrogen Antiporter NHE1 in generation of SC acidity, the colocalization of enzymatic lipid processing activity in the SC with acidic domains of the SC, and the timing and localization of emerging acidity in the SC of newborns. Together, these results have led to an improved understanding of the SC pH, its distribution, origin, and regulation. Future uses for this method include measurements of other ions important for epidermal processes, such as Ca2+, and a quantitative approach to topical drug penetration.

Although basal permeability barrier function is established at birth, the higher risk for infections, dermatitis, and percutaneous absorption of toxic agents may indicate incomplete permeability barrier maturation in the early neonatal period. Since stratum corneum (SC) acidification in adults is required for normal permeability barrier homeostasis, and lipid processing occurs via acidic pH dependent enzymes, we hypothesized that, in parallel with the less acidic surface pH, newborn SC would exhibit signs of incomplete barrier formation. Fluorescence lifetime imaging reveals that neonatal rat SC acidification first becomes evident by postnatal day 3, in extracellular "microdomains" at the SC- stratum granulosum (SG) interface, where pH-sensitive lipid processing is known to occur. This localized acidification correlated temporally with efficient processing of secreted lamellar body contents to mature extracellular lamellar bilayers. Since expression of the key acidifying mechanism NHE1 is maximal just prior to birth, and gradually declines over the first postnatal week, suboptimal SC acidification at birth cannot be attributed to insufficient NHE1 expression, but could instead reflect reduced NHE1 activity. Expression of the key lipid processing enzyme, beta-glucocerebrosidase (beta-GlcCer'ase), develops similar to NHE1, excluding a lack of beta-GlcCer'ase protein as rate limiting for efficient lipid processing. These results define a postnatal development consisting of initial acidification in the lower SC followed by outward progression, which is accompanied by formation of mature extracellular lamellar membranes. Thus, full barrier competence appears to require the extension of acidification in microdomains from the SC/SG interface outward toward the skin surface in the immediate postnatal period.

The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na(+)/H(+) antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 -/- animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform gradient, but through the progressive accumulation of acidic microdomains. These findings not only visualize the spatial distribution of the SC pH gradient, but also demonstrate a role for NHE1 in the generation of acidic extracellular domains of the lower SC, thus providing the acidification of deep SC interstices necessary for lipid processing and barrier homeostasis.

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.

Fluorescent probes have found widespread use in biomedical sciences. Particularly since they can be targeted to cellular compartments and further more can report on the properties of their environment such as calcium concentration. Near infrared ultrafast lasers find increasing use for fluorescence applications since femtosecond pulses with a few milliwatts of average power are sufficient to induce significant two photon fluorescence from the probe when focussed into typical samples. The nonlinear optical excitation process allows sectioned imaging of 3-D samples without use of a confocal pinhole. In this paper we describe two aspects of multiphoton microscopy: the two-photon excitation cross section and the fluorescence lifetime. Of interest is the wavelength characterization of two-photon excitation cross-sections of fluorescence probes. We slowly modulate (∼500Hz) the intensity envelope of the input laser pulse train and analyze the emission signal in terms of the amplitude and phase of the harmonics of this modulation. In effect this is a power study that allows separation of different order effects. An application of ultrafast laser excitation that exploits many of the features outlined above is measurement of pH gradients in the skin. This is essential to skin barrier function and disruption of the gradient is though to be a indicating factor in many skin diseases. A probe for which the fluorescence lifetime varies with pH is used. We thus are able to tackle problems associated with inhomogeneous labeling. We have developed a two-photon laser-scanning lifetime microscope and present pH maps of skin obtained with this instrument. © 2002 SPIE · 0277-786X/02/$15.00.

At birth, human stratum corneum (SC) displays a near-neutral surface pH, which declines over several days to weeks to months to an acidic pH, comparable to that of adults. Recent studies suggest that an acidic pH is required for normal permeability barrier homeostasis and SC integrity/cohesion. We assessed here the basis for postnatal acidification in the neonatal rat, where SC pH, as measured with a flat surface electrode, declines progressively from near-neutral levels (pH 6.63) on postnatal days 0 to 1 to adult levels (pH 5.9) or even below over the subsequent 7 to 8 d. The postnatal decline in SC pH was paralleled by a progressive activation of a pH-dependent hydrolytic enzyme, β-glucocerebrosidase. Because SC acidification could not be linked to commonly implicated exogenous factors, such as bacterial colonization, or the deposition of sebaceous gland products. We next assessed whether changes in one or more of three endogenous mechanisms demonstrate postnatal activity changes that contribute to the progressive development of an acidic SC pH. Although the histidine-to-urocanic acid pathway has been implicated in acidification of the adult SC, surface pH is completely normal in histidase-deficient (his/his, Peruvian) mice, ruling out a requirement for this mechanism. In contrast, when sodium/hydrogen antiporter-1 (NHE1), which predominantly acidifies membrane domains at the stratum granulosum–SC interface, is inhibited, postnatal acidification of the SC is partially blocked. Likewise, SC secretory phospholipase A2 (sPLA2) activity, measured with a fluorometric assay, is low at birth, but increases progressively (by 66%) over the first 5 d after birth, and inhibition of sPLA2 between days 0 to 1 and days 5 to 6 delays postnatal SC acidification. Together, these results describe a neonatal model, in which the development of an acidic surface pH can be ascribed, in part, to progressive SC acidification by two endogenous mechanisms, namely, sPLA2 and NHE1, which are known to be important for acidification of adult rodent SC. Conversely, the impaired acidification of neonatal SC, which has important functional and clinical consequences, can be explained by the relatively low activities of one or both of these mechanisms at birth. [ABSTRACT FROM AUTHOR]