ABSTRACT Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 (mu)m) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and6)-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 (<=50 (mu)m x 50 (mu)m) are acquired every 1.7 (mu)m from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 (mu)m 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 (mu)m 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.
INTRODUCTION
Within the 10-20 cell layers of the uppermost epidermis (stratum corneum) of human skin, the hydrogen ion concentration decreases 100-1000-fold (Ohman and Vahlquist, 1994). The surface of the skin is acidic, ranging between pH 4.5 and pH 6 depending upon body site, sex, and species, forming what is termed the acid mantle (Dikstein and Zlotogorski, 1994; Ohman and Vahlquist, 1994). In contrast, the first viable epidermal layer (stratum granulosum) below the stratum corneum, ~10 (mu)m below the surface, reaches neutrality. Current research shows that pH greatly influences the barrier nature of the stratum corneum. Thus, understanding both the effect of pH and its origin upon the skin barrier is synonymous with improving topical drug delivery and understanding barrier-influenced diseases like dermatitis and icthyosis. To determine how pH affects barrier function, a method for detecting pH in the stratum corneum on the subcellular level is first needed.
Tape stripping has allowed measurements of pH as a function of stratum corneum depth using a flat electrode (Ohman and Vahlquist, 1994, 1998). These measurements have determined that pH increases with each deeper corneocyte layer showing that a pH gradient exists between the surface and the deepest stratum corneum. There are two drawbacks to tape-stripping measurements. First, it is intrinsically destructive. Once perturbed, the skin naturally undergoes barrier recovery, which may in turn alter the pH of the stratum corneum. Therefore, such measurements do not necessarily measure the pH at equilibrium. Second, using a flat electrode on the stratum corneum provides a bulk measurement of pH over an extended area (square centimeters). The electrode method cannot identify at the subcellular level those compartments, such as the extracellular matrix and/or intracellular spaces, that contribute to the dramatic pH differences observed across the stratum corneum.
Microscopy in conjunction with pH-sensitive fluorescent probes offers a method to determine pH with the required spatial resolution (Hanson et al., 2000). In general, these fluorophores report their local pH through a shift in excitation spectrum and change in the intensity/spectrum of emission as the probe changes between an acid and a base form. These spectral changes are often accompanied by a change in the fluorescence lifetime (Rink et al., 1982; Szmacinski and Lakowicz, 1993). Quantitative use of these probes in a cellular environment presents many challenges. Purely optical absorbance methods for determination of pH are generally not used because of difficulties in measuring the spectrum of a necessarily dilute stain against a complex cellular background absorption. Emission intensity methods have more than adequate sensitivity of detection for probe concentrations thought not to disturb normal cell behavior; however, because of inhomogeneous labeling, simple intensity measurements cannot be used to determine pH in the cellular environment. In such cases, either excitation ratio or emission ratio methods can be used. For lamp-based systems it is possible to rapidly change excitation filters to facilitate ratio imaging of samples that are not opaque.
In this work, three-dimensional information upon skin with subcellular resolution is desired. This suggests the use of confocal microscopy techniques. In this case, the availability of suitable laser lines poses a difficulty. In addition, it is difficult to change between laser lines rapidly, and it is difficult to retain the exact depth of focus of the different excitation wavelengths. These problems make excitation ratiometric methods cumbersome and unattractive. Emission ratio methods are also possible in the case of acquiring data within the relatively thin (~15 (mu)m) stratum corneum. However, for thicker samples, quantification using emission ratio imaging is complicated by wavelength-dependent inhomogeneous absorption and scattering as the fluorescence light leaves the skin sample in its path to the detector (Jacques, 1996). More specifically in the case of measuring pH within the stratum corneum, a single probe is not commercially available to date to detect pH over the pH range of the stratum corneum by emission ratio methods. Multiple probes would be required to determine pH by this method within the stratum corneum.
Fluorescence lifetime imaging offers a solution to these problems and is compatible with confocal microscopy. The lifetime is independent of probe concentration and inhomogeneities in excitation and emission light paths. Scatter will delay the emitted light in reaching the detector, but this effect is negligible (picoseconds) compared with typical fluorescence lifetimes (nanoseconds) for the tissue penetration in this study.
When working with bulk tissue, the penetration of the excitation light must be considered. Two-photon excitation using near-infrared light and without a confocal pinhole in the emission path has been shown to allow sectioned imaging at greater depths into a tissue sample compared with ultraviolet confocal excitation (Masters et al., 1997). Because in two-photon microscopy no pinhole is used, subsequent scatter of the fluorescent emission does not result in rejection by the confocal pinhole as in one-- photon excitation.
A number of fluorescein-derived dyes are available for measurement at near physiological pH. For this work we have chosen to use 2',7'-bis-(2-carboxyethyl)-5-(and-6)-- carboxyfluorescein (BCECF) with a pK^sub a^ of 7.0 (Rink et al., 1982; Szmacinski and Lakowicz, 1993; Haughland, 1998). The emission from BCECF has a maximum at 535 nm. The broad tuning range of the mode-locked Ti:sapphire laser used in this study allows selection of an excitation wavelength that minimizes contributions from autofluorescence.
MATERIALS AND METHODS
CONCLUSIONS
Two-photon fluorescence lifetime-resolved imaging microscopy was selected because it affords submicron spatial resolution and submillimeter depth penetration into tissue using one excitation wavelength. This technique provides an excellent method for imaging cellular processes on the submicron scale within all layers of the epidermis.
The primary issue in determining pH in the skin accurately has been one of calibration (Hanson et al., 2000). Lifetime imaging circumvents this difficulty because lifetime measurements are independent of concentration, which allows for a straightforward calibration. Intensity measurements are difficult to calibrate because the pH-sensitive dyes are unevenly distributed. This is because of the heterogeneous environment and efficient barrier properties of the stratum corneum. As Fig. 4 a shows, a region of bright intensity may indicate either a neutral pH or simply the presence of a greater number of dye molecules relative to another area within the skin. Ratiometric measurement techniques can circumvent the issue of uneven dye distribution. The dye, BCECF, that we have used in our lifetime-resolved experiments, has been used to detect intracellular pH differences using intensity excitation-ratio measurements (Rink et al., 1982). Because the excitation spectrum of BCECF spectrally shifts with pH, a fluorescence emission intensity ratio can be formed by exciting at two wavelengths. However, this is experimentally inconvenient; with two-photon FLIM only one excitation wavelength is needed. This avoids the movement of the excitation beam within the focal plane that results when excitation wavelengths are changed, which is a serious problem in ratiometric methods. The ratiometric measurements have also proven to be difficult to calibrate within the skin (Turner et al., 1998). However, with the development of new probes that spectrally shift over the entire pH range of the stratum corneum, emission ratio imaging of stratum corneum pH may prove comparable in ease of use to two-photon FLIM.
Several mechanistic theories have been published to explain the origin of the stratum corneum acid mantle. Passive mechanisms proposed to date include the accumulation of the ultraviolet chromophore trans-urocanic acid (Krien and Kermici, 2000), sweat by-products lactate and lactic acid (Patterson et al., 2000), or acidic free fatty acids (Lieckfeldt et al., 2000). These mechanisms differ from active pathways (sodium/hydrogen antiporter) that may influence pH by actively regulating the hydrogen ion concentration. We are currently using two-photon FLIM to determine the effect of active and passive mechanisms upon the origin of acidic microdomains within the stratum corneum.
The Laboratory for Fluorescence Dynamics at the University of Illinois is supported by National Institutes of Health PHS P41-RR03155. K.H. is supported by the Cancer Research Foundation of America and the Skin Cancer Foundation.
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[Author Affiliation]
Kerry M. Hanson,* Martin J. Behne,^ Nicholas P. Barry,* Theodora M. Mauro,^ Enrico Gratton,* and Robert M. Clegg*
*Laboratory for Fluorescence Dynamics, Department of Physics, University of Illinois, Urbana-Champaign, Illinois 61801, and ^Dermatology Service, Veterans Affairs Medical Center and Department of Dermatology, University of California, San Francisco, California 94110 USA
[Author Affiliation]
Submitted January 8, 2002, and accepted for publication May 1, 2002. K.M.H. and NT .B. contributed equally to this work.
Address reprint requests to Dr. Kerry M. Hanson, Laboratory for Fluorescence Dynamics, University of Illinois, 1110 W. Green Street, Urbana, IL 61801. Tel.: 217-244-5620; Fax: 217-244-7187; E-mail: khanson@uiuc.edu.
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