2 2 JO RNAL OF MET'1C CI.ENCE (a) 9) 100 1!'.;l :m (b) .. I 100 150 ,!lj (c) igur 6,, hanr:ieoi TP1r kin ima�t:-s of p 9 nt z = µm that were (c'"Xposecl to Soluri.oil v 00 we gl ): (a)--SRB fluo ceo ai in moflu nc (gr n dwnn I) ·t1 he linear fluoresc nee inrensity dec.11s:truct1011 re. •ion mark d by che yellow rectan •l .. The r d and the gr en line in rt b rl!prest!m norm. li�ed . SR.B fiuon:sc::tnce nd "k·n 1u1 ,oJl.uoresc:tnce spcc:t 1, n:s_pt�ri tl)•. The c ior .1 b r. i ndi care an ioc ren. e in · RB i ni: nsi ry for rhe red- hatmel i m e ( ) anl an increas in kin autofluorescenGf intensity for the grnfo chann ·l image (c), as th.e coJor progrl"s l"S from a low , lue d.o ·e to zt.ro, black) to a] igb Lue (red/.gr n). K, y: C--or11u:ocyre l ·c Lin in _re a - 25 µm. cyte keratin·, but that it also disrupts the intercellular lipid bifo)ers, ther,eby inducing RB penetration. into 11:he cornen ,,res (the brick) a.nd into the lipid bilayer (the mortar) in the C

VISUALIZATION OF SKIN BARRIER PERTURBATION 283 In Figure 4e, the autofluorescence spectrum (green line) of the p-FTS sample exposed to solution ii (1 wt% SDS + 10 wt% glycerol) exhibits two major peaks, at x = 50 pixels and x = 145 pixels, and three minor peaks at x = 1 pixel, x = 73 pixels, and x = 254 pixels. These five peaks again correspond to the positions of the intercellular lipid bilayer regions (see above). The SRB probe intensity spectrum peak heights (see the red line in Figure 4e) are almost identical to those corresponding to the skin autofluorescence peaks (see the green line in Figure 4e). This indicates that contacting solution ii does not induce any significant SRB penetration into the lipid bilayers of the SC. The peak widths corresponding to the peaks at x = 1 pixel and x = 145 pixels are larger for the SRB probe fluorescence intensity spectrum (the red line in Figure 4e) relative to the skin autofluo­ rescence intensity spectrum (the green line in Figure 4e). This indicates penetration of the SRB probe into these regions, which are, in fact, the corneocyte regions of the SC. Therefore, these TPM results indicate that adding 10 wt% glycerol to an aqueous 1 wt% SDS contacting solution mitigates SDS-induced intercellular lipid bilayer disruption, but does not eliminate SDS-induced SRB penetration into the corneocyte regions of the SC. In Figure 56, the autofluorescence spectrum (green line) of the p-FTS sample exposed to solution iii (1 wt% SCI) exhibits two major peaks, at x = 1 pixel and x = 107 pixels, and three minor peaks at x = 57 pixels, x = 158 pixels, and x = 210 pixels. These five peaks again correspond to the positions of the intercellular lipid bilayer regions (see above). The SRB probe intensity spectrum peak heights (see the red line in Figure 56) are higher than those corresponding to the skin autofluorescence peaks (see the green line in Figure 56). This indicates that contacting solution iii promotes SRB penetration into the intercellular lipid bilayers of the SC. However, upon comparing Figure 5a-c with Figure 4a-c, which correspond to 1 wt% SDS, it is clear that 1 wt% SCI induces a lower SRB penetration into the intercellular lipid bilayers relative to 1 wt% SDS. 11 This finding is consistent with transmission electron microscopy (TEM) studies on the effect of SCI on the skin barrier reported by Ananthapadmanabhan et al. (6,43). Specifically, these authors found that SCI does not induce skin dryness relative to SDS, because SCI disorders the intercellular lipid bilayer structure in the SC to a lesser extent than SDS (6,43,45). The SRB probe intensity spectrum peak widths (see the red line in Figure 56) are slightly larger than the skin autofluorescence intensity spectrum peak widths (see the green line in Figure 56). This difference between the SRB probe fluorescence and the skin autofluorescence intensity peak widths provides evidence of SCI-induced SRB penetration into the corneocytes, albeit to a lesser extent than that induced by SDS (see above). 12 In Figure 5e, the peaks corresponding to the skin autofluorescence intensity spectrum are lower in height than those corresponding to the SRB probe fluorescence intensity spectrum. This indicates that when p-FTS samples are exposed to the PBS control 11 Comparing Figure 4d-f with Figure Sa-c, one can also observe that contacting solution iii containing 1 wt% SCI promotes a lesser extent of SRB penetration into the intercellular lipid bilayers relative to contacting solution ii containing 1 wt% SDS + 10 wt% glycerol. 1 2 Indeed, SDS induces a larger difference in the SRB fluorescence and skin autofluorescence peak widths than SCI (compare Figures 46 and Sb). This is because SDS, being a harsh surfactant, interacts more strongly with the corneocyte envelopes and corneocyte keratins than SCI, which is a mild surfactant (6,43,45).