See related commentary on pg 2131

ORIGINAL ARTICLE

The Human Skin Barrier Is Organized as Stacked

Bilayers of Fully Extended Ceramides with
Cholesterol Molecules Associated with the
Ceramide Sphingoid Moiety
fverstedt4,
Ichiro Iwai1,2, HongMei Han1,8, Lianne den Hollander1,8, Stina Svensson1,3, Lars-Goran O
5
6
6
1
1,7
Jamshed Anwar , Jonathan Brewer , Maria Bloksgaard , Aurelie Laloeuf , Daniel Nosek , Sergej Masich1,
Luis A. Bagatolli6, Ulf Skoglund4 and Lars Norlen1,7
The skin barrier is fundamental to terrestrial life and its evolution; it upholds homeostasis and protects against the
environment. Skin barrier capacity is controlled by lipids that fill the extracellular space of the skins surface
layerthe stratum corneum. Here we report on the determination of the molecular organization of the skins lipid
matrix in situ, in its near-native state, using a methodological approach combining very high magnification cryoelectron microscopy (EM) of vitreous skin section defocus series, molecular modeling, and EM simulation. The
lipids are organized in an arrangement not previously described in a biological systemstacked bilayers of
fully extended ceramides (CERs) with cholesterol molecules associated with the CER sphingoid moiety. This
arrangement rationalizes the skins low permeability toward water and toward hydrophilic and lipophilic
substances, as well as the skin barriers robustness toward hydration and dehydration, environmental temperature
and pressure changes, stretching, compression, bending, and shearing.
Journal of Investigative Dermatology (2012) 132, 22152225; doi:10.1038/jid.2012.43; published online 26 April 2012

INTRODUCTION
The skin constitutes a barrier between the body and the
environment (Elias and Friend, 1975). By preventing water
loss via evaporation it upholds homeostasis, and by preventing penetration of exogenous substances it protects against
the environment. The skins barrier capacity is a function
of the molecular architecture of the lipid structure in the
extracellular space between the cells of the stratum corneum
(Bouwstra et al., 1991). The lipids consist of a heterogeneous
1
Department of Cell and Molecular Biology (CMB), Karolinska Institutet,
Stockholm, Sweden; 2Shiseido Research Center, Yokohama, Japan; 3Center
for Image Analysis, Swedish University of Agricultural Sciences, Uppsala,
Sweden; 4Structural Cellular Biology Unit, Okinawa Institute of Science and
Technology, Okinawa, Japan; 5Institute of Pharmaceutical Innovation,
University of Bradford, Bradford, United Kingdom; 6Department of
Biochemistry and Molecular Biology, Membrane Biophysics and
Biophotonics group/MEMPHYSCenter for Biomembrane Physics, University
of Southern Denmark, Odense, Denmark and 7Dermatology Clinic,
Karolinska University Hospital, Stockholm, Sweden
8

These authors contributed equally to this work.

Correspondence: Lars Norlen, Department of Cellular and Molecular Biology

(CMB), Karolinska Institutet, von Eulers v 1, 171 77 Stockholm, Sweden.
E-mail: lars.norlen@ki.se
Abbreviations: CEMOVIS, cryo-electron microscopy of vitreous skin section;
CER, ceramide; CHOL, cholesterol; EM, electron microscopy; FFA, free
fatty acid; GP, generalized polarization; LAURDAN, 6-dodecanoyl-2dimethylaminonaphthalene
Received 12 May 2011; revised 12 December 2011; accepted 17 December
2011; published online 26 April 2012

& 2012 The Society for Investigative Dermatology

mixture of saturated, long-chain ceramides (CERs), free fatty

acids (FFAs), and cholesterol (CHOL) in a roughly 1:1:1 molar
ratio (Wertz and Norlen, 2003). In the CER fraction alone,
more than 300 different species have been identified
(Masukawa et al., 2009).
Ever since the discovery in the early 1970s (Breathnach
et al., 1973; Elias and Friend, 1975) that the stratum corneum
extracellular space is filled with lipid material, the skin lipids
have been a subject of much activity. However, the
molecular organization of these lipids remains unresolved.
By using conventional electron microscopy (EM) on ruthenium tetroxidestained mouse skin, Madison et al. (1987)
reported that the stratum corneum lipid material, or lipid
matrix, displays 13-nm repeating units of broad:narrow:
broad electron lucent bands. Using small-angle X-ray
diffraction on isolated human stratum corneum, Garson
et al. (1991) reported the presence of one 4.5-nm and one
6.5-nm diffraction peak related to lipids. Concomitantly,
White et al. (1988) and Bouwstra et al. (1991) reported the
presence of a 13-nm repeating unit in mouse and human
isolated stratum corneum, respectively. Later, McIntosh
(2003) observed an asymmetric distribution of CHOL within
model systems composed of reconstituted stratum corneum
lipids extracted from pig skin. On the basis of these data,
as well as on data obtained from different in vitro lipid
model systems, six theoretical models for the molecular
arrangement of the extracellular lipid matrix have been
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I Iwai et al.
The Human Skin Barrier

proposed. These are based on either triple-band repeating

units and/or bands with the same width (Supplementary
Figure S1 online).
We have used a novel methodological approach based on
cryo-electron microscopy of vitreous skin section (CEMOVIS)
defocus series combined with molecular modeling and EM
simulation to investigate the molecular organization of the
human skins lipid matrix in its near-native state. In CEMOVIS,
the native tissue is preserved down to the molecular level, and
the micrograph pixel intensity is directly related to the local
electron density of the specimen (Dubochet et al., 1988; AlAmoudi et al., 2004, 2007; Norlen et al., 2009). Biomolecules
generally possess small intermolecular and intramolecular
differences in electron density, as they are essentially
composed of atoms with similar atomic weight (carbon,
nitrogen, and oxygen). However, for orderly arranged
molecular assemblies, such as lipid tails and headgroups in
membranes, even small differences in shape and atomic
composition may be amplified because of interference effects
that appear in the image phase contrast. During image
acquisition, phase contrast is made visible using defocus
ktem, 2008).
(compare e.g., Fanelli and O
Here we show that the human skin barriers CEMOVIS
pattern is characterized by an asymmetric B11-nm repeating
unit consisting of alternating narrow (B4.5 nm) and broad
(B6.5 nm) bands. Further, at, and only at, very high
magnification (pixelsize p3.31 A), complex interference
patterns can be resolved in the CEMOVIS micrographs.
Exploiting this, CEMOVIS micrographs were recorded repeatedly at very high magnification at the same position of the skin
sample while increasing stepwise the microscopes defocus,
thus ensuring that differences in the recorded micrographs
were due exclusively to the different defocuses used.
Simulated electron micrographs were then generated at
corresponding defocuses for different skin lipid models, and
compared with the CEMOVIS micrographs. This approach was
found to be highly discriminating between different models.
The lipid organization that emerges from the analysis is a
stacked bilayer structure of CERs in the fully extended
(splayed chain) conformation with CHOL associated with
the CER sphingoid moiety.
RESULTS

breaks, the intensity is displaced away from and perpendicular

to the mainstream intensity bands, giving rise to thin dark
cross-stripes (Figure 1d, white open arrows; Supplementary
Figure S4A online).
The averaged, image intensity profile for the stacked lipids
(Figure 1e) was obtained by fuzzy distance-based image
analysis and is shown in Figure 1b and c. Lamellar regions
with 2, 4, 6, 8, 10, or 12 dark lines (Figure 2af, left column)
can be observed between adjacent stratum corneum cell
plasma membranes (also referred to as lipid envelopes).
The dark lines are arranged in a periodic pattern with an
asymmetric B11-nm (1012 nm) repeating unit consisting of
alternating narrow (B4.5 nm) and broad (B6.5 nm) bands,
where the 4.5-nm bands express a higher averaged image
intensity (i.e., they are darker) than the 6.5-nm bands (Figure
2af, right column). A low-intensity region (i.e., a light band)
is present in the mid-plane of the 6.5-nm bands (Figure 1b,
solid black arrows; Supplementary Figure S4A online). Small
shoulders centrally on each slope of the corresponding
averaged intensity profiles delimit its extension (Supplementary Figure S4B online, open arrows). No interindividual or
intersite (forearm vs. abdomen) variation was recorded
(Supplementary Figure S5 online).
Power spectra extracted along the lamellar stacks revealed
that the 6.5-nm region, around its low-intensity mid-plane, is
somewhat ordered, whereas the other regions appear to be
unstructured (Figure 3ag). The 6.5-nm region is characterized by peaks equating to repeat distances of 2.33.0 nm and
1.51.7 nm (Figure 3d).
Generalized polarization (GP) function measurements
obtained from multi-photon excitation images of 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) labeled nearnative skin (Figure 4a and b) show that the stratum corneum
lipid matrix is in a condensed state and contains little, if any,
water at the lipid headgroup/hydrocarbon interface regions.
Furthermore, the LAURDAN GP function was not affected by
hydration (Figure 4a and b). Similarly, the CEMOVIS intensity
profiles of the extracellular lipid matrix remained unaltered
upon hydration (Figure 4c and d), whereas the intracellular
keratin intermediate filament network at the same locations
swelled extensively (Figure 4c; compare Supplementary
Figure S2A online) compared with normal stratum corneum
(Figure 1a; compare Supplementary Figure S2B online).

Skin lipids CEMOVIS pattern

The CEMOVIS data reported here represent over 1,000

original observations obtained from the left volar forearm
and abdomen of five Caucasian men in the age group of
4050 years with no history of skin disease.
The CEMOVIS data demonstrate that the stratum corneum
extracellular lipid matrix is composed of a stack of layers
(Figure 1a). They display a meandering pathway (Figure 1a) and
folds/unfolds on hydration/dehydration (Supplementary Figure
S2AC online). This implies that these layers are malleable. At
high magnification (Figure 1d), the distinct dark lines of the
lamellar stacks (Figure 1b) are revealed to be not entirely
continuous. There are regular breaks in intensity, implying
some disorder at the microstructural level (Figure 1d, white
solid arrows; Supplementary Figure S4A online). At these
2216 Journal of Investigative Dermatology (2012), Volume 132

Modeling and simulation

The key aspect of the lipid matrix CEMOVIS image intensity

profile is the asymmetric B11-nm repeating unit. This consists
of two peaks with a peakpeak distance of B4.5 nm separated
by a shallow trough of low intensity (Figure 2). The twin-peak
repeats are separated by a deeper (low intensity) trough with a
peakpeak distance of B6.5 nm. The peaks are expected to
correspond to the lipid headgroup regions, which are
characterized by the heavier polar atoms nitrogen and
oxygen. The low-intensity troughs are expected to correspond
to the carbon-dominated lipid alkyl chains.
Given these characteristics of the data, we decided to
combine molecular modeling and EM simulation (Figure 5) to
identify a lipid organization that would be consistent not only

I Iwai et al.
The Human Skin Barrier

5 nm

6.5 nm
4.5 nm

6.5 nm
4.5 nm

5 nm
4.5 nm

Figure 1. The cryo-electron microscopy of vitreous skin section (CEMOVIS) intensity pattern of the stratum corneum extracellular lipid matrix consists of
folded stacked layers. (a) Medium-magnification CEMOVIS micrograph of the interface between two cells in the mid-part of the stratum corneum. Note that in
CEMOVIS the tissue is unstained, and that the pixel intensity is directly related to the local electron density of the sample. The stacked lamellar pattern represents
the extracellular lipid matrix. Dark B10-nm dots represent keratin intermediate filaments filling out the intracellular space. Note that the extracellular
lipid matrix locally expresses extensive folding. (b) High-magnification CEMOVIS micrograph of the extracellular space in the mid-part of the stratum corneum.
The intensity profile of the lipid matrix was obtained by fuzzy distance-based image analysis. The red stars in (b) represent the manually chosen start and
end points for fuzzy distance-based path growing. (c) The red line represents the traced-out path. Stacked lines mark extracted intensity profiles. (d) Enlarged area
of the central part of (b). Note that the electron-dense bands are composed of dark 1- to 3-nm dots from which thin, weak lines protrude 23 nm into the
lucent areas. The same pattern is also evident in (b). (e) Reversed averaged pixel intensity profile obtained from the extracted area in (c). Peaks in (e) correspond
to dark bands and valleys to lucent bands in (d). Black arrows in (b) denote electron lucent narrow bands at the center of the 6.5-nm bands. Section
thickness: B50 nm (ad). Bar (a): 100 nm. Pixel size in (ad): 6.02 A.

with the observed intensity profile but also with the

CEMOVIS intensity interference pattern changes observed
on changing the microscopes defocus during image acquisition (Figure 6).
Rather than considering the full complexity of the stratum
corneum lipid composition, we focused on the three main
components (Wertz and Norlen, 2003; Masukawa et al.,
2009), namely CER NP (C18-phytosphingosine-non-hydroxyC24:0), CHOL, and lignoceric acid (C24:0), because these will
likely determine the main features of the lipid organization,
and variation in chain lengths will serve only to modulate the
structure. Polyunsaturated o-hydroxyacid CERs were not
included, as these correspond to o15% of the total lipid
mass extracted from the stratum corneum and are structurally
different from the remaining stratum corneum lipids.
Further, water was not included in the models as neither
the LAURDAN GP (Figure 4a and b) nor the CEMOVIS
(Figure 4c and d) swelling experiments expressed signs of the
presence of water in the lipid matrix.
For each of the three lipid components, we built molecular
models with the atoms located at their ideal bond distances,
angle, and torsions. The torsions in the headgroup region of the
CER molecules were chosen to yield either a fully extended

(the splayed chain conformation; compare Supplementary

Figures S6S7 online) or a fully folded (the hairpin conformation; compare Supplementary Figure S8 online) structure.
EM image intensity patterns were then simulated for a
variety of molecular models at three radically different
defocuses (!0.5, !2, and !5 mm; Figure 6df; Supplementary
Figures S6S8 online) using a newly developed EM simulator
(Rullgard et al., 2011). The different stratum corneum lipid
models were thus discriminated by how closely their
corresponding simulated EM patterns mirrored the CEMOVIS
patterns of near-native skin, and how closely they reflected
the CEMOVIS interference pattern changes observed upon
radically changing the microscopes defocus during image
acquisition (Figure 6af; Supplementary Figures S6S8 online).
For the best-fitting model (Supplementary Figure S6D online),
EM images were simulated at !1, !2, and !3-mm defocus
(Figure 6jl) and compared with a series of CEMOVIS
micrographs obtained sequentially at the same location
at !1, !2, and !3-mm defocus (Figure 6gi).
Remarkably, of the various molecular organizations
considered, a model based on bilayers of fully extended
CERs with CHOL selectively localized to the CER sphingoid
part was found to account for all the major features of the
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I Iwai et al.
The Human Skin Barrier

4.5 nm

6.5 nm
4.5 nm

4.5 nm

6.5 nm
4.5 nm

6.5 nm
4.5 nm

4.5 nm

4.5 nm

6.5 nm

4.5 nm

6.5 nm

4.5 nm

6.5 nm

4.5 nm

6.5 nm

4.5 nm

6.5 nm

4.5 nm

6.5 nm

4.5 nm

6.5 nm
4.5 nm

6.5 nm

4.5 nm

6.5 nm

4.5 nm

6.5 nm

4.5 nm

4.5 nm

6.5 nm

4.5 nm

6.5 nm

4.5 nm

4.5 nm

Figure 2. An asymmetric B11-nm repeating unit characterizes the cryo-electron microscopy of vitreous skin section (CEMOVIS) intensity profile of the
lipid matrix. Left column: high-magnification CEMOVIS micrographs of the extracellular space in the mid-part of the stratum corneum of vitrified epidermis.
Right column: corresponding intensity profiles obtained from fuzzy distance-based image analysis. (a) Two high-intensity (dark) lines between stratum
corneum cell borders. (b) Four high-intensity lines. (c) Six high-intensity lines. (d) Eight high-intensity lines. (e) Ten high-intensity lines. (f) Twelve high-intensity
lines. Section thickness: B50 nm (af). Pixel size in (a): 3.31 A, and in (bf): 6.02 A.

CEMOVIS images at all defocuses (Figure 6). None of the

other lipid models could account for any major feature of the
CEMOVIS images at any defocus. For reference and
comparison, the simulated electron micrographs for all the
lipid models tested are given in Supplementary Figures S6S8
online. From these simulated images, it is clear that only
bilayer models in which the CER molecules are in the fully
extended conformation show any similarity to the observed
CEMOVIS data (Supplementary Figure S6 online). The folded
(hairpin) CER conformation and stacked monolayers of
extended CERs are readily discounted (Supplementary
Figures S7 and S8 online).
We specifically explored whether the simulated images
are sensitive to how CHOL is distributed within the lipid
model, given the reported asymmetric distribution of CHOL
within model systems composed of reconstituted stratum
corneum lipids (McIntosh, 2003). We considered models
2218 Journal of Investigative Dermatology (2012), Volume 132

(i) without CHOL (Supplementary Figure S6E online), (ii) with

CHOL selectively localized to the CER sphingoid part
(Supplementary Figure S6D online), (iii) with CHOL selectively localized to the CER fatty acid part (Supplementary
Figure S6F online), and (iv) with CHOL homogenously
distributed between the CER fatty acid and sphingoid parts
(Supplementary Figure S6GJ online). The simulations reveal
that the CEMOVIS patterns are remarkably sensitive to CHOL
distribution and suggest that CHOL is selectively localized to
the CER sphingoid part (Supplementary Figure S6D online).
DISCUSSION
The lipid organization that emerges from the analysis is a
bilayer structure of fully extended CERs with the sphingoid
moieties interfacing. Both CHOL and the FFA appear
to be selectively distributed: CHOL at the CER sphingoid
end and the FFA at the CER fatty acid end. The characteristic

I Iwai et al.
The Human Skin Barrier

1,000
30

be

20

nu
m

0.02
0.04

1 1

10

St
r ip

r
Powe

800
600
400
200
0

0.06
0.08

1,000
800
600
Power

Power

600
400

200

200
0

400

0
0

0.02 0.04 0.06 0.08 0.10

1

200

Power

Power

600

400

0.02 0.04 0.06 0.08 0.10

1 1

600

400
200

0.02 0.04 0.06 0.08 0.10

1 1

0.02 0.04 0.06 0.08 0.10

1 1

Figure 3. Cryo-electron microscopy of vitreous skin section (CEMOVIS) micrograph power spectra analysis indicates different molecular packing orders
in different regions within the lamellar structure. (a) Surface plot of all power spectra obtained along the lamellar pattern of a CEMOVIS micrograph
(b, c, Figure 5a) of a unit of one central 4.5-nm band with two adjacent 6.5-nm bands. The power spectra of the 6.5-nm bands, besides their mid-plane,
show recurring characteristics, represented by one 2.3- to 3.0-nm peak and one 1.5- to 1.7-nm peak (d). The 6.5-nm bands, besides their low-intensity
mid-plane, are thus somewhat ordered. The power spectra extracted from the remaining regions are more amorphous with nonreproducible characteristics (eg).
(e) Power spectra obtained from four strips located within the mid-plane of the 6.5-nm bands. (f) Power spectra obtained from five strips located within the
central 4.5-nm band. (g) Power spectra obtained from four strips located within the high-intensity (dark) headgroup bands. The 4.5-nm-thick band, including its
mid-plane, and the headgroup bands are thus amorphous but have, at the same time, some intrinsic order with recurring characteristics of low reproducibility
(f, g). The mid-plane low-intensity region of the 6.5-nm-thick bands is amorphous (e). In (dg) the extracted power spectra are connected to their respective
sampling locations (two-pixel-wide strips) in the CEMOVIS micrograph (c). Pixel size in (b, c): 3.31 A.

6.5-nm distance is consistent with two apposing C24 amidebound fatty acid chains plus one CER headgroup (48C !
1.27 A 5.1 A 66 A; C24 fatty acids dominate in stratum
corneum (Masukawa et al., 2009)). The 4.5-nm distance is
consistent with two apposing C18 sphingoid backbone
chains plus one CER headgroup (36C ! 1.27 A
5.1 A 51 A; C18 sphingoid backbones dominate in
stratum corneum (Masukawa et al., 2009)).

The proposed lipid organization is optimal in terms of

packing, with the fatty acid chain lengths matching the CER
fatty acid chains and CHOL matching the CER sphingoid
chains. This hydrophobic matching minimizes the potential energy of the molecular configuration. The segregation
of CHOL and FFAs to the respective ends of the CERs may be
expected, as CHOL and stratum corneum FFAs do not mix in
model systems (Norlen et al., 2007). CHOL, however, does
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I Iwai et al.
The Human Skin Barrier

0.8

Max

FI

GP

Min

0.6

GP 0.4

0.2

0.0

Normal Hydrated

20 m

5 nm

6.5 nm
4.5 nm

6.5 nm
4.5 nm

5 nm
4.5 nm

Figure 4. The stratum corneum lipid matrix is in a condensed state and remains unaltered upon hydration. (a) 6-Dodecanoyl-2-dimethylaminonaphthalene
(LAURDAN) fluorescence intensity (left) and LAURDAN generalized polarization (GP) function images (right) of normal near-native skin (top panel) and of skin
exposed to hydration for 2 hours in vivo (bottom panel). The images were obtained at the level of mid-part of the stratum corneum using a multi-photon
excitation microscope. (b) Bar graphs (averageSD) showing the LAURDAN GP values of normal and hydrated skin. The high (40.5) LAURDAN GP values
show that the extracellular lipid matrix is in a condensed state and displays little, if any, water dipolar relaxation at the lipid headgroup interface regions.
(c) High-magnification cryo-electron microscopy of vitreous skin section (CEMOVIS) micrograph of the extracellular space in the mid-part of the stratum
corneum after hydration. (d) Intensity profile obtained from fuzzy distance-based path growing of the area marked by a white box in (c). Note that the intensity
profile after skin hydration (d) is identical to that of normal skin (compare Figure 1e). Also note the loose arrangement of keratin intermediate filaments
(open arrows) in the swollen stratum corneum cells as compared with their condensed arrangement in normal stratum corneum (compare Figure 1a).
The CEMOVIS intensity profile of the extracellular lipid matrix thus remains unaltered on hydration. Pixel size in (c): 3.31 A.

mix with sphingosine and its derivatives (Garmy et al., 2005).

The hydrophobic match between CHOL and saturated alkyl
chains is optimized between 14 and 18C chain lengths,
which correspond to the length of the CHOL molecule
(Ouimet and Lafleur, 2004). Further, wide-angle X-ray
diffraction experiments on isolated stratum corneum indicate
crystalline-like hydrocarbon chain packing with characteristic distances of 3.73.8 A and 4.14.2 A (Wilkes et al., 1973;
White et al., 1988; Garson et al., 1991; Bouwstra et al., 1992;
Hatta et al., 2006), which would not appear if CHOL were
distributed throughout both lipid hydrocarbon chains of the
CERs. However, a stratum corneum lipid matrix in which
CHOL and FFAs are segregated into different bands does
allow for crystalline-like hydrocarbon chain packing on the
fatty acid sides of the stacked extended CER bilayer system. It
thus rationalizes how a biological lipid system composed of
30% CHOL could express crystalline-like wide-angle X-ray
diffraction patterns without signs of lateral crystal domain
formation detectable by high-resolution CEMOVIS.
2220 Journal of Investigative Dermatology (2012), Volume 132

The power spectra obtained along the lamellar plane

reflect the lateral lipid packing at a resolution of 12 nm,
which characterizes our CEMOVIS micrographs. The following comments therefore refer to the microstructure rather than
the molecular-level packing of headgroups or alkyl chains.
Overall, the microstructure is predominantly disordered,
but there are some recurring characteristics detectable in
the power spectra (Figure 3). These characteristics were
most evident around the mid-plane of the 6.5-nm bands, i.e.,
the CER fatty acid moiety region, and corresponded to
periodicities of 2.33.0 nm and 1.51.7 nm (Figure 3d). They
correspond roughly to the distance between (24 nm) and
width of (11.5 nm) the cross stripes observed within
the 6.5-nm bands of the CEMOVIS micrographs (Figure 1d,
open white arrows). These periodicities may therefore
reflect the average repeat distance and width of coherent
lipid clusters separated by defect regions. Remarkably,
the perpendicular cross-stripe pattern could be recreated
in the simulated electron micrographs by randomly rotating

I Iwai et al.
The Human Skin Barrier

CEMOVIS micrograph

Averaged intensity profile

along line pattern

Schematic 2D model

Atomic 3D model repeating unit

Electron scattering potential 3D map
of model subunit composed of
one extended ceramide,
one free fatty acid, and
one cholesterol molecule

One out of 20 stacked layers of the total model

electron scattering potential 3D map used for
the model EM simulation

Simulated electron micrograph

Figure 5. Electron microscopy (EM) simulation of the stratum corneum extracellular lipid matrix. (a) High-magnification cryo-electron microscopy of vitreous
skin section (CEMOVIS) micrograph of the extracellular space in the mid-part of the stratum corneum. (b) Corresponding intensity profile obtained by fuzzy
distance-based path growing (compare Figure 1b and c). (c) Schematic two-dimensional (2D) illustration of ceramides (CERs) (tetracosanylphytosphingosine
(C24:0)) in fully extended conformation with cholesterol (CHOL) associated with the CER sphingoid part, and free fatty acids (FFAs) (lignoceric acid
(C24:0)) associated with the CER fatty acid part. (d) Atomic three-dimensional (3D) model of the repeating unit composed of two mirrored subunits, each
composed of one fully extended CER, one CHOL, and one FFA molecule. (e) Calculated electron scattering potential of one model subunit. (f) Calculated
electron scattering potential 3D map of the topmost layer out of 20 superimposed layers used to generate the simulated electron micrograph (g). Defocus
(a, g): !2.5 mm. Pixel size in (a, g): 3.31 A.

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I Iwai et al.
The Human Skin Barrier

UF: 0.5 m

UF: 2 m

UF: 5 m

UF: 1 m

UF: 2 m

UF: 3 m

CEMOVIS
first exposure
micrographs

Model simulation

CEMOVIS
underfocus series
micrographs

Model simulation

Figure 6. Electron microscopy (EM) simulation of alternating fully extended ceramides (CERs) with selective localization of cholesterol to the CER
sphingoid part accurately accounts for the observed cryo-electron microscopy of vitreous skin section (CEMOVIS) intensity pattern, as well as for the
interference pattern changes observed in sequential CEMOVIS micrograph defocus series obtained at very high magnification (p1.88 A pixel size).
(ac) High-magnification CEMOVIS micrographs (first-exposure images) of the extracellular space in the mid-part of the stratum corneum obtained at
(a) !0.5 mm, (b) !2 mm, and (c) !5 mm defocus. At very low defocuses (!0.5 mm) (a) CEMOVIS intensity patterns can only be observed at very high magnification
(p1.88 A pixel size). At very high defocuses (!5 mm) (c) image resolution is low but still allows for resolution of the B11-nm repeating unit. The slightly
larger lamellar repeat distance in (b) (B12 nm) compared with (a) and (c) (B11 nm) may be due to more pronounced compression of the vitreous skin section
during cryo-sectioning along the lamellar plane in (b) compared with that in (a) and (c). (df) represents corresponding atomic three-dimensional (3D)
model (compare Figure 5) EM simulation images recorded at (d) !0.5 mm, (e) !2 mm, and (f) !5 mm defocus. (gi) Sequential CEMOVIS micrograph
defocus series obtained at very high magnification (1.88 A pixel size) at (g) !1 mm, (h) !2 mm, and (i) !3 mm defocus. Note the fine changes in interference
patterns caused by gradually increasing the microscopes defocus during repeated image acquisition at a fixed position. Owing to electron beam damage after
repeated electron exposure, the image contrast is lower in micrographs (h, i) compared with micrograph (g), which was acquired first. In micrograph (i), some
shrinkage can be observed. This is probably due to mass loss during repeated electron exposure. In addition, the curvature of the lamellar pattern is slightly
increased in micrographs (h, i) compared with that in micrograph (g), which may similarly be caused by nonhomogeneous mass loss during repeated electron
exposure. (jl) Represents corresponding atomic 3D model (compare Figure 5) EM simulation images recorded at (j) !1 mm, (k) !2 mm, and (l) !3 mm defocus. It
is shown that the atomic 3D model in Figure 5 accurately accounts not only for the major features of the CEMOVIS micrographs (af) but also for the interference
intensity pattern changes observed upon varying the microscopes defocus during image acquisition at very high magnification (gl). UF, under focus. Pixel size
.
in (c, f): 3.31 A, in (b, e): 6.02 A, and in (a, d and gl): 1.88 A

the individual lipid molecules along their length axes

and displacing them 1 A in the x, y, and z directions
(Figure 5g).
A particularly notable feature of the proposed lipid
structure is that it comprises a bilayer rather than an
arrangement of stacked monolayers. Energetically, either
arrangement should be feasible, given that leaflets of extended
CER structures present alkyl chains at both ends, and therefore
both organizations exclusively involve hydrocarbon interactions between leaflets. The preference for the bilayer
organization in the stratum corneum lipid matrix could result
2222 Journal of Investigative Dermatology (2012), Volume 132

from the biological processes involved in its formation. The

lipid matrix is formed from stacked bilayers of glycosylceramides in the hairpin conformation in a hydrated environment, after deglycosylation and dehydration (Holleran et al.,
1993; Caspers et al., 2001; Norlen, 2001; Al-Amoudi et al.,
2005). During its formation, the extended CER bilayer
organization will therefore require a transformation from
hairpin to splayed chain conformation, involving a flip of one
of the two CER alkyl chains. For glycosylceramides, which
bind 510 water molecules per lipid molecule (Bach et al.,
1982; Bach and Miller, 1998), flip-flop is slow (half-time of

I Iwai et al.
The Human Skin Barrier

hours (Buton et al., 2002)). For CERs, which only bind 01

water molecules per lipid molecule (Faure et al., 1998), the
flip-flop movement is considerably faster (half-time o1
minute (Lopez-Montero et al., 2005)). For CHOL, flip-flop is
rapid (half-time o1 second (Steck et al., 2002)). The initial
hairpin glycosylceramide bilayer organization may therefore
carry over into the extended CER bilayer organization by a flip
of one of the two CER alkyl chains together with CHOL. The
driving force could be the deglycosylation and dehydration
encountered during the formation process. Once the strongly
hydrated glycosyl groups of the glycosylceramides have been
removed (Holleran et al., 1993) and the water evacuated from
the extracellular space (Al-Amoudi et al., 2005), the extension
of the two hydrocarbon chains in opposite directions aids the
separation of both the ill-matched CHOL and FFAs (Norlen
et al., 2007), and the ill-matching CER C24 fatty acid and C18
sphingoid moities, into different bands within an optimally
close-packed stratum corneum bilayer structure. Speculatively, being a rapid membrane flipper, CHOL may facilitate
the extension of the CERs by dragging the sphingoid moiety
with it during the lipid matrix reorganization from a hairpin to
an extended bilayer structure.
The major physiological consequence of a condensed,
fully extended CER bilayer organization is that the lipid
matrix will be largely impermeable to water, as well as to both
hydrophilic and lipophilic substances, because of the
condensed structure and the presence of alternating
lipophilic (alkyl chain) and hydrophilic (headgroup) regions
(compare Figure 5c). It will be resistant toward both
hydration and dehydration because of the absence of
exchangeable water between lipid leaflets (compare Figure 4).
Further, the proposed structure accounts for stratum
corneum cell cohesion without advocating desmosomal
cell-adhesion structures, and hence allows for the possibility
of sliding of stratum corneum cells to accommodate skin
bending. Finally, as the interaction between the individual
layers of the lipid structure involves only hydrocarbons, the
individual layers are relatively free to slide with respect to
each other, making the matrix pliable. The lipid matrix thus
meets the barrier needs of skin by being simultaneously robust
and impermeable.
How does our CEMOVIS data reconcile with earlier
studies? The B11-nm repeating unit fits with the 12- to
13-nm repeating unit observed in small-angle X-ray diffraction
by White et al. (1988), Bouwstra et al. (1991), Hou et al.
(1991), and Schreiner et al. (2000) in isolated mouse and
human stratum corneum, as well as with the 13-nm repeating
unit observed by Madison et al (1987) and Hou et al. (1991) in
ruthenium tetroxidestained mouse skin, given a preparation
(dehydration)-induced slight straightening of lipid alkyl chains
in these studies. Further, in ruthenium tetroxidestained
stratum corneum (compare Supplementary Figure S9A online),
the electron intensity pattern of the extracellular lipid matrix
expresses a broad:narrow:broad electron lucent band pattern
(Madison et al., 1987), which is consistent with our data given
that ruthenium tetroxide not only associates with lipid
headgroups but also penetrates to some extent between the
lipid leaflets when facilitated by local liquid-like disordering

(Supplementary Figure S9C online). In the CEMOVIS micrographs, we observed a mid-plane low-intensity region in the
6.5-nm bands but not in the 4.5-nm bands (Figure 1b and d;
Supplementary Figure S4A, B online. This translates to an
increasingly lower atomic density toward the distal parts of the
CER fatty acid and FFA alkyl chains, which would suggest a
liquid-like disordered region close to terminal methyl groups.
This is further supported by molecular dynamic simulations,
which point at a skin lipid hydrocarbon chain arrangement
involving a gradual change between condensed and liquid
along the chain axes (Das et al., 2009).
We are unable to comment on previous wide-angle X-ray
diffraction and attenuated total reflection-Fourier transform
infrared spectroscopy studies (Bouwstra et al., 1992; Damien
and Boncheva, 2010), particularly with respect to alkyl chain
packing as to whether it is hexagonal or orthorhombic or
both, as the CEMOVIS data are limited to a resolution of
around 12 nm.
To summarize, the stratum corneum lipid matrix is,
according to our analysis, organized as stacked bilayers
of fully extended CERs with CHOL molecules associated
with the CER sphingoid moiety. Further, despite its crystalline-like character, it is malleable. This condensed but pliable
fully extended CER bilayer organization with asymmetric
CHOL distribution rationalizes the skins low permeability
toward water and toward hydrophilic and lipophilic substances, as well as the skin barriers robustness toward
hydration and dehydration, environmental temperature
and pressure changes, stretching, compression, bending,
and shearing.
Final remarks

Until now, progress in skin barrier replacement, skin barrier

repair, skin permeability enhancement, and skin protection
has largely resulted from empirical efforts, treating the
stratum corneum lipid matrix as a black box. The molecular
description of the lipid matrix presented here may constitute
a molecular platform for the above research goals, and in
particular for in silico approaches such as molecular
simulations, as well as for in vitro modeling, to underpin
interactions of the lipid matrix with drugs and other
chemicals. For example, it is foreseeable that this knowledge
of the lipid organization will now enable in silico screening
to identify molecules for enhancing skin penetration for drug
delivery and to find tightening or repair molecules for
the stratum corneum lipid matrix. Further, it may constitute a
reference for engineering artificial skin for the treatment of
wounds and burn injuries.
More generally, the novel methodological approach we
have used, that combines very high magnification CEMOVIS
defocus series with molecular modeling and EM simulation,
may open the way for determining the near-native molecular
organization of other structures in normal and diseased cells
and tissues.
MATERIALS AND METHODS
A more detailed description of experimental procedures is available
in Supplementary Information.
www.jidonline.org 2223

I Iwai et al.
The Human Skin Barrier

Skin biopsy specimens were collected from the human volar

forearm and abdomen of five Caucasian males in their 40s50s with
no history of skin disease. The samples were immediately vitrified
using a Leica EMPACT2 high-pressure freezer and cryosectioned at
!1401C. Images were recorded at !0.5 to !5 mm defocus, at
120 kV, at a dose of 1,00017,000 electrons nm!2 per image and a
pixel size of 6.02, 3.31, and 1.88 A, respectively, in a FEG CM200
FEI microscope equipped with a cooled slow-scan 2048 " 2048
TVIPS TemCam-F224 HD CCD camera.
The averaged intensity profiles of the cryo-electron micrographs
were obtained by fuzzy distance-based image analysis. A path was
automatically extracted based on the shortest intensity-weighted
distance between two points. Along the path, perpendicular intensity
profiles were extracted (Figure 1c) and then averaged (Figure 1e).
To accurately estimate the distance between intensity peaks,
numerical analysis was used, based on least-squares fitting between
the central part of the measured intensity peaks and a Gaussian
model (Supplementary Figure S3 online).
To check the degree of the packing order of the 6.5- and 4.5-nm
bands, power spectra of 2-pixel-wide strips parallel to the bands in
the micrographs were extracted (Figure 3).
To evaluate the native phase state and the hydration behaviour of
the stratum corneum lipid matrix, multi-photon excitation microscopy LAURDAN GP measurements were performed on excised
forearm skin before and after hydration in vivo (Figure 4a and b).
Simulated CEMOVIS images for the different atomic models
(Supplementary Figures S6D3-J5, S7D3-J5, and S8D3-E5) were created
with a computer program simulating the interaction between the
potential map and the electron beam, the optical transformation
effect of the lens system of the microscope, and the image formation
on the detector (Rullgard et al., 2011). The software is freely
available at http://tem-simulator.sourceforge.net/.
CONFLICT OF INTEREST
The authors state no conflict of interest.

ACKNOWLEDGMENTS
We thank Bertil Daneholt for unwavering support and encouragement for more
ktem, Jacques Dubochet, Massimo Noro,
than a decade. We also thank Ozan O
Chinmay Das, Emma Sparr, Johan Engblom, Philip Wertz, Ichiro Hatta, Jonathan
Hadgraft, Richard Guy, Marek Haftek, Richard Mendelsohn, Stephen Hoath,
David Moore, Jennifer Thewalt, Walter Holleran, Gopi Menon, Michael Roberts,
Fred Frasch, Gabriel Wittum, Jim Riviere, Samir Mitragotri, Jesus Perez Gil,
Thomas McIntosh, Reinhard Neubert, and our reviewers for invaluable
comments on the manuscript. The present work was made possible by the
generous support from the Swedish Medical Society, Swedish Research Council,
Oriflame, Shiseido Japan, LEO Pharma, Wenner-Gren foundation, Welander
Foundation, European community (3D-EM Network of Excellence), Visualization
Program by Knowledge Foundation, Vardal Foundation, Foundation for Strategic
Research, VINNOVA, Invest in Sweden Agency, Forskningsradet for Sundhed og
Sygdom (FSS, Denmark), and the Danish National Research Foundation. The
Knut and Alice Wallenberg Foundation is acknowledged for support of the
electron microscope facility. II developed and performed high-resolution
(pixelsize p3.31 A; nominal section thickness 2030 nm) CEMOVIS and
designed the in vivo hydration/dehydration experiment. HH and LdH performed

CEMOVIS. SS developed the fuzzy distance-based analysis method. L-GO

performed the EM model simulations. JA constructed the atomic threedimensional models, formulated the alternative models with LN, and contributed
to the writing of the paper. JB, MB, and LAB performed the multi-photon
excitation microscopy LAURDAN GP analysis. AL and DN assisted the
CEMOVIS data analysis. SM supported the high-resolution CEMOVIS development and data collection. US performed the numerical curve fitting analysis and
the power spectra analysis. LN designed the study, analyzed the data, formulated
the model, and wrote the paper.

2224 Journal of Investigative Dermatology (2012), Volume 132

SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at http://
www.nature.com/jid

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This work is licensed under the Creative Commons

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www.jidonline.org 2225

MATERIALS AND METHODS

All human studies were approved by the authors' Institutional Review Board and
followed the Declaration of Helsinki protocols. All subjects involved gave their written
informed consent.

Skin sample preparation

Skin biopsies (area, 1x1 mm2; thickness, 100-150 m) for CEMOVIS were collected,
before as well as after hydration in-vivo, from the left volar forearm and abdomen of 5
male Caucasians in their 40s-50's with normal skin and no history of skin disease, as
judged from dermatological evaluation. The skin area used had not been exposed to any
detergents, treatments or skin care products for one month prior to experimentation. To
avoid sample dehydration, the biopsies were instantly immersed in 1-hexadecane
(MERCK). Subsequently, the samples were placed in the cavity of a cylindrical gold cup
prefilled with 1-hexadecane, and immediately vitrified using a Leica EMPACT2 high
pressure freezer (Leica, Wien, Austria).

Skin hydration and dehydration

To swell the skin, two filter papers (2 cm x 2 cm, Whatman no.1001090, Maidstone,
England) soaked with 300 l deionised water were put on the volar forearm and occluded
for two hours with Tegaderm (3M Health Care, Germany). Biopsies were then taken and
high-pressure frozen either immediately or after 24 hours acclimatization at 21C at 93%
RH, in air in equilibrium with a saturated solution of KNO3 (cf. Middleton, 1968).

Cryo sectioning
The vitreous skin samples were trimmed with a trimming diamond blade (Diatome, Biel,
Switzerland) and cryosectioned at -140C with a nominal section thickness of 25-50 nm
using a 35 diamond knife (Diatome, Biel, Switzerland) with a clearance angle of 6.
Cutting speed was set to 0.2-1.0 mm/s. The sections were transferred to copper grids with

1000 mesh, using an eyelash glued to a wooden stick. Subsequently they were pressed
with a stamping tool and stored in liquid nitrogen. A facemask was used throughout the
cutting and section transfer procedure to minimize ice-crystal contamination. Air flow,
temperature (21C) and humidity (<25%RH) were controlled in the work room.

CEMOVIS
Two major advantages of CEMOVIS are that the native, hydrated tissue is preserved
down to the molecular level and that the image pixel intensity is directly related to the
local electron density of the sample. For more detailed accounts of CEMOVIS, see
Dubochet et al., (1988), Al-Amoudi et al. (2004) and Norln et al. (2009).
We used a GATAN model 626 cryo-holder (GATAN, Pleasanton, CA) at -180C.
Images were collected at 120kV in a FEG CM200 FEI microscope, equipped with a
cooled slow scan 2048x2048 TVIPS TemCam-F224 HD CCD camera (pixel size 14m).
Images were generally recorded at a dose of 1.000-17.000 electrons/nm2 per image and a
total magnification of 27.500 X (pixel size: 6.02 ), 50.000 X (pixel size: 3.31 ), and
88.000 X (pixel size: 1.88 ), respectively. During image acquisition, a defocus of about
-2 m was regularly employed as it yields a good compromise between relatively high
image resolution (low defocus) and relatively high image contrast (high defocus). To
discriminate different molecular lipid models, images were also acquired at very low
defocus (-0.5 m), yielding images with high resolution but with very low contrast (cf.
Fig. 6A), and at very high defocus (-5 m), yielding images with high contrast but with
very low resolution (cf. Fig. 6C). In addition, defocus series were acquired at very high
magnification (pixel size: 1.88 ) (cf. Fig. 6G-I). In this way, fine changes in interference
patterns caused by gradually increasing the microscope's defocus during repeated image
acquisition at a fixed position could be used to verify the selected model (Fig. 6J-L).

Image analysis
For the image analysis, we developed a semi-automatic program using MATLAB and
C++. As a preprocessing step, we used the edge preserving smoothing algorithm

anisotropic diffusion filtering (Perona and Malik, 1990). In this way small variations,
largely corresponding to noise, are suppressed while significant features are left
untouched. We then treated the image locally as a fuzzy membership map, where the
membership corresponds to degree of belongingness to a lucent band of the lamellar
intensity pattern. The procedure is as follows. Two points are marked on one lucent band.
A path is then automatically extracted from one point to the other based on the shortest
intensity-weighted distance between the two points. We used the intensity-weighted
distance introduced by Levi and Montanari (1970), for which the mean of the intensities
of two neighbouring points is multiplied by the spatial distance between them. Intensityweighted distance is described in a theoretical framework and denoted fuzzy distance by
Saha et al (2002). The result is that a path is placed along the highest ridge between the
two points. It thus becomes centrally located on the lucent band. For each point along the
path, perpendicular intensity profiles were extracted using the improfile method in
MATLAB. Finally, the profiles were averaged.

Numerical curve fitting

Averaged perpendicular intensity profiles (cf. Fig. 1C) were used for the numerical curve
fitting. The goals were to accurately estimate the distance between intensity peaks and to
detect systematic deviation from Gaussian distribution. In figure S3, seventeen profiles
were used, each with 201 grid points. The grid unit was 3.31 and corresponded to the
image pixel size. As seen in Figure 1D, the electron micrographs vary systematically in
intensity in lucent regions (a fine stripe pattern perpendicular to the lamellar plane),
while lamellar peak intensity positions remain stable. The average intensity is thus
suitable for accurate determination of peak distance. The least squares based fit to the
central part of the measured intensity peaks was excellent (Fig. S3). This indicates that
Gaussian modelling is sufficient to establish peak positions accurately. Two distances
emerge, one of ~ 4.5 nm and one of ~ 6.5 nm. Deviations from Gaussian distribution
were confined to the intensity peak peripheries (Fig. S3). The bilateral broadening of the
peaks towards the valley mid planes is largely an effect of deviation from the Gaussian
model. As the deviation is equally pronounced on the 4.5 nm valley side and the 6.5 nm

valley side of the intensity peaks (Fig. S3), the higher intensity of the 4.5 nm valley midplanes with respect to the 6.5 nm valley mid-planes is partly due to more pronounced
intensity curve overlapping between the more closely separated peaks (4.5 nm) compared
to between the more widely separated peaks (6.5 nm).

Power spectra analysis

To check the degree of order or amorphous character of the ~6.5 nm and ~4.5 nm bands
we analysed power spectra of 2 pixel wide strips parallel to the bands in the micrographs
(Fig. 3A). Individual power spectra obtained from different regions of the layered
structure are presented in figures 3D-G.
The power spectra show that each strip-region has some intrinsic order with
recurring characteristics. However, at the same time the intrinsic order seems to vary
considerably when comparing the spectra from other strips in a similar region. The only
region with somewhat reproducible spectra across its sampled strips is seen in (D). The
conclusion is that the (D) region, besides its low intensity mid-plane (E), is somewhat
ordered, while the other regions are more amorphous.
The ~4.5 nm thick band including its mid-plane (F), as well as the head-group
bands (G), are amorphous but have at the same time some intrinsic order with recurring
characteristics of low reproducibility.

LAURDAN GP before and after hydration in-vivo

The LAURDAN generalized polarization (GP) function reflects the wavelength

dependence of LAURDANs emission spectrum (Parasassi et al., 1998). The LAURDAN

emission spectrum is sensitive to the extent of the water dipolar relaxation process that
occurs in the probes microenvironment (this probe is insoluble in water and shows a
high partition to membranes). Since the extent of water dipolar relaxation at the
membrane interface (where LAURDAN is located) is highly connected with the packing

of the host membrane, this probe is able to discriminate different lipid phases (Bagatolli,
2006). For example, a 50 nm shift is observed in the fluorescence emission spectrum of
LAURDAN when phospholipid membranes undergo solid ordered/liquid disordered
phase transition (Parasassi et al., 1998). The GP function was defined analogously to the
fluorescence polarization function as:

GP =

IB IR
IB + IR

(1)

where IB and IR correspond to the intensities at the blue and red edges of the emission
spectrum (440 and 490 nm) using a given excitation wavelength (Parasassi et al., 1990).
In lipid bilayers high LAURDAN GP values (0.45 0.60) correspond to ordered phases
whereas low LAURDAN GP values (below 0.15) correspond to fluid disordered-like
phases (Bagatolli, 2006). Ordered phases have been reported previously in mixtures
composed of purified human stratum corneum lipid extracts (Plasencia-Gil et al., 2007)
and in pig skin ex-vivo (Carrer et al., 2008).
A total of 96 GP values were calculated from normal and hydrated volar forearm
skin (one from each of 6 images, for each of 4 samples, for each of 2 preparations, for
each of 2 individuals) (Fig. 4A-B). The subjects were both male Caucasians in their 40s
with normal skin and no history of skin disease, as judged from dermatological
evaluation. The skin area used had not been exposed to any detergents, treatments or skin
care products for one month prior to experimentation. The GP value obtained per image
was an average GP value obtained from over 20 regions using a ROI routine. After
excision, the skin samples were immediately immersed in a hexadecane solution of
LAURDAN (1.4 mM) for 2 hours to avoid sample dehydration and ensure proper
LAURDAN incubation. As a control, experiments were also performed after 5 and 30
minutes of LAURDAN incubation. After LAURDAN incubation, the samples were
rinsed in pure hexadecane and mounted in a microscope slide for obtaining the GP
images. A custom-built multi-photon excitation microscope (Brewer et al., 2010) was
used in our experiments. This microscope is controlled by Globals for Images SimFCS
(Laboratory for fluorescence dynamics, University of Irvine). The excitation light source

was a femtosecond*Ti:Sa*laser*(Mai Tai DeepSeeTM,*Spectra*Physics,*Mountain*View,*

CA)* operated* at* a* wavelength of 780 nm. For the LAURDAN GP measurements the
total fluorescence signal was divided between two detection channels (each of them
equipped with a Hamamatsu H7422P-40 photomultiplier) using a dichromatic mirror
(splitting below and above 475 nm). One detection channel contain a bandpass filter of
438 12nm whereas the other contain a bandpass filter of 494 10 nm (corresponding
respectively to IB and IR in equation 1).

The calculation of the GP function was

performed from Globals for images software and a GP standard was used as previously
described (Brewer et al., 2010).

Atomic 3D modelling
The atomic model comprised the 3 key components of the known lipid composition,
namely a C24 ceramide NP (the predominant ceramide in the stratum corneum),
cholesterol, and a C24 free fatty acid (the predominant free fatty acid in the stratum
corneum). Models of the individual molecules were generated using a molecular-model
building procedure with the atoms being located at their ideal bond distances, angle and
torsions.

The

ceramide

molecule

was

rotated

about

the

bonds

CH(OH)CH(CH2OH)NH and CH(CH2OH)NHCOCH to yield a fully extended

conformation. The three component molecules were then rotated to lie lengthwise along
the z-axis and translated relative to each other to give a close packed assembly with the
cholesterol molecule lying adjacent to the sphingoid chain of ceramide NP and the fatty
acid adjacent to the ceramide fatty chain. The full assembly containing fully extended
ceramides gave a unit subcell with dimensions of approximately 1.0 x 1.1 x 5.25 nm.

Cryo-EM simulation
Simulated images from generated atomic models were created with a TEM simulator
program recently developed by H. Rullgrd, L.-G. fverstedt and O. ktem (Rullgrd et
al., 2011). The first part of the program is a phantom generator that can read one or more
atomic models in the RCSB Protein Data Bank (PDB) format and construct a model

scenario with molecules at defined positions. An electron scattering potential map is then
generated with a background structure and potential corresponding to that of vitrified
water. The second part of the program simulates the interaction between the potential
map and the electron beam, the optical transformation effect of the lens system of the
microscope and the image formation on the detector. Parameters defining optical
properties of the microscope, e. g. acceleration voltage, aberration constants and defocus,
and the point spread function of the detector can be set to mimic the conditions in a real
experiment. Thus, simulated images (Fig. 5J, 6D-F, 6J-L, S6D3-J5, S7D3-J5, and S8D3E5) were generated with conditions corresponding to the CEMOVIS images.

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Richter, K. Sartori Blanc, N. Studer, D. Dubochet, J (2004) Cryo-electron microscopy of
vitreous sections. EMBO J. 15;23(18):3583-8
Bagatolli, LA (2006) To see or not to see: lateral organization of biological membranes
and fluorescence microscopy. Biochim Biophys Acta 1758:1541-1556
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Carrer, DC. Vermehren, C. Bagatolli, LA (2008) Pig skin structure and transdermal
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(1988) Cryo electron microscopy of vitrified specimens. Q Rev Biophys. 21 (2): 129-228.
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*

SUPPLEMENTARY FIGURE LEGENDS

Figure S1. Molecular models for the lipid organization of the stratum corneum
extracellular space. Models proposed by Schrter et al. (2009) (A), by McIntosh (2003)
(B), by Hill and Wertz (2003) (C), by Bouwstra et al., (2001) (D), by Forslind (1994) (E), by
Swartzendruber et al., (1989) (F). Six different models for the molecular organization of the
stratum corneum extracellular lipid matrix have been proposed. Swartzendruber et al. (1989)
presented the first model (F). It was based on the broad:narrow:broad electron lucent band
pattern observed in RuO4 stained stratum corneum (cf. Suppl. fig. 8A), and consisted of
triple-band units with ceramides in fully extended conformation (i.e., with the ceramide
sphingoid- and the fatty acid parts pointing in opposite directions). Each triple-band unit
was composed of one narrow central band and two broad peripheral bands. The central band
expressed hydrocarbon chain interdigitation and contained no cholesterol. The peripheral
bands expressed no chain interdigitation but contained cholesterol (F). Later, Forslind
(1994) presented a model (E) based on lateral lipid domain segregation. It consisted of
stacked lipid bilayers composed of ceramides in folded conformation (i.e., with the ceramide
sphingoid- and the fatty acid parts pointing in the same direction). Each bilayer expressed
thickness changes due to laterally segregated crystalline and liquid crystalline domains (E).
Combining data derived from conventional electron microscopy and SAXD, Bouwstra et al.
(2001) presented a model (D) consisting of triple-band units with a 13 nm repeat period.
Each triple-band unit was composed of one narrow central band and two broad peripheral
bands. The central band expressed interdigitating omega-esterified polyunsaturated fatty
acids of ceramide EOS, short chain ceramides in folded conformation and cholesterol. The
two peripheral bands expressed long chain ceramides in folded conformation and cholesterol
(D). Reinterpreting the band patterns of RuO4 stained stratum corneum, Hill and Wertz
(2003) proposed a model (C) similar to that of Bouwstra et al (2001), but with uniform
thicknesses of the three bands constituting the triple-band units. Based on SAXD
experiments on skin lipid model systems in-vitro, McIntosh (2003) presented a model (B)
consisting of twin-band (double bilayer) units with a 13 nm repeat period, with an
asymmetric cholesterol distribution, and with equal thicknesses of the constituent bands.
Note that McIntosh's model does not state whether the constituent ceramides are all in
folded conformation or, alternatively, as depicted in (B), both in folded and fully extended
conformation. Recently, Schrter et al. (2009) presented a model (A) based on neutron

scattering experiments on skin lipid model systems in-vitro. It consisted of lamellar units of
equal thickness, composed of mixed ceramides in folded and extended conformation, and
with a homogenous cholesterol distribution. All bands were spanned by the fatty acid chains
of ceramide EOS (A). LE: 'lipid envelope' (or stratum corneum cell plasma membrane).
Note that the schematically sketched molecular organization of the lipid envelopes is not
part of the models (A-E) and is included only to put the models in their proper context.

Figure S2. The lipid matrix is, despite its crystalline-like character, malleable. The
stratum corneum extracellular lipid matrix is folded locally. The folding decreases on
hydration and increases on dehydration. Low magnification CEMOVIS micrographs of
stratum corneum after hydration in-vivo (A), at normal in-vivo conditions (B), and after
hydration in-vivo followed by dehydration ex-vivo (C). The lower panel illustrates the
folded pattern of the extracellular space. Image side lengths: 5 m.

Figure S3. Numerical curve analysis was performed to accurately estimate the distance
between intensity peaks. (A) Averaged electron intensity line (grey) obtained from 17
profiles extracted from figure S4A. Gaussian curve peaks (red) numerically least squares
fitted to the electron intensity peaks (black). Intensity difference (green) between measured
(black) and calculated (red) peaks. The least squares based fit to the central part of the
measured intensity peaks was excellent. This indicates that Gaussian modelling is sufficient
to establish peak positions accurately. Two alternating distances emerge, one of
approximately 4.5 nm and one of approximately 6.5 nm.

Figure S4. The 6.5 nm bands express a mid-plane low intensity region. (A) High
magnification CEMOVIS micrograph of the extracellular space in the mid-part of stratum
corneum. (B) Intensity profile obtained from fuzzy distance based path growing of the area
shown in (A). Note the "shoulders" (open arrows) centrally on each slope delimiting the
mid-plane low intensity region of the 6.5 nm bands. Black arrows (A) denote their
corresponding positions in the cryo-electron micrograph. (C) Schematic representation of a
stacked ceramide alternating monolayer organization with cholesterol localized to the
regions corresponding to the 4.5 nm bands and with FFA localized to the regions

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corresponding to the 6.5 nm bands. Square brackets mark regions with presumed liquid-like
disordering. Pixel size in (A): 3.31 .

Figure S5. No inter-individual or inter-site (forearm versus abdomen) variation was

recorded. Left column: high magnification CEMOVIS micrographs of the extracellular
space in the midpart of stratum corneum of vitrified epidermis obtained from left volar
forearm (A, C-E) and abdomen (B). Right column: corresponding intensity profiles obtained
from fuzzy distance based image analysis. Section thicknesses ~50 nm (A-E). Pixel size in
(A): 6.02 , and in (B-E): 4.35 .

Figure S6. Electron microscopy simulation results from 7 fully extended ceramide
bilayer models with varying cholesterol distribution. (A-C) CEMOVIS micrographs of
the stratum corneum extracellular lipid matrix acquired at -5 m (A), -2 m (B), and -0.5
m (C) defocus. (D3-J5) Corresponding simulated electron micrographs obtained from 7
fully extended ceramide models. (D1-J1) Repeating units for each simulated model. (D2-J2)
Calculated electron scattering potential 3D maps of the topmost layer out of 20
superimposed layers used to generate each individual simulated micrograph (D3-J5). In
model (D), cholesterol is selectively localized to the ceramide sphingoid part. In model (E),
cholesterol has been removed to evaluate whether the simulation method could discriminate
the presence (D) or absence (E) of cholesterol. In model (F), cholesterol is selectively
localized to the ceramide fatty acid part. In models (G-J), cholesterol is homogenously
distributed between the ceramide sphingoid and fatty acid parts. Contrary to models (G, J),
models (H, I) express axial headgroup displacement of cholesterol and free fatty acids.
Models (H, I) differ in that model (H) expresses a pair-wise lateral distribution of ceramides
while model (I) expresses a homogeneous lateral distribution of ceramides. Note that except
for the position of the lipid headgroups, the localization of cholesterol within the fully
extended ceramide structure largely determines the electron scattering properties of the
models.

Figure S7. Electron microscopy simulation results from 7 fully extended ceramide
stacked monolayer models with varying cholesterol distribution. (A-C) CEMOVIS

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micrographs of the stratum corneum extracellular lipid matrix acquired at -5 m (A), -2 m

(B), and -0.5 m (C) defocus. (D3-J5) Corresponding simulated electron micrographs
obtained from 7 stacked fully extended ceramide models. (D1-J1) Two repeating units for
each simulated model. (D2-J2) Calculated electron scattering potential 3D maps of the
topmost layer out of 20 superimposed layers used to generate each individual simulated
micrograph (D3-J5). In model (D) cholesterol is selectively localized to the ceramide
sphingoid part. In model (E) cholesterol has been removed to evaluate whether the
simulation method could discriminate the presence (D) or absence (E) of cholesterol. In
model (F), cholesterol is selectively localized to the ceramide fatty acid part. In models (GJ), cholesterol is distributed homogenously between the ceramide sphingoid and fatty acid
parts. Contrary to models (G, J), models (H, I) express axial headgroup displacement of
cholesterol and free fatty acids. Models (H, I) differs in that model (H) expresses a pair-wise
lateral distribution of ceramides while model (I) expresses a homogeneous lateral
distribution of ceramides. Note that except for the position of the lipid headgroups, the
localization of cholesterol within the fully extended ceramide structure largely determines
the electron scattering properties of the models.

Figure S8. Electron microscopy simulation results from 2 folded ceramide bilayer
models with and without the presence of cholesterol. (A-C) CEMOVIS micrographs of
the stratum corneum extracellular lipid matrix acquired at -5 m (A), -2 m (B), and -0.5
m (C) defocus. (D3-E5) Corresponding simulated electron micrographs obtained from 2
folded ceramide models. (D1-E1) Repeating units for each simulated model. (D2-E2)
Calculated electron scattering potential 3D maps of the topmost layer out of 20
superimposed layers used to generate each individual simulated micrograph (D3-E5). In
model (D), cholesterol is present. In model (E), cholesterol has been removed to ascertain if
the simulation method could distinguish the presence (D) or absence (E) of cholesterol. Note
that the presence of cholesterol within the folded ceramide structure largely determines the
electron scattering properties of the models.

Figure S9. Electron intensity pattern in ruthenium tetroxide stained neonatal mouse
skin. (A) RuO4 staining pattern of the stratum corneum extracellular space. (B) Enlarged
view of the area marked by a white box in (A). (C) Schematic model for the RuO4 staining
!

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pattern in (B). (D) RuO4 staining pattern of lamellar body inside cell in stratum granulosum.
(E) Enlarged view of the area marked by a white box in (D). (F) Schematic model for the
RuO4 staining pattern in (E). A transition from folded to stretched ceramide bilayer
conformation during skin barrier formation can explain the ruthenium tetroxide staining
pattern of both 'lamellar bodies' (i.e., the precursor system of the stratum corneum
extracellular lipid matrix) and the stratum corneum lipid matrix. Inside lamellar bodies in
topmost viable epidermis just beneath the stratum corneum, glycosylceramides (i.e., the
precursors of the stratum corneum ceramides) sometimes form aggregates of irregularly
stacked bilayers (Norln et al., 2003; Al-Amoudi et al., 2005). The corresponding electron
density pattern in ruthenium tetroxide stained skin is characterised by alternating broad
(strong) and thin (weak) dark lines. The broad dark lines correspond to lipid headgroups, as
they can be seen to change in thickness locally, reflecting local bilayer separation in the
aqueous protein-enriched environment inside the lamellar bodies (D-F). On the contrary, the
thin dark lines are constant in thickness throughout (D-E). They therefore likely correspond
to the mid-plane of the lipid bilayer, reflecting a change in the hydrocarbon chain region
between liquid-like disordered (closer to the bilayer mid-plane) and ordered (closer to the
lipid headgroups) carbons. This indicates that ruthenium tetroxide not only associates with
lipid headgroups but also penetrates to some extent between lipid leaflets, especially when
facilitated by local liquid-like hydrocarbon chain disordering. This makes the interpretation
of the broad:narrow:broad electron lucent band pattern in ruthenium tetroxide stained
stratum corneum (A) straightforward. The central narrow electron lucent band then
corresponds to the mid-plane of the cholesterol-enriched sphingoid side (C), and the broad
electron lucent bands to the proximal part of the fatty acid side (C), of the extended
ceramide bilayers. The broad (strong) dark lines thus correspond to lipid headgroups and the
thin (weak) dark lines to the fatty acid mid-plane (C). The staining mechanism in the
ceramide-based stretched-out bilayer system of the stratum corneum extracellular space (AC) thus becomes identical to that in the glycosylceramide-based bilayer system of the
lamellar bodies (D-F). Figures (A-B, D-E) are adapted from Madison et al. (1987), with
permission.

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