Biochimica et Biophysica Acta 1798 (2010) 703–718

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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m e mReview

Surface topography of membrane domains
Marie-Cécile Giocondi a,b, Daisuke Yamamoto c, Eric Lesniewska d, Pierre-Emmanuel Milhiet a,b,
Toshio Ando c, Christian Le Grimellec a,b,⁎aInstitut National de la Santé et de la Recherche Médicale, Unité 554, Montpellier, France
Université de Montpellier, Centre National de la Recherche Scientiﬁque, UMR 5048, Centre de Biochimie Structurale, Montpellier, France
Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japand
Institut Carnot de Bourgogne, Centre National de la Recherche Scientiﬁque, UMR 5209, Université de Bourgogne, Faculté Sciences Mirande, Dijon, Francebca r t i c l e

i n f oArticle history:
Received 8 July 2009
Received in revised form 11 September 2009
Accepted 20 September 2009
Available online 29 September 2009
Keywords:
Membrane domain
Langmuir Blodgett ﬁlm
Supported lipid bilayer
Atomic force microscopya b s t r a c t
Elucidating origin, composition, size, and lifetime of microdomains in biological membranes remains a major
issue for the understanding of cell biology. For lipid domains, the lack of a direct access to the behaviour ofsamples at the mesoscopic scale has constituted for long a major obstacle to their characterization, even insimple model systems made of immiscible binary mixtures. By its capacity to image soft surfaces with aresolution that extends from the molecular to the microscopic level, in air as well as under liquid, atomic
force microscopy (AFM) has ﬁlled this gap and has become an inescapable tool in the study of the surface
topography of model membrane domains, the ﬁrst essential step for the understanding of biomembranes
organization. In this review we mainly focus on the type of information on lipid microdomains in model
systems that only AFM can provide. We will also examine how AFM can contribute to understand dataacquired by a variety of other techniques and present recent developments which might open new avenues
in model and biomembrane AFM applications.
© 2009 Elsevier B.V. All rights reserved.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
AFM characterization of lipid ﬁlms and membrane hemi-leaﬂets . . . . . . . . . . . . . . .
2.1.
Single lipid and binary mixtures of lipids . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Lipid mixtures related to microdomains enriched in sphingolipids and cholesterol. . . .
2.3.
Lipid interaction with peptides and proteins can create domains in monolayers . . . . .
3.
AFM characterization of supported bilayers . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Formation of SLB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
AFM imaging at room temperature of domains in SLB . . . . . . . . . . . . . . . . .
3.3.
AFM imaging of lipid–peptide and lipid–protein interaction in SLB . . . . . . . . . . .
3.4.
Temperature-dependence of AFM membrane domains imaging. Interaction SLB-support .
4.
AFM recent developments and perspectives . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Elucidation of biomembranes molecular structure remains a major
challenge for cell biology. As for simpler biological objects, like soluble⁎ Corresponding author. Nanostructures and Membrane Complexes, Centre deBiochimie Structurale, INSERM U554, 29 rue de Navacelles, 34090 Montpellier Cedex,
France. Tel.: +33 467 41 79 07; fax: +33 467 41 79 13.
E-mail address: clg@cbs.cnrs.fr (C. Le Grimellec).
0005-2736/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamem.2009.09.015

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703
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707
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712
712
714
714

proteins, understanding of structure–function relationships in cell
membranes represents an essential step in the development of drugs
directed not only against pathologies resulting from membrane
dysfunction but also against all the intracellular processes which
require drugs internalization. In plasma membrane, the slow
transmembrane movement of most polar lipids, in particular inabsence of energy source (ﬂip-ﬂop), has early allowed to establish, via
chemical labelling or enzymatic treatment, their distribution between
the cytoplasmic (inner) and external (exoplasmic, outer) membrane

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M.-C. Giocondi et al. / Biochimica et Biophysica Acta 1798 (2010) 703–718

Fig. 1. Phase behaviour of phospholipids. Upon hydration, a majority of phospholipids
form lipid bilayers that can exist in two distinct physical states, gel (Lβ and Lβ’) and ﬂuid
(Lα, Ld), according to the temperature. In the gel phases, molecules are tightly packed ina quasi-hexagonal array. The acyl chains are extended and parallel to each other. Intraand intermolecular motions are slow as compared to the ﬂuid, liquid disordered phase
where the acyl chains are highly mobile and the molecules undergo fast rotational andlateral (D) diffusion. For pure phospholipid species, the gel to ﬂuid transition ischaracterized by a melting temperature, Tm, recorded as a sharp peak by differential
scanning calorimetry. It is accompanied by a thinning of the bilayer. Addition ofcholesterol induces a concentration dependent decrease in the sharp peak until it issuppressed and the formation of a new phase, the liquid ordered phase (Lo). In the Lophase, the acyl chains are ordered and mostly extended but the molecules have a highrotational and lateral mobility (adapted from Ref. [42]).

leaﬂets [1]. In contrast, the fast ﬂip-ﬂop of cholesterol (Chol), in thesecond time scale [2], has so far led to contradictory results about its
transmembrane distribution, a situation expected for other neutral
lipids. For the same kinetic reasons, the in plane dynamics ofmembrane constituents is responsible for our limited knowledge ofthe membrane lateral molecular organization. Even in a gel phase, theslowest rate for a freely diffusing single phospholipid is still N100 nm2
s− 1 (Fig. 1) [3,4]. For the last three decades, existence of membrane
microdomains resulting from either lipid–lipid, lipid–protein orprotein–protein interactions [5–11] was the object of animateddebates. Thermotropic transitions starting around room temperatureand ending between ∼ 39 and 42 °C were early characterized either in
puriﬁed apical brush border membranes (BBM) from renal andintestinal epithelial cells [12–14], or in situ [15], by differential
scanning calorimetry (DSC), ﬂuorescence polarization and electron
spin resonance (ESR). These data strongly suggested that a sphingolipid-dependent [16] lipid phase separation could account for the

existence of a category of membrane domains. The biochemical andbiophysical characterization of detergent resistant plasma membrane
fractions (DRMs) isolated from MDCK cells [17,18] and the concept ofrafts, a category of microdomains enriched in sphingolipids (SL) andcholesterol (Chol) [8], as functional membrane platforms controlling alarge variety of cell functions [19], have generated a huge amount ofwork on membrane heterogeneity in a great number of cell types.
Determination of the lipid composition of the ﬁrst DRMs led to a SL/
Chol/glycerophospholipids (GPL) molar ratio close to 1/1/1 (Table 1)[17]. With this Chol concentration, DRMs membrane lipids are
expected to be in a liquid ordered phase (Lo) (Fig. 1). The Lo phase isformed by the interaction of phospholipids with Chol [20–22]. It ischaracterized by a high degree of acyl chains order associated withlateral diffusion properties close to those determined for lipids in theliquid-crystalline or ﬂuid phase (Lα or Ld for lipid-disordered) where
the acyl chains are kinked and loosely packed. For lipids in the gelphase (Lβ' or s for solid), acyl chains are even more ordered than in the
Lo phase but lateral diffusion (D) is much slower (Fig. 1). The
formation of Chol-enriched domains would be driven by a Lo-Lα phaseseparation process in which Lo SL/Chol-enriched lipid domains are
surrounded by a ﬂuid Lα matrix enriched in more unsaturated GPL
species. Despite all this information on various membrane domains,
many questions about microdomains like the existence of families ofdomains sharing the enrichment in SL and Chol but with large
individual variations in acyl chains and polar headgroup composition,
the coupling between the exoplasmic and cytoplasmic leaﬂets, theirkinetics of formation, lifetime, and their size range(s), remain open.
Thus, domains in the micrometer range were reported using
ﬂuorescence photobleaching recovery (FRAP), ﬂuorescence digital
imaging microscopy and single molecule ﬂuorescence microscopy[23–25], while sizes ∼ 20 nm [26,27] or b5 nm [28] for domains
associated with glycosylphosphatidylinositol (GPI) anchored proteins
were estimated using the photonic force microscopy (PFM), stimulated emission depletion (STED) far ﬁeld ﬂuorescence microscopy andﬂuorescence resonance energy transfer (FRET) methods, respectively.
Difﬁculty in characterizing lipid-dependent domains of cell membranes is not surprising considering that the phase behaviour of lipid
mixtures is still poorly understood [29]. Elucidating the properties ofthese mixtures with, or without, inserted peptides or proteins, istherefore of primary importance, even considering that in model
membranes lipid domains are at, or close to, equilibrium which
contrasts with the transient non equilibrium structures found inbiomembranes [30]. The obvious ﬁrst step is the study of Langmuir/
Langmuir Blodgett (LB) monolayers properties [31] since a biomembrane can be considered as two “weakly” coupled monolayers (seehowever [32]) and because in plasma membranes SL, a key
constituent of SL/Chol-enriched microdomains, is practically exclusively found in the exoplasmic leaﬂet. The next step is thecharacterization of lipid mixtures in bilayers which, since Gorter and
Grendel [33], has followed the development of new tools as well as of

Table 1
Lipid composition of DRMs and apical membranes of intestinal and renal epithelial cells (brush border membranes, BBM).
Composition (mol%)

DRMs
1

Total
Sphingolipids (SL)GSL
SM
Glycerophospholipids (GPL)
Cholesterol (Chol)
SL/GPL ratio
Chol/(SL + GPL) ratio
Chol/GPL ratio

22
14
33
32
1.09
0.47
1.03

Intest. BBM
3Out.Leaf. (calculated)
∼ 40
∼ 30
–
∼ 30
∼ 0.43

2

Total

Renal. BBM
Out.Leaf. (calculated)

37

∼70

32
31
1.16
0.45
0.98

–
∼30
∼0.43

3

Total4
6

b2
23
36
38
0.72
0.59
0.62

Out.Leaf. (determined)5
3
47
12
38
4.17
0.59

Total lipid composition of DRMs, intestinal and renal brush border membranes taken from literature. For each membrane, the second column gives the corresponding composition of
the exoplasmic leaﬂet (Out.Leaf.) either calculated from the known sphingolipid asymmetry or determined experimentally.
From :1[17]; 2[254]; 3Estimation assuming that GSL and SM are localized on the external leaﬂet [1]; 4[255]; 5[256,257]; 6[258].

M.-C. Giocondi et al. / Biochimica et Biophysica Acta 1798 (2010) 703–718

new models, from supported bilayers to small (SUV), large (LUV) and,more recently, micrometer size giant (GUV) unilamellar vesicles [34].
Among these new tools, atomic force microscopy (AFM) [35] has
become very popular in surface science by giving access, in air or in
liquid, to topography at a molecular scale. This holds true for soft
surfaces where the AFM high resolution of ∼ 1 nm in lateral and∼ 0.1 nm in the vertical direction was initially applied in P.Hansma
laboratory to image phospholipids in LB and supported lipid bilayer(SLB) model membranes [36,37]. Lipid domains in various binary andternary mixtures under phase separation, containing or not inserted
peptides/proteins, were thus later imaged in LBs and SLBs [38–42].
Recent reviews have exposed numerous AFM data obtained these lastyears both on the formation of solid-supported bilayers and on theimaging of domains in various lipid mixtures [43–47].
In this review we will essentially focus on the type of information
on model systems that only AFM can provide. We will then examine
how AFM can contribute to our understanding of data obtained on
similar samples by lower resolution techniques. Some of the
intriguing properties of SLBs microdomains revealed by AFM andthe new questions on membrane-substrate relationships they raise
will be discussed in a next section. Finally, we will present AFM recent
developments which might open new avenues in model andbiological membrane applications of AFM imaging.
2. AFM characterization of lipid ﬁlms and membrane hemi-leaﬂets
2.1. Single lipid and binary mixtures of lipids
Langmuir and LB ﬁlms have been extensively used to study theproperties of monolayers as models for membrane biophysics [48–50].
Following ﬂuorescence microscopy and FRAP methods [49,51–53],705

total internal reﬂection ﬂuorescence (TIRF) [54], Brewster-angle
microscopy (BAM), x-ray and neutron scattering techniques, Fouriertransform infrared (FTIR) spectroscopy, and polarization-modulated
infrared reﬂection absorption spectroscopy (PM-IRRAS) [55,56] wereapplied to monolayers studies. Together, these techniques haveprovided invaluable data of liquid expanded (LE) and liquidcondensed (LC) phases in monolayers and their dynamic properties
down to the microscopic scale (for a detailed description of structure
and phase transitions in Langmuir monolayers see [50]). In many
experiments, planar supported lipid monolayers prepared fromvertical Langmuir–Blodgett (LB) or horizontal Langmuir–Schäfer(LS) transfers from the air-water interface to a hydrophobic silanizedglass slide [48,57] have been examined under aqueous buffer. The
transfer was performed at a chosen surface pressure which, whenmodelling biological membranes, was generally comprised between
30 and 40 mN/m [58,59]. Because the dynamic properties of samples
are essentially maintained under these transfer conditions, FRAP, totalinternal reﬂection ﬂuorescence microscopy (TIRF) [60,61] andﬂuorescence correlation spectroscopy (FCS) [62] have been applied
to study lipid–lipid, lipid–protein interactions and properties of model
rafts [63,64].
In contrast with the experiments mentioned above, upward
transfer of lipid ﬁlms onto hydrophilic substrates like mica, quartz
and glass resulting in the exposure of acyl hydrophobic chains to air
has been so far practically exclusively used for AFM analysis (Fig. 2). It
is worth noting that the use of ﬂuorescently labelled phospholipid
ﬁlms presenting large phase separated domains showed that the
microscopic monolayer topology was preserved for transfer pressures
greater than 10 mN/m [49,65]. Thus, although details of moleculararrangement might be affected, the difference in packing density
before and after transfer is generally negligible for molecules closely

Fig. 2. AFM Imaging of POPC, DPPC and SM LB ﬁlms. Langmuir ﬁlm transfers were performed at 32 mN/m for POPC in LE (A), DPPC in LC (B) and SM (C) in LE + LC phases. (D) is avirtual section of (C). The (E) cartoon illustrates the sample positioning under the tip when imaging with an AFM in air. Blue headgroups correspond to LC, red to LE lipids. Bar:
500 nm.

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packed before the transfer from water interface is accomplished[66,67]. For lower surface pressures, transfer of monolayers from theLE phase or in the LE/LC coexistence region often results in theformation of a substrate (and pH)-dependent close-packed domains
in the corresponding LB ﬁlms [66,68–70] (however see [71,72]). AFM,
and more generally scanning probes techniques, have given access tothe missing essential information on monolayers organization at themesoscopic and eventually molecular scales [36,73,74]. For thesehighest resolutions imaging, care has to be taken to use scanning
forces as low as possible to prevent orientational ordering ofstructures by the AFM tip [75]. Sub-micrometer organization ofphase-separated fatty acids in the LC-LE coexistence region andcorresponding determination of local mechanical and tribologicalproperties early illustrated the usefulness of AFM in the characterization of heterogeneous soft surfaces [76,77]. As illustrated by Fig. 2A,
AFM images of ﬁlms made of a single phospholipid species in the LEphase, transferred at 32 mN/m, show a homogeneous surface of low
roughness (b0.4 for 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine, POPC). 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)ﬁlms transferred in the LC phase at the same surface pressure also
exhibit a low roughness (∼ 0.6 ) but with the presence of line defects(0.1–0.5 depth, 15–20 nm in width) at their surface (Fig. 2B, arrows),
which might correspond to a border between different domains, that
only the incredible resolution of the AFM can reveal. Transferring
DPPC ﬁlms labelled by a ﬂuorescent phospholipid probe, at a surfacepressure where LC-LE coexist, demonstrated the existence of bothlarge and mesoscopic LC domains and indicated that the presence ofthe dye reduced signiﬁcantly the total amount of the LC phase [78].
Analysis of natural sphingomyelin (SM) ﬁlms, the major SL found ineukaryotic cells, under identical transfer pressure conditions (Fig. 2C),
clearly demonstrates the existence of a marked heterogeneity of thesurface, with darker zones generally less than 200 nm in size and∼1 nm lower than the lighter surface. The presence of such domains inthe ﬁlm which, according to DSC thermograms of SM [79], correspondto LE regions surrounded by LC zones, would not be detected by
optical techniques including ﬂuorescence microscopy and their
topographical details would escape all other analysing techniques.
For SM, the height difference between domains can be explained by
the fact that the shortest or unsaturated disordered acyl chains are in
the LE phase. In the most common used contact mode for AFM
imaging, the tip remains continuously in contact with the sample
during the raster scan of the surface [80,81]. Consequently, the relative
height of surface structures can also be affected by the scanning force
applied during scanning, which has to be minimized, and the local
mechanical properties of the sample [82]. It is worth noting that when
imaging in air under ambient conditions strong adhesion forces
between the tip and the ﬁlm, linked to water condensation at the air/sample interface, impose the use of scanning forces signiﬁcantly
higher than those required for imaging under liquid. Before imaging,samples are often kept in a dry cabinet or maintained in the laboratory
atmosphere where relative humidity (RH) is generally less than 50%, avalue sufﬁcient to block the lateral diffusion of lipids in ﬁlms
transferred onto polymer cushions. For ﬁlms transferred onto glass,the lateral diffusion is at the lower resolution limit of FRAP techniqueeven at 90% RH [83]. Accordingly, in contrast with ﬁlms exposing theirpolar head group to water, AFM in air gives topographical information
on ﬁlms where lateral diffusion is blocked. Immersion of themonolayer in water increases the diffusion coefﬁcient by two ordersof magnitude and the ﬁlm remains stable even though thehydrophobic tails of the lipid molecule are exposed directly to water[83,84], allowing AFM imaging [85]. Frictions forces between the tip
and the sample [86], as well as energy dissipation which reports on

Fig. 3. Example of LE-LC phase separated binary mixture in LB ﬁlm. DOPC/DPPC (1:1) ﬁlms were transferred at 32 mN/m onto mica and examined in air with an AFM working incontact mode. A: low magniﬁcation height image: bar 5 μm, z scale 20 nm; B and C: samples from two other preparations, bar: 2 μm, z scale 15 nm; D: height image at a higher
magniﬁcation of A: bar 400 nm, z scale 7 nm. E and F, corresponding lateral force (friction) images in the forward and backward direction of the tip scanning, z: 0.2 V.

M.-C. Giocondi et al. / Biochimica et Biophysica Acta 1798 (2010) 703–718

local viscoelastic properties when using AFM oscillating modes, canalso probe the existence of phase separation in monolayers in an original
way [82,87]. So far, however, the friction and viscoelastic signals areaffected by the topography signal and, for lipid ﬁlms, give only qualitative
information. Moreover, improvement of the imaging quality by reducing
the scan force is associated with minimizing friction in contact and phaseshift in oscillating modes. AFM characterization of LC-LE coexistence
regions in LB ﬁlms made of single phospholipids species has been applied
to various binary and ternary lipid mixtures and has established theusefulness of this technique for the detection of membrane domains
ranging from the nanometer to the micrometer scale. Fig. 3 illustrates anexample of contact mode AFM imaging of a LB ﬁlm made 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC, LE)/DPPC (LC) now commonly used instudies on phase-separated binary mixtures, because their respective
transition temperature of −20 and 41 °C insures a large zone of orderdisorder phase coexistence [88–92]. A large scan, at the AFM scale, shows
the presence of two categories of light domains protruding by ∼0.6 nm
from a darker matrix (Fig. 3A). The larger ones, ∼1 to 2.2 μm in lateral size,
could have been detected by optical microscopy. This is not the case of theabundant smaller domains ∼100–400 nm in size. The taller larger
domains exhibit irregular, often linear and angular boundaries (whitearrows), indicating they correspond to LC phase domains. Imaging of twoother samples conﬁrms the general characteristics of the mixture,
showing the coexistence of large and small domains, but also indicatesthat their form and relative size can vary (Fig. 3B and C). Decreasing thescan size demonstrates that angular boundaries are also found in smaller
domains (Fig. 3D, white arrows). Local variations in friction force images(black arrows) obtained on LC domains in the forward (Fig. 3E) and the
backward scanning direction (Fig. 3F), not associated with signiﬁcantheight modiﬁcation, further suggest the existence of heterogeneity intheir physical state as a function of the distance from the boundary.
LB ﬁlms made of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE)/1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)[87], DOPE/1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)[82], 1-palmitoyl-2oleoyl-sn-glycero-3- phosphoethanolamine (POPE)/
1-palmitoyl-2oleoyl-sn-glycero-3-phospho-L-serine (POPS) and POPE/
POPS/SM [93], DPPC/1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) [94], DPPC/ POPC/cardiolipin (CL), POPE/CL [95] andPOPC/POPE [96] are among the various binary mixtures examined by
AFM. In many occasions, the detailed surface structure could not be
predicted from the other approaches. For example, this is the case of thephase topography of ceramides, Chol and free fatty acids mixtures thatmimic the lipid composition of stratum corneum [97,98].
2.2. Lipid mixtures related to microdomains enriched in sphingolipids
and cholesterol
AFM has allowed to establish the distribution of ganglioside GM1,
a glycosphingolipid (GSL) which is the natural receptor for cholera
toxin, between ordered/disordered two-phase monolayers [88,99].
GSL acts as a receptor for numerous biologically active agents and its
distribution in phase-separated lipid mixtures had been previously
extensively investigated by various indirect methods leading tocontrasting conclusions (see references in [88]). AFM examination of
LE/LC DOPC/DPPC monolayers doped by physiological, low concentrations (b5%) of GM1, shows that it forms round shaped (15–30 nm)
and ﬁlamentous nanodomains, preferentially localized in the DPPCenriched LC phase (Fig. 4), which eventually fuse to form fence-likestructures at the interface (black arrows). This indicates the existenceof a DPPC/GM1 LC/LC immiscibility, most likely driven by hydrophobic mismatch and strongly suggests that GM1 may also form
nanodomains within larger ordered microdomains. In addition, a few
GM1 nanodomains also localize in small LC microdomains (whitearrows). Although in some cases GM1 nanodomains seem to belocated in the LE phase, it cannot be excluded they are standing on LC
nanodomains (white arrows). This complex distribution at the707

Fig. 4. Topography of DPPC/DOPC LB ﬁlms containing 4% GM1. The ﬁlm was transferred
at 32 mN/m onto mica and examined in air in an AFM working in contact mode. bar:
250 nm, z scale: 5 nm; For more details see [88].

nanoscale probably explains at least partly the difﬁculties encountered before in deﬁning GM1 distribution. These studies, inagreement with preferential localization of GM1 in the ordered“rafts” domains in plasma membranes, have been extended to higher
GM1 concentrations [100] and to other gangliosides [101]. Alone,
AFM cannot answer the questions concerning the monomeric/aggregates state of GM1 in domains. The possibility to analyse
phospholipid monolayers labelled with ﬂuorescent probes by nearﬁeld scanning optical microscopy (NSOM) was early recognized[102]. Association of AFM with confocal and NSOM was applied to
DPPC LB ﬁlms and SLB [70]. Combination of AFM and NSOM in one
single equipment has further allowed to demonstrate, using Bodipylabelled GM1, that the addition of ganglioside produced signiﬁcantchanges in the phase-separation behaviour of the binary DPPC/DOPC
and the ternary DPPC/DOPC/Chol monolayers [103]. Moreover, with
the Bodipy ﬂuorophore, monomeric and aggregated gangliosides
could be distinguished, which provided new insight into thecomplexity of GM1 partitioning. This work also demonstrated that,independently of a lipid oxidation process which affects the size of
domains [104,105], the ﬂuorescent label affected the partition of GM1
between LC and LE phases.
Lipid composition of detergent resistant membrane fractions(DRMs), whose relationships with native “rafts” remain a matter of
debates [106–109], is close to that of the apical membrane of intestinal
and renal epithelial cells (Table 1). The asymmetry of SL distribution
in plasma membranes indicates that exoplasmic leaﬂet of intestinal
and renal cells BBM is essentially made by mixtures of GSL/Choland SM/GPL/Chol, with a predominant fraction of SM, respectively(Table 1). A SL/GPL/Chol ratio close to unity, often chosen to modelcells exoplasmic leaﬂet, rather corresponds to the composition of theouter leaﬂet of non polarized human erythrocytes and platelets [32].
Accordingly, LB ﬁlms made of SM/GPL ratio modelling the exoplasmic
leaﬂet of kidney BBM were examined by AFM. Because the saturated/unsaturated fatty acid ratio of phosphatidylcholine (PC) in thesemembranes is close to 1 [16], POPC was chosen rather than DOPC only
present as a minor PC species in biomembranes [110]. For these BBM
models AFM could establish the presence of concentration-dependent
LC SM-enriched nanodomains [111] which would have escaped opticaldetection. Neither the size nor the spatial distribution of these
domains could have been obtained by other approaches. The sameseries of experiments strongly suggested that LC/LE lipid phaseseparation may occur in the renal BBM exoplasmic leaﬂet in theabsence or upon depletion of Chol. Furthermore, Chol-induced
connection of nanodomains resulting in the formation of a lipidordered network was also demonstrated (Fig. 5).

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ternary and complex mixtures of biological interests. Complementarity with other recent techniques including FTIR, PM-IRRAS and mass
spectrometry imaging (TOF-SIMS) [119,120][115] will be likely
scientiﬁcally rewarding for many years to come.
3. AFM characterization of supported bilayers
3.1. Formation of SLB

Fig. 5. Branching of SM enriched nanodomains by cholesterol addition. A: height imageof SM/POPC (3:1) LB ﬁlm. B: SM/POPC (3:1) + 20 mol% Chol. Bar: 500 nm, z colour
scale: 10 nm.

2.3. Lipid interaction with peptides and proteins can create domains inmonolayers
Numerous studies on the interactions of amphipathic peptides andwater-soluble proteins with membranes start with monolayers toinvestigate the lipid-water interface step. To surface pressure andelectric potential measurements, they associate various techniques
like ﬂuorescence and Brewster angle microscopy, vibrational spectroscopy, x-ray and neutron scattering techniques and PM-IRRAS. Hereagain, AFM brings unique invaluable information. For example,
investigation of the interaction with monolayers of some amphipatic
peptides that act as very efﬁcient drug carriers has shown concentration and lipid headgroup-dependent α to β conformational transitions. AFM analysis of samples further revealed that these transitions
were associated with formation of nanoﬁlaments and nanodomains
supramolecular arrangements, providing a new interpretation ofdata [112] [113]. Similarly, the human calcitonin fragment 9–32,
another efﬁcient carrier spontaneously forms supramolecular structures which looks like ﬁlaments rolled into spirals made of α-helices[114]. AFM also contributed to the understanding of interactions
between puroindolines, plant lipid binding proteins with antifungal
properties with wheat galactolipids [115]. This also applies tothe characterization of nanoscale ﬁlm heterogeneity in models
of pulmonary surfactant [94,116,117], with an elegant experiment
performed directly on an air bubble coated with the surfactant [118].
By giving access to the nanoscale organization, AFM has played aparticularly important role for the understanding of LB ﬁlms made of

Besides being a widely used model for analysing fundamental
properties of cell membranes, supported planar lipid bilayers (SLB)[121] also offer unique possibilities for the development of nanobiosensors, nanomotors and nanotools [55,122–124]. Today, formation of(SLB) is achieved using three main classes of methods. The ﬁrst isbased on the use of a LB ﬁlm for the proximal (inner) leaﬂet facing thesolid support. Deposit of the distal (outer)-leaﬂet facing the bulk
solution is done using either LB or LS technique. Formation of SLB fromadsorption of unilamellar vesicles, SUV, LUV and GUV [43,125,126]
regroups the second category of methods and the third uses spin
coating [127,128]. In addition, some protocols belong to two different
classes like the LB/vesicle fusion (VF) method [129]. SLB formationfrom direct adsorption of detergent-phospholipid micelles was also
described [130,131].
For each class of method, AFM has brought crucial information forestablishing that differences in the experimental protocol, including
the temperature, the nature and roughness of supports (quartz,
glass, mica, gold, alumina, SiO2, TiO2,) covered or not by a polymer
layer, the use of water or various buffers, the lipid constituents etc,
can inﬂuence the bilayer properties [43]. Thus, for LB-based
protocols whose major interest is in the formation of asymmetricbilayers which mimic the situation found in cells plasma membranes,
the earliest AFM investigations suggested the use of a LC phase, DPPE,
DSPE or DPPC, which enhances the bilayer stability, as the proximallayer in building stable bilayers on mica from LB/LB deposition
processes [91,132–134]. Even under these most favourable conditions and despite good transfer ratios during their formation, AFM
examination revealed the presence of defects and holes in these SLB
that escape optical detection [132,135–137]. According to theﬂuorescence interference contrast microscopy method (FLIC),
extensive loss of transmembrane asymmetry occurs during theformation of SLB by the use of LB/LS protocol. Moreover, the use oftethered polymer support for the initial monolayer does not improvelipid asymmetry in the resulting bilayer which would be only
preserved using the LB/VF method [32,138]. Contrasting with these
LB data on methods initially developed to insure SLB asymmetry,vesicles fusion expected to provide symmetrical bilayers can result in
the formation of asymmetrical bilayers, as a function of the support,
buffer, lipid composition, vesicles size and temperature conditions
used [139,140–142]. Although not complete, the understanding of
mechanisms and parameters involved in SLB formation from lipid
vesicles has strongly beneﬁted from AFM and its association withquartz crystal microbalance dissipation monitoring (QCM-D)[43,143–146]. It is worth noting that, like for LB-based methods,
optically continuous SLB formed by vesicles fusion are most often
pierced by holes, even for lipids in Lα or Lo phases (Fig. 6)[44,136,145,147–149]. This imposes to probe different zones of eachsample with large scans for interpretation of dynamic data. Finally, it
is worth noting that the same three main classes of methods can beused to prepare supported double bilayers, [128,150–152] examinedby AFM only in a few occasions [153,154].
3.2. AFM imaging at room temperature of domains in SLB
Like LB ﬁlms data, most AFM information collected from SLB has
been based on the characterization, at room temperature, of samples
topographical height differences between ordered and disorderedM.-C. Giocondi et al. / Biochimica et Biophysica Acta 1798 (2010) 703–718

709

Fig. 6. Supported DOPC/DPPC 1:1 bilayers made by vesicle fusion. Inﬂuence of substrate. A, B, C and G: SLB on mica; D, E, F and H: SLB on glass. White arrows in (C) point at thepresence of holes in the bilayer whereas dark arrows indicate the presence of aggregates (and/or non-fused liposomes). In D and E, white arrows show the presence of patches madeof unconnected, tiny, pieces of bilayers while black arrows indicate glass zones not covered by the bilayer. The white arrow in H shows a zone of the bilayer with a different
organization. Bars: A, B, C: 2 μm; D, E, F: 5, 1, 0.3 μm. vertical z colour scale: A, B, C: 20 nm; D, E, F: 30, 30, 10 nm, respectively. Note that the bilayer surface is N 8 nm above the substrate(G and H virtual sections).

phases which depend on the acyl chains length of glycerophospholipids and sphingolipids. Various binary mixtures under Lα/Lβ'phase separation were ﬁrst characterized on asymmetrical SLB built
by LB/LB transfers where the proximal leaﬂet facing the support wasmade of a single phospholipid in a single phase. Brieﬂy, in thisconﬁguration, the topography of SLB is close to that of LB ﬁlms having
the same composition, as long as transfers were performed at thesame surface pressure. This has been established for DOPE/DSPE
mixtures were the size of LC domains in monolayers corresponded tothat of gel domains in the DSPE supported DOPE/DSPE bilayer [155].
Thus, like for LB ﬁlms examined in air, these SLB nanodomains are
expected to coexist with the larger domains which are observable by
ﬂuorescence microscopy. It is worth noting that height difference
between ﬂuid and gel phases lipids take a single value, generally
consistent with the results of X-ray diffraction studies when using low
scanning forces. Large scanning forces increase the thickness
differences because of different elastic properties of the two phases[156]. For these highly asymmetrical samples, no evidence for time-

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dependent transmembrane migration of phospholipids has beenreported, which suggests that these LB/LB bilayers were more stable
than LB/LS bilayers. Besides topography, direct visualization of thelocal surface charge in 1,2-dilauroyl-sn-glycero-3-phosphocholine(DLPC) /1,2 dimyristoyl-sn-glycero-3-phospho- L-serine (DMPS)
phase separated distal leaﬂet was obtained in aqueous solution fromthe phase shift in oscillating mode [157]. Using phase-separated ﬁlms
for both proximal and distal leaﬂet can result in LB/LB bilayers locallypresenting three quantized thickness levels (Fig. 6), as reported for
DPPC [70].
Imaging of SLB made by SUV or LUV fusion has demonstrated why
their complete characterization requires AFM, even for simple binary
mixtures. Taking the DOPC/DPPC 1:1 mixture as an example, AFM
imaging shows that, keeping the buffer composition, the fusion
temperature and the nature of the support (mica) constant, large
variations exist in the size, the spatial distribution of domains, theextent of support coverage, the presence of holes, pieces of double
bilayers and non-fused vesicles still adsorbed (Fig. 6 A, B, C and D).
Changing mica for glass signiﬁcantly modiﬁes the aspect of the surface
and enhances the heterogeneity of the topography between different
zones of the sample (Fig. 6E white and dark arrows). Consequently,
whereas the single step in height between the gel and the ﬂuid
domains indicates they are superimposed in proximal and distal
leaﬂet, i.e. that coupling between leaﬂets is complete, when using
mica as a support, it is difﬁcult to exclude the existence of a second
step in height, corresponding to asymmetric ﬂuid/gel phase superimposition, with the glass support (see the virtual section in E). With
DLPC/1,2-distearoyl-sn-glycero-3-phosphocholine DSPC mixtures in
SUV, such uncoupling between leaﬂets can be induced by modifying
the temperature of vesicles during their formation and their
incubation before deposit on mica surface [140]. In the same study,

time lapse study of SLB topography demonstrated that mixed
symmetry lipid bilayer can convert over a period of several hours toeither an asymmetric or a symmetric SLB, likely via a ﬂip-ﬂop at theinterface of domains. The rate constant for ﬂipping event wasestimated to be ∼76 h− 1 [46,140]. Phase coupling between leaﬂets
determined by AFM for a variety of binary phospholipid mixtures
under gel-ﬂuid phase separation [158] was previously observed by
FRAP on DMPC/DSPC multibilayers [159] and in GUV [64,160]. Besides
differences in the size of domains and their transmembrane coupling,
the behaviour of phase-separated binary mixtures made of synthetic
lipids leads to rather ﬂat structures. This is not the case for mixtures ofnatural SM with DOPC or DPPC which frequently adopt a variety ofmesoscopic morphologies including large stripes, tightly packed small
globular structures, branched-ﬁlaments decorated domains and gel–gel phase-separated systems surrounded by a ﬂuid phase [161]. The
diversity of structures imaged strongly evokes composition ﬂuctuations corresponding to spinodal decomposition process [162–166] andalso makes clear that small changes in experimental conditions canlead to large changes in the domains morphology. Various morphologies also coexist within the same sample in ﬂuid-Lo phase-separated
GUV containing SM [167], indicating that vesicles-support interactions are probably not at the origin of the topography diversity in SLB.
Although much smaller than in GUV, the average size of ordered
microdomains in phospholipid binary mixtures examined by AFM
remains much larger than that predicted from FRAP and ESR
experiments on ﬂat multibilayers or from Monte Carlo simulations.
Using similar DMPC/DSPC mixtures, the estimated size of gel domains
corresponded to ∼ 250 to 1500 molecules, i.e. less than 30 nm indiameter [159,168,169]. On the other hand, earliest studies using
freeze-fracture electron microscopy and electron diffraction describeddomains in the few-hundred nanometers to a few micrometer range

Fig. 7. Imaging Ripple Phase in multibilayers. Multibilayers were formed from di-C15:0 PC LUV in PBS buffer and imaged in the same buffer at room temperature. A, B and C and D andE correspond to different experiments. A, B and E are deﬂection images. C and D: height images. Bar A, B, C: 300 nm; D and E: 250 nm. White arrow in A shows the absence of ripple on
the ﬁrst bilayer. The white arrow in B strongly suggests the presence of triple bilayers.

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for liposomes made of various phase-separated binary mixtures ofphospholipids [170–172]. These differences could be at least partly
explained considering that the size of domains is markedly affected by
the thermal history of the samples, the composition of their bathing
medium [158,173,174] and by lipid oxidation [45,175]. Another uniquecontribution of AFM to the characterization of phase-separation inbinary mixtures is the study of nucleation and domain growth process
which requires the use of high resolution imaging techniques in realtime. This was ﬁrst achieved on DOPC/DPPC mixtures after a rapid
quench in temperature from 60 (miscibility state) to 23° (immiscibility region) [176]. More recently, using new facilities for controllingthe sample temperature under the microscope, nucleation rates andgrowth of domains were determined on DOPC/DSPC and DOPC/
GalCer symmetric and asymmetric bilayers and the analysis of data
further allowed to determine the values of the interfacial line tension,of the activation energy barrier and of the lateral diffusion coefﬁcientof lipid addition to a growing domain [177,178]. Differential resistanceof phase separated binary mixtures to detergent solubilisation can be
explored in situ by AFM. This has been done, for example, on DOPC/DPPC mixtures treated with Triton X-100, a detergent frequently usedfor DRMs preparation [89,179]. The behaviour of other binary mixtures
made of phospholipids more particularly present in identiﬁed cellularorganelles like mitochondria has also been characterized [95,180]. The
vertical sensitivity of AFM provided direct evidence for alcohol or
halothane-induced formation of interdigitated domains in saturated
phosphatidylcholine SLBs [181,182]. Vesicle fusion method on mica
with SUV and LUV made of a single saturated PC component or
equimolar DMPC/DSPC mixtures often results in the formation oflarge patches of supported double bilayers [154,183]. Such double
bilayers are characterized by the presence of a ripple phase whichforms at the pretransition temperature upon heating from the gelphase (Fig. 7). Ripple repeat distances corresponding to the stable(13–15 nm) and metastable (26–30 nm) ripples were directly
measured from images taken in solution. In these samples, the surfaceof the ﬁrst bilayer, closest to mica, can be ﬂat and featureless orpresents wavelike structures of low amplitude at room temperature,
likely as a function of the thickness of the aqueous ﬁlm between themica surface and this ﬁrst bilayer. These data strongly suggest that theearly AFM observation of a buffer-induced ripple phase in diC15-PC
bilayers [184] was due to the presence of double bilayers. Unfortunately, to our knowledge, there is no data on the topography of phaseseparated domains in hydrated supported double bilayers made by
the other techniques.
In biological membranes, because of the presence of Chol as amajor constituent of membrane lipids, phase diagrams strongly
suggest that microdomains formation results from Lo–Ld and,eventually, gel–Lo–Ld phase-separations [185,186]. Uncertainties
exist ﬁrstly because the distribution of Chol between the inner andouter membrane leaﬂets remains poorly known for the reasonspreviously mentioned and secondly because of the likely existence ofdifferent Chol pools which also might involve protein–lipid interactions [187,188]. Behaviour of ternary mixtures made of DOPC/SM or
POPC/SM containing various amounts of cholesterol, taken as models
for “rafts” biological membrane microdomains, has been examined by
AFM [40,79,91,189]. Again, as for simpler binary mixtures andprobably further ampliﬁed by the SM domains polymorphism [190],
various morphologies were described in accordance with theobservation reported for GUVs [167]. For models of BBM exoplasmic
membrane leaﬂet, it was established that in accordance withmonolayer studies, gel-Ld phase separation exists in the absence ofChol and that there is a range of Chol concentration (∼ 15–25 mol%)where gel/Lo domains connect over the sample surface before
disconnecting again. Finally, contrasting with corresponding LB
ﬁlms, no more domains could be detected for 33 mol% Chol [79].
AFM also allowed following in real time the consequence of in situ
manipulating Chol level on microdomains behaviour [191,192]. Three711

remarks have to be done concerning all these experiments on ternary
mixtures model microdomains. The ﬁrst is that, in most cases, AFM
can hardly discriminate a gel from a Lo phase only on the basis of theirbilayer thickness. X-ray studies on SM/Chol mixtures established thatthickness of the bilayer decreased only by 0.5 nm for temperatures
below gel to ﬂuid transition Tc upon addition of 50 mol% Chol [193].
Moreover, presence of Chol in the POPC ﬂuid phase can increase thebilayer thickness by up to 0.4 nm for 30 mol% Chol [194]. This explains
why increasing Chol concentration in the bilayer reduced the height
difference between the Ld phase and the gel–Lo/Lo phases [79]. In fact,coexistence of gel and Lo phases can be detected by the presence ofstraight and angular portions at the interface of domains protrudingfrom the ﬂuid Ld phase. As a consequence of this reduced difference
between bilayer domains thicknesses, assessing the degree ofmembrane leaﬂet coupling becomes very difﬁcult in these samples.
The second remark concerns the temperature control of commercial
AFMs sample stages which, due to the temperature sensitivity ofscanners and tips, took time to be developed. Consequently, most of
the AFM experiments on domains behaviour were done at room
temperature, i.e. ∼15 °C below the physiological temperature. Finally,
the third remark is also linked to a technical limitation i.e. that of AFM
scan rate. Whereas the “freezing” of structures in LB ﬁlms allowed todemonstrate the lateral heterogeneity of renal Brush Border Membrane containing 33 mol% Chol with the presence of small (20–70 nm)
domains [111], such small domains were not detected in corresponding SLB. It can not be excluded that they escaped detection because
they were diffusing in the ﬂuid phase during AFM imaging. Going toquaternary mixtures of lipids brought original new data and added a
supplementary level in the image interpretation complexity. Introducing ceramide (Cer), the second messenger involved in sphingolipid
signal transduction which can account up to 10% of membrane lipids[195–197], into model rafts under conditions where Ld–Lo [198] or Ld–
Lo–gel [199] phase separations occur, results in the detection of three
topographic levels. In both cases, this third level was also induced byin situ sphingomyelinase treatment suggesting it resulted from thedisplacement of a part of Chol from the Lo/Lo–gel domains to the Ldphase [198,199]. To our knowledge, there is no published AFM images
of SLB made of lipid mixtures modelling the cytoplasmic membrane
leaﬂet composition, likely because they do not show phase separation[32,200]. Unfortunately there is also no AFM report of the domain
coupling induced by models of outer leaﬂet [32].
3.3. AFM imaging of lipid–peptide and lipid–protein interaction in SLB
The interest of using simple membrane model systems like SLBs tobetter understand interactions between proteins and lipids has beenrecognized for long [201]. These model systems enable detailed
analysis of how the properties of lipids inﬂuence the structure and the
dynamics of proteins and in reverse how proteins and peptides affectthe lipid bilayer behaviour. The pioneer work of the Shao's group,
revealing the exquisite supramolecular organization adopted bygramicidin A in DPPC bilayer [147], has been followed by numerous
AFM studies on lipid–peptide and peptide–peptide interactions which
all gave fundamental information that could not have been obtainedby other techniques. For example, this was the case of WALP, KALP,
HALP [202,203], primary amphipathic [204], and fusogenic tilted
peptides [205], all forming microdomains of supramolecular structures only visible at the nanoscale in SLB. Because they are mostly
associated with membrane domains enriched in SL and Chol, theexoplasmic proteins class constituted by glycosylphosphatidylinositol-anchored (GPI) proteins was a good candidate for AFM studies.
Direct evidence for an insertion of a GPI in the most ordered domainsof binary or ternary model rafts mixtures was obtained taking
intestinal (BIAP) or placental (PLAP) alkaline phosphatases (AP-GPI)
as models [189,206]. AFM has also allowed to get direct informationon the associated transfer of lipids between phases [207] and on the712

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effect of temperature on GPI distribution [208]. Indeed, the origin ofproteins, the SLB composition, the experimental conditions chosen forprotein insertion, the imaging temperature, are all parameters capable
to inﬂuence the imaged enzyme distribution (see [209] for a recent
review). It must be kept in mind that the time required to acquire such
images (between ∼ 1 and 5 min for commercial equipments) actually
precludes the quantitative determination of ﬂuid ordered partition
coefﬁcient for diluted, rapidly diffusing molecules [210]. Interactionsof amyloids [211–214], of annexins [215,216] with SLB and of a carrier
peptide, with or without its cargo, with phase-separated model rafts[217] constitute other examples of the interest of AFM in characterizing lipid–protein interactions.
3.4. Temperature-dependence of AFM membrane domains imaging.
Interaction SLB-support
The ﬁrst, relatively recent, AFM studies where the temperature wasvaried in situ in order to characterize the SLB phase behaviour andthermotropic properties were performed on DMPC [218] and DMPC/DSPC samples [148]. Since that time, various laboratories haveinvestigated the thermal behaviour of SLB, essentially that ofdisaturated PC species. The results are presented in Table 2 which
also includes the transition temperature (Tm) determined by othertechniques. The ﬁrst observation, common to all AFM determinations,is the increase in the transition half-width, as compared to DSC
determination on multilamellar vesicles [219]. The Tm itself wasreported to be unchanged [220] or to be increased from ∼ 4 to 16 °C
depending on the acyl chain length and the leaﬂet considered,proximal or distal. For example, the upper end of DPPC melting wasreported to be as high as 52 °C for the proximal and 60 °C for the distal
leaﬂets of DPPC, [221,222]. Moreover, in these studies the melting of
the proximal leaﬂet started only after the distal leaﬂet gel to Ld phasecompletion, indicating a complete decoupling of the two leaﬂets.
These data strongly differ from those obtained by DSC, also on mica,
showing a slight Tm shift of 2 °C for the proximal and of 3 °C for the
distal leaﬂet [223], associated with a modest increase in Tm halfwidth. They also differ from those reported using FRAP for LB/LS
DMPC and DPPC SLB on oxidized silicon where no difference withliposomes data were observed [121] and from the literature on beads
and nanoparticles-supported SLB which most often indicated a slightdecrease in Tm (Table 2). Studies of thermotropic and lyotropic
mesorphism of saturated diacylphosphatidylcholines have demonstrated that the larger shifts recorded for the proximal leaﬂet would beequivalent to a marked (N50% ) dehydration of the polar head groups[224,225]. Comparing with DSC on multilamellar vesicles, the marked
widening of the transition is associated with an important decrease of
the transition cooperativity and can hardly be accounted for by aclassical ﬁrst-order process [218,226]. Taking into account AFM
experiments on supported double bilayers [154], the results presented
in Table 2 strongly suggest that the shifts in gel-Ld transition
temperature of lipids present in the proximal leaﬂet are markedly
dependent on the nature of the support, the thickness and composition of the aqueous sandwiched layer, in particular the presence or notof divalent cations, the experimental procedure followed to prepare
the SLB and, as shown by the correspondence between thermotropic
behaviour expected from DSC and temperature-dependent AFM
imaging of model rafts [209], the lipid composition. Determinationof the aqueous layer thickness by various techniques like NMR,
neutron diffraction, X-ray reﬂectivity, FLIC, gives values between ∼ 0
and 4 nm [121,138,142,151,227–229]. This is also the case of the
thicknesses estimated by AFM from the distance between the bilayer
surface and the support, see for example [79,181,184,222] and Fig. 6.
The thinner the aqueous ﬁlm, the higher the Tm shift. Why is the Tm ofthe distal leaﬂet of SLB made by vesicles fusion so much affected while
it is unchanged when using LB/LS transfers [121] remains
unexplained.
4. AFM recent developments and perspectives
The capacity to image surface topography from the nano to themicro scale, in air and under liquid, has imposed AFM as a major toolin the characterization of complex surfaces like microdomains inmodel membranes. The possibility to follow in situ, at the nanoscale,membrane modiﬁcations upon addition or insertion of drugs,
peptides, proteins, has also provided direct structural information
inaccessible to other techniques. Comparison of LB and SLB AFM data
has one more time emphasized the interest of each model tocharacterize the membrane behaviour of complex lipid mixturescontaining several components. Thus, besides being excellent model
for studying pulmonary surfactant, monolayer ﬁlms allow to determine the physico-chemical properties of one isolated membrane
leaﬂet, its ordering in two dimensions and surface interaction withpeptides, proteins or drugs introduced in the subphase as a function of
the surface pressure, keeping in mind the limitation associated withﬁlm deposition on solid substrates. As discussed above, nanometerscale details of supramolecular arrangements revealed by AFM are
obtained in air on ﬁlms where lateral diffusion is blocked and might be
actually inaccessible for freely diffusing structures at the surface of
membranes under liquid buffer. The inverted AFM system equippedwith a tip approaching the liquid-air interface from the subphasedeveloped at the ETH Zurich [230], combined with grazing-incidence
x-ray diffraction and x-ray reﬂectivity techniques[231], could provide
direct access to the nanoscale organization of Langmuir ﬁlms. SLB gavesuch information for the membrane surface exposed to the buffer.

Table 2
DMPC and DPPC transition temperature (Tm ) in liposomes and SLB.
SampleTechnique used

DMPC Tm (°C)

DPPC Tm (°C)

MLV [219]
SLB mica [222]
SLB, mica, [221]
SLB, mica [259]
SLB, mica, [220]SLB, mica, [226]
SLB, mica [218]
SLB, Au (111) [260]
SLB, mica [223]
SLB silica bead [261]
SLB glass beads [228]
SLB, SiO2 nanoparticles [262]
SLB silica bead [263]
SLB, oxidized silicon [121]

DSC
AFM
AFM
AFM
AFM
AFM
AFM
AFM
DSC
DSC
NMR
DSC
DSC
FRAP

23.9

41.4
(42–52)/(53–60)a(44.8–51.4)/(52.9–59.4)a(41–45.5)/(46.5–49.0)a
40.3–43.0(23.6–30.3)/(31.3–37.5)a(22–25)/(28–31)a(26–35)/(36–47)a28.2 (24–32)
22.5
∼21
25.4
∼22.9

Values given in brackets correspond to the lower and higher ends of the phospholipids melting temperature of uncoupled proximal/distal leaﬂets.
aLeaﬂets uncoupling.

40.4, 42.4, 43.8
∼ 38.4
39.4
39.4
40

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Local uncoupling of leaﬂets and time-dependent reorganization of
membrane surface associated with the loss of asymmetry or with theaddition of compounds that insert differently in mono and bilayers
due to hydrophobic matching are among numerous examples of
events where LB and SLB studies give complementary information.
While new imaging AC modes like phase modulation AFM [232],
higher harmonic AFM [233] and bimodal AFM [234] are expected tolead to improved resolution of topographical features and local
mechanical properties, increasing the application ﬁeld of the AFM
technique requires to widen the nature of the information collected, toaddress the question of the membrane-support relationships and, last
but not least, to decrease by at least two or three orders of magnitude
the image capture time.
In what concerns the ﬁrst requirement, ﬂuorescence-based
imaging techniques are among the most powerful approaches for
examining structure–function relationships in biology. Following theAFM coupling with far ﬁeld ﬂuorescence imaging [70], the use of
ﬂuorescence correlation spectroscopy (FCS) has conﬁrmed thecompositional/structural heterogeneity of ordered domains in aphase-separated DOPC/DPPC binary mixture [235] and has shownthat the diffusion coefﬁcients for ﬂuorescent lipids and for two
membrane proteins in model rafts SLB were comparable withdiffusion in free-standing membranes [210]. The ﬁrst images recently
obtained on DOPC/DSPC/Chol mixtures using a combined Polarized
total internal reﬂection ﬂuorescence/AFM gave access to the orderparameter in the same SLB domains characterized by AFM [236]. It canbe predicted these ﬂuorescence/AFM couplings will be helpful in theunderstanding of domains behaviour and inﬂuence of ﬂuorescent713

probes on the existence and size of domains [175,237–239]. RamanAFM [240], association of AFM with high-resolution secondary ion
mass spectrometry (Nano SIMS) which also demonstrated theheterogeneity of local composition within a single domain andbetween domains in order–disordered phase separated DLPC/DSPC
freeze-dried bilayer [241], will also provide the necessary complementary chemical analysis of domains for ternary and more complex
mixtures. Recent progress in nanoscale magnetic resonance imaging,with resolution in the nanometer range [242], will also result in thedevelopment of a new powerful tool for the understanding of
membranes structure.
There is good indication that solutions to the unpredictable effectsof support on membrane properties are under way. From the use for
AFM studies of SLB ﬂat supports pierced by holes of various diameterslike those described by Steltenkamp et al. [243], Böcker et al. [244],
and by Goncalves et al. [245], one can expect to get direct informationon the parameters involved in the support inﬂuence on lipid bilayers
and lipid domain properties. Moreover, ﬁlling these holes by gelshaving cytoplasmic-like properties, which can also promote phaseseparations [246], would also offer SLB models closer to thebiomembranes situation.
Finally, while it has the unique capability to image biologicalsamples at a nanometer resolution in physiological solutions, the timerequired to acquire one image between 0.5 and 10 min. has until
recently limited the application of commercial equipments either tothe high resolution imaging of immobile structures or to the low
resolution of fast diffusing structures or fast events. As recently
reviewed [247], the pioneers studies in the Viani et al. [248], Ando etFig. 8. Imaging SLB formation by High-speed AFM (HSAFM). Membranes were made of a ternary mixture made of DOPC/DOPS/biotin-cap-DPPE in buffer (10 mM HEPES, 150 mM
NaCl, 2 mM CaCl2, pH 7.4) and sonicated. The lipid suspension (0.5 mg/ml) was then directly diluted into the buffer present in the HSAFM liquid cell and the bilayer formation imaged
in continuous at a 975 ms/frame scanning rate (scan size 800 × 800 nm). SLB was formed from tubular lipid membranes (arrows, approximately 20 nm in height) and unruptured
liposomes (arrowheads, 7.5–30 nm in height). Z color scale: 25 nm. The corresponding ﬁlm is placed at http://www.s.kanazawa-u.ac.jp/phys/biophys/BBA/lipid.htm.

714

M.-C. Giocondi et al. / Biochimica et Biophysica Acta 1798 (2010) 703–718

al. [249] and Humphris et al. [250] groups for developing a high-speed
AFM (HS-AFM) capable to work in liquid on soft matter have now
succeeded in offering the capacity to ﬁlm in real time the structural
modiﬁcation of a functioning single molecule like the GroEL–GroES
interaction regulated by the ATPase [251]. As shown by the time lapse
study illustrated by Fig. 8 and the corresponding ﬁlm placed at
http://www.s.kanazawa-u.ac.jp/phys/biophys/BBA/lipid.htm., it is
now possible to follow the SLB formation from a ternary phospholipid
mixture with a sampling rate better than 1 image per second. This
opens a new ﬁeld of applications for the study of model membranes
including, for example, the early steps of bilayers formation and of
domains nucleation, the diffusion of nanoscale domains or of slow
diffusing proteins and lipids whose intra- and inter-molecularmotions remain important, even in the gel state [3,252,253].
Acknowledgments
We acknowledge funding by the ANR-08-NANO-010-03 and ANR08-PCVI-0003-02 programs.
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