Journal of Colloid and Interface Science227,585–587 (2000)doi:10.1006/jcis.2000.6897, available online at http://www.idealibrary.com on

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Submolecular Structures in DipalmytoylphosphatidylethanolamineLangmuir–Blodgett Films Observed by Scanning Force Microscopy

Dipalmitoylphosphatidylethanolamine (DDPE) Langmuir filmsat the air/water interface have been studied. These films exhibithigh stability. The resulting films transferred on muscovite havebeen studied by scanning force microscopy with the contact mode.At the microscopic scale, DDPE Langmuir–Blodgett films appeardensely packed with few defects. At molecular resolution the filmsappear well ordered; the double tail of the lipids has been observed.C© 2000 Academic Press

Key Words: dipalmitoylphosphatidylethanolamine; Langmuir–Blodgett films; mica; scanning force microscopy; molecular reso-lution.

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INTRODUCTION

A key problem in cell division inEscherichia coliis the formation of a proteinring constituted of FtsZ molecules in the septum zone (1–6). A major questito understand the mechanisms used for the formation of the FtsZ ring. Thismay be organized by the formation of lipid domains; conversely, the formaof the ring may induce lipid segregation (7, 8).

The Langmuir–Blodgett technique (9) is a very useful tool for modelingological membranes specially to study lipid–protein interactions (10–13)the case of FtsZ–lipid interactions, this technique may be used to study, awater/air interface, the interactions between FtsZ and a monolayer constiof two lipids able to form domains in the physical-chemical conditions ofexperiment (14).

In this paper we focus on dipalmitoylphosphatidylethanolamine (DPPwhich is the main constituent of the inner membrane. DPPE films have bpreviously studied (15) and molecular resolution has been obtained. DPPEhave been elaborated upon and studied by the classical methods to chaize a Langmuir film (pressure/area isotherms, stability, etc.). The corresponLB film obtained by its transfer on a solid substrate has been characterizeinfrared spectroscopy and atomic force microscopy. We focus here on scaforce microscopy results; in particular, we tried to obtain the highest resolupossible (16, 17) for molecular scale images of DPPE to be able, eventuto distinguish the nature of molecules (lipid, protein) that will be presendomains.

MATERIALS AND METHODS

A homemade Langmuir–Blodgett trough was utilized for the preparatiothe samples; the trough was made of PTFE; its dimensions were 50× 10.9 cm;the volume of the liquid phase was 550 cm3. This system used a couple of plunging mobile barriers for symmetrical compression of the amphiphilic molecuA Wilhelmy balance was used to measure the interfacial pressure (preci±0.1 mN/m). The trough was enclosed in a cabinet in which the temperawas maintained at 22◦C.

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The elaboration of DPPE films was achieved using a pure water subpThe water was obtained by a Millipore system involving deionization, reveosmosis, and filtration (Milli-RO+Milli-Q).

DPPE obtained from Sigma (purity, 99%) was prepared as a 10−3 M solutionin a chloroform/methanol mixture (90%/10%), and an amount of 0.1 ml waspread with a capillary micropipette (Nichiryo).

After the DPPE solution was spread, a time of 15 min was allowed for solevaporation and for the relaxation of the system. Then, the film was comprefor pressure/area isotherms, the rate of compression was 0.5 cm/min. The trwas performed according to the classical Langmuir–Blodgett technique (vemethod) at 40 mN/m, this pressure being maintained by a feedback looptransfers were performed on mica muscovite samples obtained from M´etafix(Montdidier, France) as rectangular pieces 9× 35 mm2, 200-µm thick. Mus-covite samples were freshly cleaved before any transfer and used withouparticular processing. Muscovite samples, initially in the air, were dippedthe subphase before DPPE spreading and covered by one monolayer durifinal upward translation. The rate of the vertical transfer was 1 cm/min.transfer ratio may be estimated to 0.9± 0.1.

The scanning force microscopy images presented in this paper in thetact mode were obtained with a Nanoscope II from Digital Instruments, wa 180-µm piezoelectric scanner for large-scale investigations and with a 1µmscanner for molecular resolution investigations. The cantilevers used wereacterized by a low spring constant (0.06 N/m). A standard tip of silicon nitrwas used. The measurement was performed with the feedback loop on (coforce: from 10−9 to 10−8 N). The scan rate was 2.5 Hz (scan lines per secofor large-scale imaging and around 8 Hz for high-resolution imaging. Allinvestigations were performed in the air. A basic antivibration system wasfor damping vibrations, and a thick plastic cover was used for minimizing sotrouble.

The images are presented in height mode (palette of color for height:colors for low zones and light colors for high zones) and are top-view imag

RESULTS

Pressure–Area Isotherm

A pressure–area isotherm of DPPE is presented on Fig. 1. This isothecharacterized in the more condensed states by a liquid-condensed phalowed by a solid phase. These states are separated by a phase transition38 mN/m. The solid phase collapses above 52 mN/m. Pressure-area isotare quite reproducible. In addition, DPPE Langmuir films are very stable: wi1 h, it is not possible to measure a significant area decrease at a constant prof 40 mN/m. The molecular area of DPPE, measured by extrapolation osolid phase at a surface pressure of zero, is 38 A

a2. These results are in agreeme

with the literature (13).

Scanning Force Microscopy

Large-scale imaging. When imaged at large scale, DPPE monolayerspear densely packed and very flat. On Fig. 2, a sample imaged in a 18× 18µm2

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FIG. 1. Pressure/area isotherm of a dipalmitoylphosphatidylethanolamLangmuir film.

zone appears in most parts very flat and dense; only a few small holeobservable (characteristic dimensions ranging from 100 to 200 nm). In sparts of the sample, it is possible to measure a 2.8-nm height in the larger hthis measure is consistent with the thickness of a single monolayer (15).

In some zones, it is also possible to observe multilayers which are multiof bilayers; these terraces are characterized by heights of 5.5 nm; each tehas a height corresponding to a bilayer.

FIG. 2. Scanning force microscopy (contact mode) image of a dipalmitophosphatidylethanolamine Langmuir film transferred onto muscovite at 40m; image size: 18×18µm2.

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FIG. 3. Scanning force microscopy (contact mode) image of a dipalmitophosphatidylethanolamine Langmuir film transferred onto muscovite at 40m; image size: 38× 38 nm2.

The homogeneity and the density of the package of DPPE molecules aagreement with the high stability of the DPPE film organized at the air–winterface. This is also coherent with the presence at the transfer pressuresolid phase.

Molecular-scale imaging. Molecular scale images of the DPPE LB film archaracterized by regular arrangements of molecules with alignments clevisible (Fig. 3). In addition, molecules are well individualized. In circles 1 a2 (Fig. 3) the molecules appear as a single object with a rectangular scharacterized by dimensions of 5 and 8 A

aapproximately. This single object is

characterized by dimensions consistent with a top view of the two hydrophchains; moreover, its area is in agreement with the area of DPPE moleobtained with pressure/area measurements. It seems also that domainsferent orientations of the molecules exist: in circle 1 molecules are orient“vertically” (on the image) whereas in circle 2 molecules are orientated “hizontally”. In some other zones of this image (circle 3, for example), the ttails of DPPE molecules are individualized; in circles 1 and 2, 7, or 8 individobjects are visible, whereas in circle 3, 14 individual objects are visible.correlation between this observation and the dimensions of the objects ledthe observation of the two tails of lipid molecules. In addition, in some zo(circles 4 and 5) the two chains seem to exhibit different heights, as expefrom theoretical models.

CONCLUSION

The behavior of dipalmitoylphosphatidylethanolamine (DPPE) at a wateinterface has been studied and classical results have been confirmed: prearea isotherms of DPPE are characterized at high interfacial pressure by aphase. In addition, DPPE Langmuir films are very stable.

When imaged by scanning force microscopy, DPPE monolayers apdensely packed and very flat with a few defects.

Molecular-scale images of the DPPE LB film are characterized by regarrangements of molecules.

Our major result is the observation, in some zones of the film, of the two tof DPPE molecules which are individualized. To our knowledge, there isother report of such a resolution with phospholipid molecules.

NOTE 587

a

nd1 To whom correspondence should be addressed.

REFERENCES

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Stephane Alexandre∗Veronique Derue∗Saıda Garah∗Chantal Monnier†Vic Norris†Jean-Marc Valleton∗,1

∗UMR 6522 Universite de Rouen–CNRS†UPRES EA 2123Universite de Rouen76821 Mont-Saint-AignanFrance

Received July 22, 1999; accepted April 10, 2000