Proc. Natl. Acad. Sci. USAVol. 81, pp. 3249-3253, May 1984Chemistry

Periodic structures in lipid monolayer phase transitions(two-dimensional systems/lateral diffusion/fluorescence microscopy/membranes)

HARDEN M. MCCONNELL, LUKAS K. TAMM, AND ROBERT M. WEISStauffer Laboratory for Physical Chemistry, Stanford University, Stanford, CA 94305

Contributed by Harden M. McConnell, January 16, 1984

ABSTRACT Periodic patterns are observed when sup-ported lipid monolayers doped with low concentrations of fluo-rescent lipid probes are observed with epi-fluorescence mi-croscopy. Monolayers of dipalmitoyl phosphatidylcholine wereexamined on air-water interfaces and also on alkylated glasscoverslips. The patterns are formed by periodic arrays of sol-id-phase lipid domains in equilibrium with fluid-phase lipidunder specified conditions of temperature and two-dimension-al lipid pressure. Electrostatic forces may stabilize the periodicordering of the solid domains.

In recent work we have used sensitized lipid monolayers onsolid supports as target membranes in studies of cellular im-mune response (1-4). It is now clear that immune responsecan depend strongly on the physical state of target mem-branes, in addition to depending on the incorporation of spe-cific antigens, lipid hapteps, antibodies, etc. (5-8). Suchsupported monolayers are also of interest purely from thepoint of view of the physical chemistry of two-dimensionalsystems (9-12).

Isothermal pressure-induced phase transitions in phospho-lipid monolayers have their counterpart in the main thermo-tropic chain melting phase transitions of the correspondinglipid bilayers (12). In the case of bilayers, solid and fluidphases are readily distinguished by electron microscopy, x-ray diffraction, and magnetic resonance spectroscopy (13-16). Epi-fluorescence microscopy of lipid systems contain-ing fluorescent lipid probes also permits one to distinguishbetween solid- and fluid-phase lipids, especially when thistechnique is combined with measurements of lateral diffu-sion using photobleaching-recovery techniques (17, 18).Recently, Peters and Beck have found that equilibrium

mixtures of solid and fluid phases of L-a-dipalmitoyl phos-phatidylcholine (Pam2-PtdCho) at the air-water interface arereadily distinguished using epi-fluorescence microscopy andthe fluorescent lipid probe N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-egg phosphatidylethanolamine (NBD-egg-PtdEtn) (19).Closely related results were also observed by Losche et al.using carbocyanine fluorescent probes (20). We indepen-dently observed similar epi-fluorescence features for dopedPam2-PtdCho in monolayers on alkylated quartz slides. In aneffort to relate the several observations, we have studiedsuch monolayers before and after transfer to alkylated glasscoverslips. During the course of these studies we have ob-served striking periodic patterns of solid-phase lipid domainson both supports. Such patterns provide additional insightinto the nature of the two-dimensional phase transition inthese systems, as well as an improved understanding of thepressure dependence of lateral diffusion. These patterns alsosuggest the presence of lateral electrostatic forces, whichmay account for the much-studied deviation from infinitecompressibility in the two-phase region of the isothermalpressure-area curve (12, 21).

MATERIALS AND METHODS

Phospholipids and Fluorescent Lipid Analogs. Pam2-PtdCho was purchased from Sigma and used without furtherpurification. NBD-egg-PtdEtn, N-(7-nitro-2,1,3-benzoxadia-zol-4-yl)-L-a-dipalmitoyl phosphatidylethanolamine (NBD-Pam2-PtdEtn), and 1-acyl-2-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino caproyl]phosphatidylcholine (NBD-PtdCho)were purchased from Avanti Polar Lipids (Birmingham,AL).

Phospholipid Monolayer Preparation. Phospholipid mono-layers were observed at the air-water interface with thetrough and rhombus assembly described previously (22) onthe stage of a Zeiss Photomicroscope III. Briefly, a 2-mmdeep trough milled from aluminum, 15 cm wide and 20 cmlong, supported a rhombus with a 12-cm base. Surface pres-sure was measured with a Wilhelmy plate made from a Milli-pore filter (GSTF, 0.22 gm) 1.25 cm wide. The plate wasattached to a torsion balance (Roller-Smith, Bethlehem,PA). Air current-induced lipid flow was reduced by an open-ended cylindrical collar made from a 0.1-mm-thick Teflonsheet placed over the 20x objective (22 mm in diameter)used for observation. The collar extended below the surfaceof the monolayer. A single channel, 2 mm wide, that was cutinto the cQllar permitted pressure equilibration between themoqolayer inside and outside the collar. In addition, a Plexi-glas hood was placed over the trough with access holes forthe objective and the Wilhelmy plate and to provide adjust-ment for the rhombus.Water was deionized and then glass distilled before use as

the aqueous phase. Pam2-PtdCho containing typically 2 mol% fluorescent probe was spread from a syringe as a 1.25 mMsolution in a solvent of hexane/ethanol 9:1. A sufficient timeperiod preceded compression to allow the spreading solventto evaporate. The area of the rhombus was varied manuallyin small increments, to allow pressure equilibration and ob-servation without flow. The change in surface tension wasrecorded with each area change. All measurements weremade at ambient temperature (22 + 20C).

Epi-Fluorescence Illumination and PhotQgraphy. Epi-fluo-rescence photographs were taken with the built-in micro-scope camera using ASA 1000 film (2475 recording film,Eastman Kodak). Fluorescence was excited by the 488-nmline of a Spectra Physics argon ion laser (model 164-08,Mountain View, CA). Photographic exposure times weretypically 0.5 s using a laser intensity of 300 mW focused to a200-,um sppt diameter. A much-attenuateq beam ('=4 mW)was used for observation.

Transfer of Monolayers to Alkylated Supports. Glass cov-erslips were alkylated according to a procedure similar tothat described previously (23). In addition, after the final

Abbreviations: Pam2-PtdCho, L-a-dipalmitoyl phosphatidylcholine;NBD-egg-PtdEtn, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-egg phos-phatidylethanolamine; NBD-Pam2-PtdEtn, N-(7-nitro-2,1,3-benzox-adiazol-4-yl)-L-a-dipalmitoyl phosphatidylethanolamine; NBD-PtdCho, 1-acyl-2-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino caproyl]-phosphatidylcholine.

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drying step, the coverslips were cleaned in an Argon plasmacleaner (Harrick Scientific, Ossining, NY) for 15 min. Theoctadecyltrichlorosilane (Petrarch, Levington, PA) was ei-ther freshly distilled or used from material that after distilla-tion had been placed in sealed ampules with an Argon atmo-sphere and stored at -20CC.To transfer the monolayer to the support, the coverslip

was suspended between the objective (without the Tefloncollar) and the air-water interface by a micromanipulator(Leitz) to observe the area to be coated. The coverslip wasthen placed on the interface. The supported monolayer wasnot usually re-exposed to air during subsequent handling.The monolayer on the coverslip support could then be ob-served under the microscope.

RESULTSFig. 1 shows pressure-area curves for Pam2-PtdCho withand without fluorescent lipid probe (2% NBD-egg-PtdEtn).These pressure-area curves are similar to those published(12, 22). Our results are in good agreement with those of Pe-ters and Beck (19) in that solid-phase lipid domains that ex-dude NBD-egg-PtdEtn first appear on compression at pres-sures of the order of -5 dynes/cm (region A in Fig. 1) andincrease in size and relative area until region B is reached. Inthe present work, regions designated A, B, and C are quitereproducible in terms of the shapes of the pressure-areacurves, whereas absolute values of pressure are not (perhapsdue to small temperature variations). Illustrative epi-fluores-cence photographs are shown in Fig. 2. The dark regions aresolid-plase lipid (see below), and the fluorescent regions inwhich NBD-egg-PtdEtn is dissolved represent fluid-phaselipids. In this A-B region of the pressure-area curve, patternphotobleaching measurements yield diffusion coefficients ofthe order of 10_710-8 cm2/s, typical of fluid lipids, To es-tablish that dark regions arise from solid-phase lipid (ratherthan the absence of any lipid monolayer), we employed asecond fluorescent lipid probe, NBD-Pam2-PtdEtn, whichwe expected would partition more favorably into the (crys-talline) solid-phase lipid. As illustrated in Fig. 3, this is in-deed the case-the putative solid-phase domains show sub-stantial fluorescence. Pattern photobleaching in the two-

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FIG. 1. Pressure-area isotherms for Pam2-PtdCho spread on dis-tilled water. *, Pure Pam2-PtdCho at 201C; o, Pam2-PtdCho contain-ing 2 mol % NBD-egg-PtdEtn. Data obtained on compression over aperiod of 20 min with a Langmuir trough were similar to those de-scribed elsewhere. Pressure-area curves measured on the micro-scope stage have a similar shape to those shown here but absolutevalues of the pressure are less certain.

FIG. 2. Epi-fluorescence microscope photograph showing for-mation of solid-phase domains of Pam2-PtdCho at the air-water[phosphate-buffered saline (Pi/NaCI)] interface on compression.Dark regions are solid-phase lipid domains and strongly fluorescentregions are fluid lipid in which 2 mol % of the probe NBD-egg-PtdEtn is dissolved. (a-c) Monolayers corresponding approximatelyto point A, a point two-thirds of the way from A to B, and point B(Fig. 1), respectively. The regular patterns in b and c are thought tobe stabilized by electrostatic forces. (Bar is 50 Ium.)

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shown in Fig. 2 appear to repel one another at larger separa-tions but adhere and fuse on contact. This behavior accountsfor the necklace pattern seen in Fig. 6, which results when acompressed film is decompresed.A number of experiments were carried out to compare the

effect of transferring lipid monolayers from the air-water in-

FIG. 3. Epi-fluorescence microscope photograph of a Pam2-PtdCho monolayer in the two-phase (solid-fluid) region of the pres-sure-area curve, where the fluorescent probe NBD-Pam2-PtdEtnpartitions into both phases, and the solid phase has a higher fluores-cence intensity than the fluid phase. This experiment demonstratesthat the dark areas in Fig. 2 represent lipid domains that excludeNBD-egg-PtdEtn and do not originate by the total absence of lipid.The field in this figure has been bleached with a burst of laser radia-tion given a period of -45 Am. The photograph was taken approxi-mately 5 s after this bleach burst. The fluorescence intensity in thefluid regions is uniform due to rapid diffusive recovery. Bleachedsolid domains remain dark due to slow lipid exchange between fluidand solid; some solid domains remain partially bleached due to slowdiffusion within the solid phase. (Bar is 50 Aum.)

phase region shows diffusive recovery of the fluid region andno substantial recovery in the solid-phase regions in times ofthe order of 5 s (see Fig. 3). (Note: We have observed darkdomains in highly expanded monolayers that are due to localareas depleted of all lipid.)The solid-phase domains illustrated in Fig. 2 are more uni-

form in size, are larger, and are much more uniformly spacedon the Pi/NaCl aqueous phase than they are on distilled wa-ter (data not shown). The solid domains form a quasi-hexag-onal lattice on aqueous substrates.To determine whether or not the charge on the lipid probe

has any effect on solid-phase domain formation, we incorpo-rated the neutral fluorescent probe NBD-PtdCho in thesemonolayers. This probe does not partition into the solid do-mains. The solid-phase domain patterns observed were verysimilar to those seen in Fig. 2, irrespective of whether theaqueous subphase was Pi/NaCl or distilled water. On theother hand, changing the concentration of the charged probeNBD-egg-PtdEtn (0.5-4 mol %) in the monolayer on the dis-tilled water subphase does have an effect on domain size andshape. At the higher probe concentrations, the domains be-came smaller and were c- and s-shaped. (See also Fig. 3.)Kinetic effects on solid-phase domain growth are illustratedin Fig. 4. For details, see the legend to Fig. 4.

In the higher pressure region (region B-C in Fig. 1), thefluorescence of the probes sometimes appears quite uni-form, but fluorescence redistribution after pattern photo-bleaching is clearly nonuniform during time periods of theorder of =5 s. See Fig. 5. At still higher pressures (left ofregion C in Fig. 1), lateral diffusion was uniform and low.Parallel bleach stripes separated by 45 Am were observed topersist clearly for >40 min (D < 10-1o cm2/s).As discussed later, solid-phase domains such as those

FIG. 4. Transient formation of "snowflake" solid-phase domainsfollowing a small abrupt pressure jump (3% decrease in the area perlipid). (a) Five to 10 s after pressure jump; (b) 2 min later; (c) 15 minlater. Subphase is Pi/NaCl at pH 4. The fluorescent probe is 2 mol %NBD-egg-PtdEtn. (Bar is 50 Jim.)

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FIG. 5. Epi-fluorescence microscope photograph of a Pam2-PtdCho monolayer containing 2 mol % NBD-Pam2-PtdEtn in regionB-C of the pressure-area curve (Fig. 1) 10 s following pattern pho-tobleaching. Inhomogeneous recovery demonstrates that eventhough the initial fluorescence intensity was uniform, lateral diffu-sion is inhomogeneous. (Bar is 50 Am.)

terface to alkylated glass coverslips using previously de-scribed techniques. To a good approximation, a given pat-tern of fluorescence observed at intermediate pressures atthe air-water interface is retained immediately after transfer.Fig. 7 illustrates the fact that under carefully controlled con-ditions transfer from the air-water interface to the alkylatedcoverslip can be achieved with little visible distortion in thedistribution of fluorescence. Differences are observed thatare due to subsequent time-dependent changes in the fluo-

FIG. 6. Epi-fluorescence microscope photograph of a Pam2-PtdCho monolayer containing 2 mol % of NBD-PtdCho on distilledwater immediately following decompression from above region C(-30 dynes/cm) to about point A in Fig. 1. The photograph illus-trates the binding of solid domains to one another following contactbetween these domains. (Bar is 50 ,um.) Similar patterns were ob-served with 2 mol % NBD-egg-PtdEtn.

FIG. 7. Epi-fluorescence microscope photograph of a Pam2-PtdCho monolayer containing 2 mol % NBD-PtdCho close to pointB (Fig. 1) in the pressure-area curve and immediately before trans-fer to an alkylated coverslip (a), immediately after the transfer to analkylated coverslip (b), and 20 min later (c). The photographs showthat the transfer can be accomplished with little disruption of thedistribution of fluorescence between solid and fluid domains. Atime-dependent diffusion of fluorescent lipid into solid domains isshown in c. Note that on the alkylated coverslip contacts betweensolid domains are frequent, and in c there are regions where thesolids have a square array. (Bar is 50 am.)

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rescence distribution on the alkylated glass. As illustrated inFig. 7c, there is a time-dependent infiltration of fluorescentlipid into the solid-phase domains. Note also that there arenow many contacts between solid domains, and many ofthese domains are in a quasi-square array. Lateral transla-tional diffusion over distances of several microns is typicallysignificantly reduced after transfer. The diffusion coeffi-cients of lipids on the alkylated glass coverslips are quitesimilar to those found in the various phases of lipid bilayers.

DISCUSSIONPerhaps the most striking result of the present work is theobserved periodic arrangement of the solid-phase domains inthe intermediate pressure region, where roughly equalamounts of solid and fluid co-exist. The observed quasi-hex-agonal arrangement of solid domains such as those in Fig.2c, together with the apparently independent but restrictedBrownian motion of these domains, suggests that there maybe long-range forces between them. A significant role forelectrostatic forces in domain structure is indicated by largeeffects of (i) subphase ionic strength, (ii) probe charge, and(iii) concentration of the charged probe. In the latter case,increasing the negative charge density in the fluid phase (orsolid phase) could reasonably enhance the formation ofsmaller and noncircular solid domains. Some of our photo-graphs in fact suggest a phase transition in the ordering of thesolid domains, perhaps analogous to those known forcharged latex spheres (24, 25).When solid-phase lipid is formed by compression, there is

sometimes a transient formation of snowflake structures,which gradually revert to smooth solid domains (Fig. 4). Theformation of snowflakes has been discussed by Langer (26).Even though the solid domains appear to repel one anoth-

er at large distances, they fuse and bind to one another atshort distances, as shown in Fig. 6. In these systems it isunlikely that thermodynamic equilibrium is reached eitherduring compression or expansion, and this may account forthe nonhorizontal aspect of the two-phase region seen in Fig.1, as well as the occurrence of hysteresis. Electrostatic ef-fects together with a positive solid-fluid interfacial free en-ergy may contribute to the slope of the pressure-area curveon compression.

Irrespective of these complications, it is clear that thepressure region A-B in Fig. 1 is a two-phase region, contraryto some previous interpretations of these data (22).Our experiments demonstrate that it is possible to transfer

lipids from an air-water interface to an alkylated glass cover-slip with little instantaneous distortion of the domain shapes.The most prominent change in domain distribution is the oc-currence of frequent contacts between solid domains aftertransfer. It is possible that electrostatic effects are sup-pressed on the alkylated surfaces. We suspect that quantita-tive analysis of data such as those presented here will showthat lipid monolayers on alkylated solid substrates are morerepresentative of lipids in bilayers than are lipid monolayersat the air-water interface.

We are greatly indebted to Drs. Reiner Peters and Erich Sack-mann for showing us their results prior to publication and to Dr.John Ross and Mr. M. LeVan for helpful discussions. This work wassupported by a Fannie and John Hertz Foundation Fellowship toR.M.W., a Swiss National Science Foundation Fellowship toL.K.T., and Grant NSF PCM 8021993 to H.M.M. This material isbased in part upon work supported by the U.S. Public Health Ser-vice under Grant RR01613 awarded to the University of California atBerkeley in collaboration with Stanford University.

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7. Cartwright, G. S., Smith, L. M., Heinzelmann, E. W., Rue-bush, M. J., Parce, J. W. & McConnell, H. M. (1982) Proc.Natl. Acad. Sci. USA 79, 1506-1510.

8. McConnell, H. M. (1979) in Les Houches, Session XXXIII:Membranes et Comunication Intercellulaire, eds. Balian, R.,Chabre, M. & Devaux, P. (North-Holland, Amsterdam), pp.270-292.

9. Sinha, S. K., ed. (1980) Ordering in Two Dimensions (Else-vier/North Holland, New York).

10. Cadenhead, D. A., Muller-Landau, F. & Kellner, B. M. J.(1980) in Ordering in Two Dimensions, ed. Sinha, S. K. (Else-vier/North Holland, New York), pp. 73-78.

11. Seul, M., Eisenberger, P. & McConnell, H. M. (1983) Proc.Nati. Acad. Sci. USA 80, 5795-5797.

12. Albrecht, O., Gruler, H. & Sackmann, E. (1978) J. Physique39, 301-313.

13. Grant, C. W. M., Wu, S. H. & McConnell, H. M. (1974)Biophys. Biochim. Acta 363, 151-158.

14. Tardieu, A., Luzzati, V. & Reman, F. (1973) J. Mol. Biol. 75,711-733.

15. McConnell, HI. M. (1976) in Spin Labeling: Theory and Appli-cations, ed. Berliner, L. (Academic, New York), pp. 525-560.

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7183-7187.20. Losche, M., Sackmann, E. & Mohwald, H. (1983) Ber. Bun-

senges. Phys. Chem. 87, 848-852.21. Abinet, G. & Tremblay, A.-M. S. (1983) Phys. Rev. A 27,

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421-427.24. Forsyth, P. A., Jr., Mardelja, S., Mitchell, D. J. & Ninham,

B. W. (1978) Adv. Colloid Interface Sci. 9, 37-60.25. Efremov, 1. F. & Us'yarov, 0. G. (1976) Russ. Chem. Rev.

(kingl. Transl.) 45, 435-453.26. Langer, J. S. (1980) Rev. Mod. Phys. 52, 1-28.

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