Determination ofmolecularmotioninmembranes periodic pattern … · well-delineatedfunctions(6-8). Twoofthesestudies(6, 7)in-cludedanattemptto relate thelateral mobilityofhaptensin

Proc. Nati. Acad. Sci. USAVol. 75, No. 6, pp. 2759-2763, June 1978Biophysics

Determination of molecular motion in membranes using periodicpattern photobleaching

(lipid bilayer/diffusion/fluorescence/photobleaching recovery)

BARTON A. SMITH AND HARDEN M. MCCONNELL*

Stauffer Laboratory for Physical Chemistry, Stanford University, Stanford, California 94305

Contributed by Harden M. McConnell, March 31,1978

ABSTRACT The lateral diffusion of a fluorescent phos-pholipid probe in oriented multibilayers of dimyristoylphos-phatidyicholine has been measured by observing the redistri-bution of fluorescence after photobleaching of the membranesin a periodic pattern of parallel stripes. The diffusion constantD of the fluorescent lipid was found to vary between 1.5 X 10-11cm2 sec at 9.60 and 2.0 X 10-10 at 22.50 in the monoclinic phase.Preliminary studies of dipalmitoylphosphatidylcholine lipo-somes in the Lpe and P1, phases yielded diffusion constants ofthe order of 10-1" cm2-/sec. These data are relevant to earlierdiscussions of the rate of complement activation by hapten-sensitized liposomal membranes [Br6let, P. and McConnell, H.M. (1976) Proc. NatL Acad Sci. USA 73, 2977-2981; Parce, J. W.,Henry, N. and McConnell, H. M. (1978) Proc. NatL Acad. Sci.USA 75,1515-15181 We have also used this method to study themotion of fluorescent antibodies bound to murine ET4 tumorcells. Pattern photobleaching techniques have the advantagesthat cellular or liposomal translation has no major adverse effecton the measurements, that certain nondiffusive motions can bedetected and characterized, and that diffusive or other motionscan be recorded photographically.

The relationship between the composition, distribution, andmotion of cell surface components and cell function is one ofthe major challenges of modern molecular biology. Attemptsto relate lateral motion and function have been made for intactcells (1-5) as well as for model membranes having specific,well-delineated functions (6-8). Two of these studies (6, 7) in-cluded an attempt to relate the lateral mobility of haptens inmodel membranes to the degree of complement depletion.Because the rate of lateral diffusion of phospholipids in the"fluid" state of phosphatidylcholines has been well known fromearly studies using spin labels (9-12) as well as more recentphotobleaching methods (13, 14), the initial goal of the presentwork was to obtain the diffusion constants of lipids in the "solid"phase of phosphatidylcholines; if low enough, such diffusionconstants could play a critical role in limiting complement-mediated attack on such membranes. The elegant experimentsby Wu et al. (13) clearly demonstrated a precipitous decreasein the diffusion constant of a lipid probe in dimyristoylphos-phatidylcholine at the transition temperature but did not yielda diffusion constant in the lower temperature phases, a quantityof central importance in our study.

In our new technique, an image consisting of alternatingbright and dark stripes is projected into a sample to establish,by photobleaching of fluorescent probe molecules, a periodicvariation of fluorescence intensity as a function of position.Observation of the subsequent redistribution of fluorescenceintensity in the sample yields information about the motion(e.g., diffusion) of the probe molecules.

THEORY AND APPARATUSAs a simple model, we consider the diffusion of fluorescentprobe molecules confined to a two-dimensional surface. Pho-tobleaching of the probe molecules by light in a periodic patternof parallel stripes produces a corresponding periodic variationin the concentration (C) along the direction (x) perpendicularto the direction (y) of the stripes. Therefore, ?C/?by = 0, andwe need only consider diffusion of fluorescent molecules in thex direction. The diffusion equation is

IC(xt) =DM2C(Xt)ait oX2 [1]

D is the diffusion coefficient of the probe molecule in thesample. The initial conditions after photobleaching are

C(x,0) = A + B sin ax + E sin 3ax + F sin 5ax +.... [2]

The parameters A, B, E ... are determined by the concentra-tion of the probe prior to the bleaching burst of light, the du-ration and intensity of the bleaching burst, and the contrast andresolution of the stripe image in the sample. The parameter ais the spatial frequency of the pattern and is equal to 2wr/P inwhich P is the period of the pattern.The solution to Eq. 1 satisfying the initial conditions given

by Eq. 2 is

C(x,t) = A + Be-Da2t sin ax+ Ee-9Da2t sin Sax + Fe 25a2t sin 5ax +.... [3]

Eq. 3 states that the period of the striped pattern remains con-stant and that the amplitude of the pattern decays with time.Because the higher spacial frequency terms decay much morerapidly than the second term, the concentration can be de-scribed after a time t > 0.1/Da2 by only the first two terms ofEq. 3. In other words, regardless of the extent to which theinitial pattern approximates a square wave, the pattern quicklybecomes sinusoidal with an amplitude that decays as a singleexponential. The diffusion coefficient D can then'be calculatedfrom the measured time constant r of this decay,

D = 1/a2r. [4]The apparatus for this experiment consists of a laser, a mi-

croscope equipped for photomicrography, and optics for di-recting the laser beam through a Ronchi ruling into the mi-croscope. The laser was a Spectra Physics model 164-03argon-ion laser. The microscope was a Zeiss photomicroscopeIII with epifluorescence condenser IIIRS. The Ronchi rulingsconsisted of evenly spaced opaque parallel lines on a transparent

Abbreviation: NBD-PE, N-4-nitrobenz-2-oxa-1,3-diazole phosphati-dylethanolamine.* To whom reprint requests should be addressed.

2759

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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2760 Biophysics: Smith and McConnell

ILASER BEAM

CONVERGING'LENS

RONCHIRULING

MICROSCOPEOBJECTIVE

- / SAMPLEFIG. 1. Diagram of optics used for pattern photobleaching. Light

from an argon-ion laser is directed through a Ronchi ruling into themicroscope objective which focuses an image of the ruling onto thesample. Details of the microscope optical system have been omit-ted.

substrate, with linewidth equal to the space between lines.Ronchi rulings were purchased from Edmund ScientificCompany with frequencies of 50-200 lines per inch.The Ronchi ruling was mounted on the microscope in a

real-image plane of the microscope optical system-i.e., a planeonto which the microscope projects a real image of the sample.Thus, a real image of the ruling, illuminated by the laser, isprojected onto the sample by the microscope objective. Fig. 1is a diagram of this optical arrangement. The laser beam canbe directed into the microscope either through the epifluo-rescence condenser or through an accessory camera port on thetop of the. microscope. We have tried both arrangements buthave used the former for most of these experiments in order toleave the accessory camera port available for an image-inten-sifier. The ruling is mounted on a sliding holder attached to theepifluorescence condenser which allows it to be removed fromthe optical path after the photobleaching burst and be replacedby an attenuator for uniform illumination of the sample forphotography.An image intensifier tube (NI-TEC, Inc.) is mounted on the

accessory camera port. This tube amplifies low-intensity imagesto allow photography of weakly fluorescent samples at levelsof excitation low enough to avoid undesirable photobleach-ing.

MATERIALS AND METHODSPhospholipids. Dimyristoylphosphatidylcholine and di-

palmitoylphosphatidylcholine were purchased from Sigma.These compounds contained no impurities as determined bythin-layer chromatography on silica gel G with chloroform/methanol/concentrated acetic acid/water, 70:30:2:3 (vol/vol).Purity of the fatty acid in these compounds was verified bygas/liquid chromatography of the fatty acid methyl esters.Zlf B~~~~WN-

N

4t

FIG. 2. Fluorescence photomicrograph of a multibilayer sample (dimyristoylphosphatidylcholine doped with NBD-PE) immediately afterpattern photobleaching. The period of the striped pattern is 13 Alm. Defects in the macroscopic ordering of the sample, which exhibit birefringencewhen viewed between crossed polarizers, appear as bright lines when viewed by fluorescence. The striped pattern has been placed in a regionfree of such defects.

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Proc. Natl. Acad. Sci. USA 75 (1978) 2761

FIG. 3. Densitometer tracing from four successive photomicrographs, the first of which is shown in Fig. 2. The trace represents optical densityof the film (increasing toward the top of the page) as a function of position (left to right). The photographs were taken at 13, 250, 500, and 750sec after pattern photobleaching. The decay of the amplitude of the periodic pattern as a function of time is exponential with time constantequal to 550 sec.

The fluorescent lipid probe N-4-nitrobenz-2-oxa-1,3-diazolephosphatidylethanolamine (NBD-PE) prepared from egglecithin was purchased from Avanti Biochemicals. TheNBD-PE showed only one spot on thin-layer chromatographyas described above.

Lipid Multibilayers and Liposomes. Hydrated phospho-lipids can be oriented in macroscopically ordered multibilayersby established procedures (9, 13, 15, 16). For each sample, asolution of 1.0 Amol of dimyristoylphosphatidylcholine andapproximately 5 nmol of NBD-PE in 100 Al of chloroform wasplaced as a single drop onto a cleaned glass microscope slide at400 and the solvent was allowed to evaporate. Dry nitrogen waspassed over the lipid film (still at 400) for 30 min to remove alltraces of solvent. The slide was next placed in a closed containerover distilled water in an oven at 450 for at least 12 hr; then, acleaned 1.8-cm-square microscope cover glass was placed overthe lipid film and the slide was returned to the 450 water-sat-urated atmosphere for another 12-24 hr. The slide was placedon a metal block at 400, and a ground flat 40-g weight wasplaced on the cover glass for 5 min. The edges of the cover glasswere sealed to the slide with paraffin to prevent dehydrationof the sample during experiments. Samples were stored in awater-saturated atmosphere at room temperature.

Multilamellar dipalmitoylphosphatidylcholine liposomescontaining approximately 1 mol% of the fluorescent probeNBD-PE were made in phosphate-buffered saline by themethod of Brulet and McConnell (6).

Diffusion Coefficient Measurements. The 476.5-nmwavelength laser line was used for all photobleaching experi-ments. For measurements on the multibilayer samples, the laserwas operated at the maximum obtainable power, approximately750 mW. Stripe periods of 4 to 22 ,m were used to measurediffusion coefficients in the dimyristoylphosphatidylcholinemultibilayer samples, with X40 and X10 objectives. The stripeperiod was varied roughly inversely with the change in diffu-sion coefficient with temperature so that the time required toperform the measurements would be in the convenient rangeof 100-600 sec. Each measurement was made as follows. Thesample was examined under differential interference contrast,and a uniform, defect-free region was selected. The region wasalso examined between crossed polarizers to be sure that it hadthe uniform, dark appearance characteristic of a properly ori-

ented multibilayer. The selected region of the sample was ex-posed to the projected image of the Ronchi ruling which wasilluminated by a burst of laser light of sufficient duration (onthe order of 0.1 see) to bleach 50-90% of the probe moleculesin the brightly illuminated regions. Five successive fluorescencephotomicrographs were taken of the sample at regular timeintervals beginning about 10 see after the photobleaching.

Experiments on liposomes and cells were performed as aboveexcept that the stripe period was 3.6,gm and the photographswere taken with the aid of the image intensifier.

Photography. Kodak recording film 2475 was used for allexperiments. For the direct fluorescence photomicrographs,

1091~~~~~~~~

E A-U

5 10 5 20 25TEMPERATURE (*C)

FIG. 4. Diffusion coefficient of NBD-PE in dimyristoylphos-phatidylcholine multibilayer samples as a function of temperature.No hysteresis was detected in the diffusion coefficients in the tem-perature range 9.6-23.70. The strong dependence of the diffusioncoefficient on temperature illustrated by the straight line correspondsto an apparent "activation energy" for diffusions of 36 kcal/mol. Notethat the largest diffusion coefficient at 23.70 is in the region of thechain melting phase transition and the diffusion coefficients 1 or 2degrees higher are >108 cm2/sec. See text 0, Increasing temperature;A, decreasing temperature.

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2762 Biophysics: Smith and McConnell

FIG. 5. Fluorescence photomicrograph of an azide-treated single murine EL-4 tumor cell labeled first with H-2b alloantiserum followed byfluoresceinated rabbit anti-mouse antiserum. The photomicrograph was taken immediately after stripe bleaching. Prior to bleaching, the patchedpattern seen in the unbleached areas was randomly distributed over the entire cell surface. The cell was photobleached with a stripe period of3.6 Mm.

the film was developed at an ASA speed rating of 8000 withKodak DK-50 developer and Factor 8 speed additive. For theimage-intensifier photographs, the film was developed as rec-

ommended by the manufacturer with DK-50 (ASA 1000).Optical density versus exposure curves for the film and for thefilm-intensifier combination were determined with a Kodakcalibrated step tablet.Film Analysis. Optical density as a function of position on

the film was measured on a Transidyne General RFT scanningdensitometer. The response of the densitometer was calibratedagainst the same calibrated step tablet used to determine thefilm response curves.

* Data Reduction. The film response curves were used toconvert optical density of the film to relative exposure of thefilm as a function of position. Exposure of the film was assumedto be directly proportional to the concentration of the un-

bleached fluorescent probe molecules in the sample. The log-arithm of the amplitude of the sinusoidal variation of concen-tration as a function of position in each photograph was plottedas a function of the time at which the photograph was taken,and linear regression was performed to find the time constantfor decay of these amplitudes.

Stripe Period Measurements. A stage micrometer was used

to calibrate the magnification of the microscope in the photo-micrographs. The periods of the stripes were than calculatedfrom measurements on the film. We estimate these measure-ments to be accurate to within 1% for the X10 objective, 2% forthe X40 objective, and 5% for the X63 objective.

RESULTS AND DISCUSSIONFig. 2 is a fluorescence photomicrograph of an orientedmultibilayer sample of dimyristoylphosphatidylcholine con-taining 0.5 mol% phospholipid fluorophore NBD-PE, thephotomicrograph being taken 13 sec after a laser bleach lastingapproximately 0.1 sec. The amplitude of the periodic fluo-rescent pattern decays with a time constant equal to 550 sec,as illustrated in the series of densitometer tracings shown in Fig.3.

Plots of the logarithm of peak-minus-valley fluorescenceintensity differences showed a simple exponential decay, typ-ically for a range of two time constants. The conformity of thedecay to a single exponential during the observation period usedis consistent with the theoretical discussion presented above andthe assumed linearity of fluorescence intensity with concen-tration. For additional discussion of the properties of NBD-PE,see Wu et al. (13). Diffusion coefficients measured in four

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separate multibilayer samples as a function of temperature aredisplayed in Fig. 4. The slope of the straight line in Fig. 4 cor-responds to an "activation energy" of 36 kcal/mol (151 kJ/mol),an energy so large that it clearly does not represent a singlemolecule activation energy. From studies by freeze-fractureelectron microscopy and x-ray diffraction it is known that onedimension of the monoclinic unit cells of phosphatidylcholinesis on the order of 100 or more A (17, 18). The large unit cellprobably contains lipids with hydrocarbon chains having dif-fering degrees of order (E. J. Luna, J. Owicki, and H. M.McConnell, unpublished data). A strong temperature depen-dence of the fraction of lipids having low order might very wellgive rise to a strong temperature dependence of the diffusionconstant. The one measurement at 23.7° is within the chainmelting transition region of this phospholipid; at 24.5° thediffusion coefficient was >10-8 cm2/sec. [Diffusion coefficientsof the probe in fluid dimyristoylphosphatidylcholine have beenmeasured by Wu et al. (13) and found to be between 0-7 and5 X 10-8 cm2/sec above the chain melting transition temper-ature.]

In connection with the experiments on complement depletionto be discussed below, we also made preliminary measurementsof the lateral diffusion of NBD-PE in dipalmitoylphosphati-dylcholine liposomes in the temperature region near 320 [closeto the LO'-Pf phase transition temperature (17)]. The diffusionconstant was found to be of the order of 10-11 cm2/sec.

In previous work (6, 7) it was found that, at low hapten sur-face densities, complement depletion by hapten-sensitized li-posomes was substantially more efficient in fluid liposomes(dimyristoylphosphatidylcholine at 320) than in solid liposomes(dipalmitoylphosphatidylcholine at 32°). It was suggested thatone source of this difference might be differences in the lateralmobilities of the haptens in the two membranes (6). Althoughthe present work demonstrates that the differences in thesemobilities are indeed substantial, the diffusion constant of thehaptens in the dipalmitoylphosphatidylcholine liposomes is stillso high that it is difficult to imagine that this diffusion can berate-limiting for complement activation. This conclusion is nowin complete accord with the recent results of Parce et al. (19)who have reported that Clq binds equally well to hapten-sen-sitized fluid and solid lipid vesicles.The present results are also in accord with previous extensive

studies of lateral phase separations in binary mixtures ofphospholipids in that the spin-label, freeze-fracture, and ca-lorimetric data indicate that the derived phase diagrams de-scribe states of solid and fluid phase thermodynamic equilibria,requiring appreciable lateral diffusion in the solid phases(20-23).The present pattern bleaching technique can also be used for

studies of molecular motion in cells-for example, motion ofplasma membrane components. Fig. 5 shows an azide-treated(H-2b) murine tumor cell labeled with anti-H-2b alloantiserumfollowed by fluoresceinated rabbit anti-mouse IgG. The spatialbleaching period is 3.6 um. In such cells, redistributions ofmembrane components such as the patches seen in Fig. 5 arereadily observed (to be reported in detail elsewhere in collab-oration with W. Clark).

Note Added in Proof. It is possible that the lateral mobilities of anti-bodies specifically bound to lipid haptens in solid and fluid membranesare not simply related to the ratios of the diffusion constants of theindividual lipid haptens in these membranes.

We are indebted to Dr. J. Spudich for the use of his scanning den-sitometer, to Dr. H. McDevitt for the anti-H-2b antiserum, and to Dr.W. Clark for his collaboration in preparing Fig. 5. Mr. J. Sheats hasdeveloped a new method of measuring lateral diffusion coefficientsof spin-labeled lipids using paramagnetic resonance and a laser inducedperiodic pattern of photochemical reactions (24); he has provided muchhelp and advice with our sample preparations. We have benefited fromthe able assistance of Mr. F. Zweers in the Stanford Center for MaterialsResearch. This work has been supported by National Institutes ofHealth Grant 5RO1 A113587.

1. Segal, D. M., Taurog, J. D. & Metzger, H. (1977) Proc. Nati. Acad.Sci. USA 74,2993-2997.

2. Mendoza, G. R. & Metzger, H. (1976) Nature 264,548-550.3. Schlessinger, J., Webb, W. W., Elson, E. L. & Metzger, H. (1976)

Nature 264,550-552.4. Edelman, G. (1976) Science 192, 218-226.5. Nicolson, G. (1976) Biochim. Blophys. Acta 457,57-108.6. Bru'let, P. & McConnell, H. M. (1976) Proc. Natl. Aced. Sci. USA

73,2977-2981.7. Bruilet, P. & McConnell, H. M. (1977) Biochemistry 16, 1209-

1217.8. Baumann, G. & Mueller, P. (1974) J. Supramolec. Struct. 2,

538-557.9. Kornberg, R. D. & McConnell, H. M. (1971) Proc. Natl. Acad.

Sci. USA 68,2564-2568.10. Devaux, P. & McConnell, H. M. (1972) J. Am. Chem. Soc. 94,

4475-4481.11. Trduble, H. & Sackmann, E. (1972) J. Am. Chem. Soc. 94,

4499-4510.12. Brnlet, P. & McConnell, H. M. (1975) Proc. Natl. Acad. Sci. USA

72, 1451-1455.13. Wu, E. S., Jacobson, K. & Papahadjopoulos (1977) Biochemistry

16,3935-3941.14. Fahey, P. F., Koppell, D. E., Barak, L. S., Wolf, D. E., Elson, E.

L. & Webb, W. W. (1976) Science 195,305-306.15. Badley, R. A., Martin, W. G. & Schneider, H. (1973) Biochemistry

12,268-275.16. Gaffney, B. J. & McConnell, H. M. (1974) J. Mag. Res. 16,

1-28.17. Luna, E. J. & McConnell, H. M. (1977) Biochim. Blophys. Acta

466, 381-392.18. Janiak, M. J., Small, D. M. & Shipley, G. G. (1976) Biochemistry

15,4575-4580.19. Parce, J. W., Henry, N. & McConnell, H. M. (1978) Proc. Nati.

Acad. Sci. USA 75, 1515-1518.20. Shimshick, E. J. & McConnell, H. M. (1973) Biochemistry 12,

2351-2360.21. Grant, C. W. M. & McConnell, H. M. (1974) Proc. Nati. Aced.

Sci. USA 71,4653-4657.22. Grant, C. W. M., Wu, S. H. W. & McConnell, H. M. (1974) Bio-

chim. Biophys. Acta 363, 151-158.23. Mabrey, S. & Sturtevant, J. M. (1976) Proc. Natl. Acad. Sci. USA

73,3862-3866.24. Sheats, J. R. & McConnell, H. M. (1978) Biophys. J. 21, No. 3,

p. 126a.

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