to allow photography of weakly fluorescent samples at levels of excitation low ... NBD-PE showed only one spot on thin-layer chromatography as described ...
Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp. 2759-2763, June 1978
Biophysics
Determination of molecular motion in membranes using periodic pattern 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
The lateral diffusion of a fluorescent phosABSTRACT pholipid probe in oriented multibilayers of dimyristoylphosphatidyicholine has been measured by observing the redistribution of fluorescence after photobleaching of the membranes in a periodic pattern of parallel stripes. The diffusion constant D of the fluorescent lipid was found to vary between 1.5 X 10-11 cm2 sec at 9.60 and 2.0 X 10-10 at 22.50 in the monoclinic phase. Preliminary studies of dipalmitoylphosphatidylcholine liposomes in the Lpe and P1, phases yielded diffusion constants of the order of 10-1" cm2-/sec. These data are relevant to earlier discussions of the rate of complement activation by haptensensitized 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 the motion of fluorescent antibodies bound to murine ET4 tumor cells. Pattern photobleaching techniques have the advantages that cellular or liposomal translation has no major adverse effect on the measurements, that certain nondiffusive motions can be detected and characterized, and that diffusive or other motions can be recorded photographically.
The relationship between the composition, distribution, and motion of cell surface components and cell function is one of the major challenges of modern molecular biology. Attempts to relate lateral motion and function have been made for intact cells (1-5) as well as for model membranes having specific, well-delineated functions (6-8). Two of these studies (6, 7) included an attempt to relate the lateral mobility of haptens in model 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 from early studies using spin labels (9-12) as well as more recent photobleaching methods (13, 14), the initial goal of the present work was to obtain the diffusion constants of lipids in the "solid" phase of phosphatidylcholines; if low enough, such diffusion constants could play a critical role in limiting complementmediated attack on such membranes. The elegant experiments by Wu et al. (13) clearly demonstrated a precipitous decrease in the diffusion constant of a lipid probe in dimyristoylphosphatidylcholine at the transition temperature but did not yield a diffusion constant in the lower temperature phases, a quantity of central importance in our study. In our new technique, an image consisting of alternating bright and dark stripes is projected into a sample to establish, by photobleaching of fluorescent probe molecules, a periodic variation of fluorescence intensity as a function of position. Observation of the subsequent redistribution of fluorescence intensity in the sample yields information about the motion (e.g., diffusion) of the probe molecules. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
THEORY AND APPARATUS As a simple model, we consider the diffusion of fluorescent probe molecules confined to a two-dimensional surface. Photobleaching of the probe molecules by light in a periodic pattern of parallel stripes produces a corresponding periodic variation in the concentration (C) along the direction (x) perpendicular to the direction (y) of the stripes. Therefore, ?C/?by = 0, and we need only consider diffusion of fluorescent molecules in the x direction. The diffusion equation is IC(xt) DM2C(Xt) =
ait
oX2
[1]
D is the diffusion coefficient of the probe molecule in the sample. 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 concentration of the probe prior to the bleaching burst of light, the duration and intensity of the bleaching burst, and the contrast and resolution of the stripe image in the sample. The parameter a is the spatial frequency of the pattern and is equal to 2wr/P in which 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 constant and that the amplitude of the pattern decays with time. Because the higher spacial frequency terms decay much more rapidly than the second term, the concentration can be described after a time t > 0.1/Da2 by only the first two terms of Eq. 3. In other words, regardless of the extent to which the initial pattern approximates a square wave, the pattern quickly becomes sinusoidal with an amplitude that decays as a single exponential. The diffusion coefficient D can then'be calculated from the measured time constant r of this decay, D = 1/a2r. [4] The apparatus for this experiment consists of a laser, a microscope equipped for photomicrography, and optics for directing the laser beam through a Ronchi ruling into the microscope. The laser was a Spectra Physics model 164-03 argon-ion laser. The microscope was a Zeiss photomicroscope III with epifluorescence condenser IIIRS. The Ronchi rulings consisted of evenly spaced opaque parallel lines on a transparent Abbreviation: NBD-PE, N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylethanolamine. * To whom reprint requests should be addressed.
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ZlfB~WN-
Biophysics: Smith and McConnell
ILASER BEAM
CONVERGING
'LENS
RONCHI RULING
MICROSCOPE OBJECTIVE
- / SAMPLE FIG. 1. Diagram of optics used for pattern photobleaching. Light from an argon-ion laser is directed through a Ronchi ruling into the microscope objective which focuses an image of the ruling onto the sample. Details of the microscope optical system have been omitted.
substrate, with linewidth equal to the space between lines. Ronchi rulings were purchased from Edmund Scientific Company with frequencies of 50-200 lines per inch. The Ronchi ruling was mounted on the microscope in a
Proc. Nati. Acad. Sci. USA 75 (1978)
real-image plane of the microscope optical system-i.e., a plane onto which the microscope projects a real image of the sample. Thus, a real image of the ruling, illuminated by the laser, is projected onto the sample by the microscope objective. Fig. 1 is a diagram of this optical arrangement. The laser beam can be directed into the microscope either through the epifluorescence condenser or through an accessory camera port on the top of the. microscope. We have tried both arrangements but have used the former for most of these experiments in order to leave the accessory camera port available for an image-intensifier. The ruling is mounted on a sliding holder attached to the epifluorescence condenser which allows it to be removed from the optical path after the photobleaching burst and be replaced by an attenuator for uniform illumination of the sample for photography. An image intensifier tube (NI-TEC, Inc.) is mounted on the accessory camera port. This tube amplifies low-intensity images to allow photography of weakly fluorescent samples at levels of excitation low enough to avoid undesirable photobleaching. MATERIALS AND METHODS Phospholipids. Dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine were purchased from Sigma. These compounds contained no impurities as determined by thin-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 by gas/liquid chromatography of the fatty acid methyl esters.
N
4t
FIG. 2. Fluorescence photomicrograph of a multibilayer sample (dimyristoylphosphatidylcholine doped with NBD-PE) immediately after pattern photobleaching. The period of the striped pattern is 13 Alm. Defects in the macroscopic ordering of the sample, which exhibit birefringence when viewed between crossed polarizers, appear as bright lines when viewed by fluorescence. The striped pattern has been placed in a region free of such defects.
Biophysics: Smith and McConnell
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 density of 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 750 sec after pattern photobleaching. The decay of the amplitude of the periodic pattern as a function of time is exponential with time constant equal to 550 sec.
The fluorescent lipid probe N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE) prepared from egg lecithin was purchased from Avanti Biochemicals. The NBD-PE showed only one spot on thin-layer chromatography as described above. Lipid Multibilayers and Liposomes. Hydrated phospholipids can be oriented in macroscopically ordered multibilayers by established procedures (9, 13, 15, 16). For each sample, a
solution of 1.0 Amol of dimyristoylphosphatidylcholine and
approximately 5 nmol of NBD-PE in 100 Al of chloroform was placed as a single drop onto a cleaned glass microscope slide at 400 and the solvent was allowed to evaporate. Dry nitrogen was passed over the lipid film (still at 400) for 30 min to remove all traces of solvent. The slide was next placed in a closed container over distilled water in an oven at 450 for at least 12 hr; then, a cleaned 1.8-cm-square microscope cover glass was placed over the lipid film and the slide was returned to the 450 water-saturated atmosphere for another 12-24 hr. The slide was placed on a metal block at 400, and a ground flat 40-g weight was placed on the cover glass for 5 min. The edges of the cover glass were sealed to the slide with paraffin to prevent dehydration of the sample during experiments. Samples were stored in a water-saturated atmosphere at room temperature. Multilamellar dipalmitoylphosphatidylcholine liposomes containing approximately 1 mol% of the fluorescent probe NBD-PE were made in phosphate-buffered saline by the method of Brulet and McConnell (6). Diffusion Coefficient Measurements. The 476.5-nm wavelength laser line was used for all photobleaching experiments. For measurements on the multibilayer samples, the laser was operated at the maximum obtainable power, approximately 750 mW. Stripe periods of 4 to 22 ,m were used to measure diffusion coefficients in the dimyristoylphosphatidylcholine multibilayer samples, with X40 and X10 objectives. The stripe period was varied roughly inversely with the change in diffusion coefficient with temperature so that the time required to perform the measurements would be in the convenient range of 100-600 sec. Each measurement was made as follows. The sample was examined under differential interference contrast, and a uniform, defect-free region was selected. The region was also examined between crossed polarizers to be sure that it had the uniform, dark appearance characteristic of a properly ori-
ented multibilayer. The selected region of the sample was exposed to the projected image of the Ronchi ruling which was illuminated by a burst of laser light of sufficient duration (on the order of 0.1 see) to bleach 50-90% of the probe molecules in the brightly illuminated regions. Five successive fluorescence photomicrographs were taken of the sample at regular time intervals beginning about 10 see after the photobleaching. Experiments on liposomes and cells were performed as above except that the stripe period was 3.6,gm and the photographs were taken with the aid of the image intensifier. Photography. Kodak recording film 2475 was used for all
experiments. For the direct fluorescence photomicrographs, 1091~~~~~~~~
E U
A-
5
10 5 20 25 TEMPERATURE (*C) FIG. 4. Diffusion coefficient of NBD-PE in dimyristoylphosphatidylcholine multibilayer samples as a function of temperature. No hysteresis was detected in the diffusion coefficients in the temperature range 9.6-23.70. The strong dependence of the diffusion coefficient on temperature illustrated by the straight line corresponds to an apparent "activation energy" for diffusions of 36 kcal/mol. Note that the largest diffusion coefficient at 23.70 is in the region of the chain melting phase transition and the diffusion coefficients 1 or 2 degrees higher are >108 cm2/sec. See text 0, Increasing temperature; A, decreasing temperature.
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Biophysics: Smith and McConnell
Proc. Nati. Acad. Sci. USA 75 (1978)
FIG. 5. Fluorescence photomicrograph of an azide-treated single murine EL-4 tumor cell labeled first with H-2b alloantiserum followed by fluoresceinated rabbit anti-mouse antiserum. The photomicrograph was taken immediately after stripe bleaching. Prior to bleaching, the patched pattern seen in the unbleached areas was randomly distributed over the entire cell surface. The cell was photobleached with a stripe period of 3.6 Mm.
the film was developed at an ASA speed rating of 8000 with Kodak DK-50 developer and Factor 8 speed additive. For the image-intensifier photographs, the film was developed as recommended by the manufacturer with DK-50 (ASA 1000). Optical density versus exposure curves for the film and for the film-intensifier combination were determined with a Kodak calibrated step tablet. Film Analysis. Optical density as a function of position on the film was measured on a Transidyne General RFT scanning densitometer. The response of the densitometer was calibrated against the same calibrated step tablet used to determine the film response curves. * Data Reduction. The film response curves were used to convert optical density of the film to relative exposure of the film as a function of position. Exposure of the film was assumed to be directly proportional to the concentration of the unbleached fluorescent probe molecules in the sample. The logarithm of the amplitude of the sinusoidal variation of concentration as a function of position in each photograph was plotted as a function of the time at which the photograph was taken, and linear regression was performed to find the time constant for decay of these amplitudes. Stripe Period Measurements. A stage micrometer was used
to calibrate the magnification of the microscope in the photomicrographs. The periods of the stripes were than calculated from measurements on the film. We estimate these measurements to be accurate to within 1% for the X10 objective, 2% for the X40 objective, and 5% for the X63 objective. RESULTS AND DISCUSSION Fig. 2 is a fluorescence photomicrograph of an oriented multibilayer sample of dimyristoylphosphatidylcholine containing 0.5 mol% phospholipid fluorophore NBD-PE, the photomicrograph being taken 13 sec after a laser bleach lasting approximately 0.1 sec. The amplitude of the periodic fluorescent 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 fluorescence intensity differences showed a simple exponential decay, typically for a range of two time constants. The conformity of the decay to a single exponential during the observation period used is consistent with the theoretical discussion presented above and the assumed linearity of fluorescence intensity with concentration. For additional discussion of the properties of NBD-PE, see Wu et al. (13). Diffusion coefficients measured in four
Biophysics: Smith and McConnell separate multibilayer samples as a function of temperature are displayed in Fig. 4. The slope of the straight line in Fig. 4 corresponds to an "activation energy" of 36 kcal/mol (151 kJ/mol), an energy so large that it clearly does not represent a single molecule activation energy. From studies by freeze-fracture electron microscopy and x-ray diffraction it is known that one dimension of the monoclinic unit cells of phosphatidylcholines is on the order of 100 or more A (17, 18). The large unit cell probably contains lipids with hydrocarbon chains having differing degrees of order (E. J. Luna, J. Owicki, and H. M. McConnell, unpublished data). A strong temperature dependence of the fraction of lipids having low order might very well give rise to a strong temperature dependence of the diffusion constant. The one measurement at 23.7° is within the chain melting transition region of this phospholipid; at 24.5° the diffusion coefficient was >10-8 cm2/sec. [Diffusion coefficients of the probe in fluid dimyristoylphosphatidylcholine have been measured by Wu et al. (13) and found to be between 0-7 and 5 X 10-8 cm2/sec above the chain melting transition temperature. ] In connection with the experiments on complement depletion to be discussed below, we also made preliminary measurements of the lateral diffusion of NBD-PE in dipalmitoylphosphatidylcholine liposomes in the temperature region near 320 [close to the LO'-Pf phase transition temperature (17)]. The diffusion constant was found to be of the order of 10-11 cm2/sec. In previous work (6, 7) it was found that, at low hapten surface densities, complement depletion by hapten-sensitized liposomes was substantially more efficient in fluid liposomes (dimyristoylphosphatidylcholine at 320) than in solid liposomes (dipalmitoylphosphatidylcholine at 32°). It was suggested that one source of this difference might be differences in the lateral mobilities of the haptens in the two membranes (6). Although the present work demonstrates that the differences in these mobilities are indeed substantial, the diffusion constant of the haptens in the dipalmitoylphosphatidylcholine liposomes is still so high that it is difficult to imagine that this diffusion can be rate-limiting for complement activation. This conclusion is now in complete accord with the recent results of Parce et al. (19) who have reported that Clq binds equally well to hapten-sensitized fluid and solid lipid vesicles. The present results are also in accord with previous extensive studies of lateral phase separations in binary mixtures of phospholipids in that the spin-label, freeze-fracture, and calorimetric data indicate that the derived phase diagrams describe 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 of plasma membrane components. Fig. 5 shows an azide-treated (H-2b) murine tumor cell labeled with anti-H-2b alloantiserum followed by fluoresceinated rabbit anti-mouse IgG. The spatial bleaching period is 3.6 um. In such cells, redistributions of membrane components such as the patches seen in Fig. 5 are readily observed (to be reported in detail elsewhere in collaboration with W. Clark).
Proc. Natl. Acad. Sci. USA 75 (1978)
2763
Note Added in Proof. It is possible that the lateral mobilities of antibodies specifically bound to lipid haptens in solid and fluid membranes are not simply related to the ratios of the diffusion constants of the individual lipid haptens in these membranes.
We are indebted to Dr. J. Spudich for the use of his scanning densitometer, 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 has developed a new method of measuring lateral diffusion coefficients of spin-labeled lipids using paramagnetic resonance and a laser induced periodic pattern of photochemical reactions (24); he has provided much help and advice with our sample preparations. We have benefited from the able assistance of Mr. F. Zweers in the Stanford Center for Materials Research. This work has been supported by National Institutes of Health 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, 12091217. 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) Biochim. 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.
