Hindered Diffusion in Polymer-Tethered Phospholipid Monolayers at

The lateral mobility of fluorescence-labeled phospholipids in mixtures of .... M.A. Deverall , E. Gindl , E.-K. Sinner , H. Besir , J. Ruehe , M.J. Sa...
1 downloads 0 Views 136KB Size
Download PDF
5076

Langmuir 2001, 17, 5076-5081

Hindered Diffusion in Polymer-Tethered Phospholipid Monolayers at the Air-Water Interface: A Single Molecule Fluorescence Imaging Study Pu Chun Ke and Christoph A. Naumann* Department of Chemistry, Indiana UniversitysPurdue University Indianapolis, 402 North Blackford Street, Indianapolis, Indiana 46202 Received March 19, 2001. In Final Form: May 29, 2001 Recently, our group was the first to apply the technique of single molecule fluorescence imaging toward homogeneous phospholipid monolayers at the air-water interface to study the lateral diffusion properties within these systems at the single molecule level.1 Here, we present measurements on Langmuir monolayers of amphiphiles at the air-water interface of a specific heterogeneity to explore problems of hindered lateral diffusion of biomolecules observed in complex biomembranes. The lateral mobility of fluorescence-labeled phospholipids in mixtures of phospholipids and lipopolymers was investigated via single molecule tracking at different lipopolymer molar concentrations. In agreement with recent fluorescence recovery after photobleaching experiments,2 we found that the diffusion behavior of phospholipids within polymertethered monolayers is characterized by different regions depending on the lateral mobility of lipopolymer molecules. At a low lipopolymer molar concentration, the diffusion coefficient of phospholipids is independent of the concentration of tethered lipids (lipopolymers), thereby showing no signs of obstructed diffusion. Clear signs of obstructed diffusion are observed, however, if polymer chains of adjacent lipopolymers interact with each other. Our experiments showed that single molecule fluorescence imaging is a powerful experimental tool to study obstructed diffusion in model membranes.

Introduction Biomembranes can be seen as complex, self-assembled structures held together by mostly weak, competing molecular forces. It is the subtle balance between these forces which gives biomembranes their peculiar dynamic properties resulting in lateral and rotational diffusion of phospholipids and proteins. The lateral diffusion of membrane molecules is of particular importance because it facilitates intramembrane protein signaling. It is, therefore, not surprising that many efforts have been made to analyze the lateral diffusion of phospholipids and proteins in biomembranes.3 The lateral diffusion in model membranes is well described. While the two-dimensional form of the free area model of Cohen and Turnbull fits the case when solute and solvent molecules are of similar size,4-6 the hydrodynamic continuum model introduced by Saffman and Delbrueck7,8 should be used if the size of the solute is larger than that of the solvent. Analogous studies on native biomembranes revealed, furthermore, that the diffusion coefficient determined for a specific molecule is typically 1-2 orders of magnitude smaller than the corresponding value for the same molecule in a model system of simpler composition.9-11 * To whom correspondence should be addressed. Email: [email protected]. Tel: 317-278-2512. (1) Ke, P. C.; Naumann, C. A. Langmuir 2001, 17, 3727. (2) Naumann, C. A.; Knoll, W.; Frank, C. W. Submitted for publication in Biomacromolecules. (3) Saxton, M. Curr. Top. Membr. 1999, 48, 229 and references therein. (4) Cohen, M. H.; Turnbull, D. J. Chem. Phys. 1959, 31, 1164. (5) Traeuble, H.; Sackmann, E. J. Am. Chem. Soc. 1972, 94, 4499. (6) Galla, H. J.; Hartmann, W.; Theilen, U.; Sackmann, E. J. Membr. Biol. 1979, 48, 215. (7) Saffman, P. G.; Delbrueck, M. Proc. Natl. Acad. Sci. U.S.A. 1975, 73, 3111. (8) Saffman, P. G. J. Fluid Mech. 1976, 73, 593. (9) Abney, J. R.; Scalettar, B. A. Biophys. J. 1995, 55, 817. (10) Webb, W. W.; Barak, L. S.; Tank, D. W.; Wu, E.-S. Biochem. Soc. Symp. 1981, 46, 191.

Still, progress in this area is limited because of the complex nature of the biomembrane architecture. For example, the complicating factors are (1) the complex composition of the biomembrane consisting of cytoskeleton, phospholipid bilayer, and glycocalix, (2) the parallel occurrence of different diffusion processes, (3) the uncertainty about the molecular areas of membranes constituents, and (4) the limited knowledge about membrane heterogeneities acting as obstacles for the phospholipid and protein diffusion. Obviously, research in this area would benefit from studies on novel model systems under controlled area and/or area fraction conditions, which mimic the complex composition of biomembranes in a more realistic manner. One promising model system, which meets these criteria, is the polymer-supported phospholipid bilayer stabilized either by attractive electrostatic forces12,13 or by covalent coupling (tethering).14,15 In the latter case, silane-functionalized polymers14 or photo-cross-linkerfunctionalized glass substrates16 provide the covalent attachment of the polymer cushion to the substrate, whereas lipid membrane and polymer cushion are tethered via lipopolymer molecules. Recent FRAP (fluorescence recovery after photobleaching) experiments have shown that transmembrane proteins retain their long-range lateral mobility if embedded into such phospholipidpolymer composites.14 They also verified that the lateral mobility of phospholipids is critically dependent on the configuration of the underlying polymer layer14 or on the (11) Edidin, M. Curr. Top. Membr. 1996, 43, 1. (12) Wong, J. Y.; Majewski, J.; Seitz, M.; Park, C. K.; Israelachvili, J. N.; Smith, G. S. Biophys. J. 1999, 77, 1445. (13) Wong, J. Y.; Park, C. K.; Seitz, M.; Israelachvili, J. N. Biophys. J. 1999, 77, 1458. (14) Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400. (15) Naumann, C. A.; Prucker, O.; Lehmann, T.; Ruehe, J.; Knoll, W.; Frank, C. W. Submitted for publication in Biomacromolecules. (16) Prucker, O.; Naumann, C. A.; Ruehe, J.; Knoll, W.; Frank, C. W. J. Am. Chem. Soc. 1999, 121, 8766.

10.1021/la010408p CCC: $20.00 © 2001 American Chemical Society Published on Web 07/06/2001

Hindered Diffusion in Monolayers

level of stabilization at the polymer-phospholipid interface (tethering density).15 A complementary model system to the polymer-supported phospholipid bilayer is the polymer-supported phospholipid monolayer at the air-water interface, which is stabilized via covalent tethers at its polymer-phospholipid interface.2 These Langmuir films, formed by a mixture of amphiphilic phospholipids and lipopolymers, allow for an accurate control over the molecular areas of its constituents. Recent FRAP experiments on such polymer-tethered phospholipid monolayers have indicated that the lateral diffusion of phospholipids is critically dependent on the strength of polymer-polymer interactions among adjacent lipopolymers. While the diffusion coefficient, D, remains unchanged in the case of noninteracting or weakly interacting polymer chains (D ∼ 7 µm2/s), a significant decrease was found for polymer chains that interact in a medium (D ∼ 4-5 µm2/s) and strong (D ∼ 1 µm2/s) manner.2 In addition to their relevance as bioartificial membranes, polymer-tethered phospholipid monolayers and bilayers represent interesting model systems for the study of the problems of hindered two-dimensional diffusion. To address such problems experimentally, single molecule detection techniques, which do not average over a huge ensemble of molecules, would be advantageous. Unlike FRAP, single molecule experiments can, for example, resolve molecules in their different dynamic states, as shown by findings on anomalous diffusion in solidsupported model membranes.17 Recently, our group applied single molecule fluorescence imaging to phospholipid monolayers at the air-water interface.1 These initial measurements showed that the surface flow could be suppressed successfully. Furthermore, it was found that the diffusion behavior of phospholipids can be described by a free area model, which was, for example, not observed by single molecule imaging experiments using gold-labeled phospholipids.18 Here, we present single molecule fluorescence imaging experiments on phospholipid (DMPC)-PEG lipopolymer mixtures at the air-water interface. This study is aimed to answer the following questions: (1) How does the tethering concentration of PEG lipopolymers affect the diffusion coefficient of phospholipids if the area per phospholipid is kept constant? (2) How well can the free area model, used in the case of phospholipid monolayers at the air-water interface, be applied for phospholipid-lipopolymer mixtures at a given lipolymer molar concentration but different area per phospholipid? (3) Can single molecule fluorescence imaging resolve different states of phospholipid diffusion? Materials and Methods The phospholipid studied was 1,2-dimyristoyl-3-glycero-phosphocholine (DMPC), while the lipopolymer was 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DSPE-EO45). Both amphiphiles were purchased from Avanti Polar Lipids (Alabaster, AL). The fluorescence label, N-(6tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine triethylammonium salt (TRITCDHPE), was purchased from Molecular Probes (Eugene, OR). The chemical structures of the amphiphiles used are illustrated in Figure 1. In addition, chloroform was used as a spreading solvent for preparing the polymer-tethered monolayers at the air-water interface. Milli-Q water (pH ) 5.5, 18 MΩ cm resistivity) was the subphase material for all experiments. Our single molecule fluorescence imaging setup is illustrated in Figure 2 and was previously described in more detail.1 In (17) Schuetz, G. J.; Schindler, H.; Schmidt, T. Biophys. J. 1997, 73, 1073. (18) Forstner, M. B.; Kaes, J.; Martin, D. Langmuir 2001, 17, 567.

Langmuir, Vol. 17, No. 16, 2001 5077

Figure 1. Molecule structures of the phospholipid DMPC, lipopolymer DSPE-EO45, and the fluorescence-labeled phospholipid TRITC-DHPE.

Figure 2. Schematic diagram of the experimental setup of fluorescence microscopy (for more details see Materials and Methods). short, a 200 mW frequency doubled Nd:YAG laser (wavelength: 532 nm) was used as an excitation source. The laser beam was spatially filtered and delivered to the EPI port of an inverted microscope (Zeiss Axiovert S100TV). Then the beam was reflected by a dichroic mirror (Omega XF1051) and was focused by a microscope objective (Olympus, water immersion, 40× numerical aperture ) 1.15). To control photobleaching of the sample irradiated by the laser beam, a Uniblitz shutter (VMM-D1) of 3 mm open aperture was utilized. The fluorescence signal, centered at 566 nm, was refocused to an intensified CCD camera (iPentaMAX 512EFT, Princeton Instruments) mounted at the TV port of the microscope. The excitation light was blocked out by the combination of a Raman filter (Omega 540ELP) and the dichroic mirror. The temperature and the gain of the camera were set at -22 °C and 67.5, respectively. The exposure time and the frame rate of the CCD camera were chosen to be 15 ms and 11 frames/s while synchronized with the Uniblitz shutter. Image recording and single molecule tracking were acquired using Isee imaging software (Inovision Corp.) running on a Linux platform. The mean-square-displacement (MSD) for individual phospholipid molecules was obtained based:

5078

Langmuir, Vol. 17, No. 16, 2001 MSD(t) )

∑

Ke and Naumann

|r(t) - r(t - t0)|2

0et-t0e0.9s

where t0 ) 0.1 s is the time interval between two subsequent frames and r(t) is a position vector at time t. No more than 11 frames were chosen for each individual track. The lateral diffusion coefficient, D, can be derived using formula:

D ) MSD(t)/4t For each sample, 105 tracks were analyzed. The induced error in the determination of the value of D is less than 12% according to the theoretical model.19 Single molecule fluorescence imaging was performed by tracking single molecules of fluorescence-labeled TRITC-DHPE within mixed monolayers of DMPC/DSPE-EO45 at the air-water interface (dye concentration: 10-5mol/mol). A representative image, including intensity profile from a similar sample geometry (TRITC-DHPE in a DMPC monolayer at the air-water interface), was recently presented elsewhere.1 The observed on-off blinking, which is a single molecule-specific phenomenon,20 ensured that single molecules were imaged. We should emphasize that it is reasonable to assume that TRITC-DHPE and DMPC, which are characterized by a slight mismatch of their acyl chain lengths, show the same lateral diffusion because the lipid diffusion is described by a two-dimensional free area model that predicts no dependence of lipid diffusion on acyl chain length.5,6,21

Figure 3. Pressure-area isotherms of the phospholipidlipopolymer mixtures at the air-water interface at different lipopolymer molar concentrations of 5, 10, 20, and 30 mol %. The darker markers correspond to the experimental conditions when Alipid ) 65 Å2. The lighter markers on the curve when the lipopolymer molar concentration is 30 mol % represent the experimental conditions when Alipo ) 146, 180, 196, and 230 Å2.

Results and Discussion Effect of Lipopolymer Molar Concentration. Figure 3 illustrates the pressure-area isotherms of monolayers of DSPE-EO45/DMPC mixtures at different lipopolymer molar concentrations of 5, 10, 20, and 30 mol %. The plateaus found at around 10 mN/m are a direct indication of the amphiphilic character of poly(ethylene oxide) chains. For film pressures less than the plateau value, the polymer chains are predominantly located at the air-water interface. At the plateau pressure, however, they become more and more submerged into the aqueous solution.22 In the case of single molecule imaging experiments, different lipopolymer molar concentrations were, therefore, studied well above the plateau pressure of 10 mN/m, as marked in Figure 3. The lateral mobility of phospholipids at the air-water interface is critically dependent on the area per molecule and can be described by a free area model.1,23,24 Since we are interested in the effect of the lipopolymer molar concentration on the lateral mobility of phospholipids, experiments were performed at a constant area per phospholipid, Alipid, of Alipid ) 65 Å2, but for different areas per lipopolymer, Alipo. Alipo and Alipid are related via Alipo ) Alipid(n + 1) with n being the phospholipid-lipopolymer molar ratio. Figure 4A-E shows histograms of MSD for the different lipopolymer molar concentrations of 0, 5, 10, 20, and 30 mol % at Alipid ) 65 Å2. The histogram of MSD for pure DMPC (Figure 4A) represents an important reference for the remaining data. In this case, the observed distribution of MSD is stochastic because the phospholipid monolayer, which is in its liquid-expanded phase state, is characterized by unobstructed diffusion of phospholipids. Within the investigated range of lipopolymer molar concentrations (Figure 4B-E), there is no indication that the histograms change significantly in shape with respect to that of DMPC (19) Qian, H.; Sheetz, M. P.; Elson, L. Biophys. J. 1991, 60, 910. (20) Dickson, R. M.; Cubitt, A. B.; Tsien, R. Y.; Moerner, M. E. Nature 1997, 388, 355. (21) Vaz, W. L. C.; Clegg, R. M.; Hallmann, D. Biochemistry 1985, 24, 781. (22) Baekmark, T. R.; Elender, G.; Lasics, D. D.; Sackmann, E. Langmuir 1995, 11, 3975. (23) Peters, R.; Beck, K. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 7183. (24) Kim, S.; Yu, H. J. Phys. Chem. 1992, 96, 4034.

Figure 4. (A-E) Distribution histograms of MSD obtained from tracks based on 11 subsequent frames (t ) 1 s). (F-J) The corresponding averaged MSD(t) for different lipopolymer molar concentrations of 5, 10, 20, and 30 mol %, respectively. Alipid ) 65 Å2. Approximately 105 tracks were counted in each case.

(Figure 4A), even though the center of those histograms shifts with higher lipopolymer concentration to smaller

Hindered Diffusion in Monolayers

Langmuir, Vol. 17, No. 16, 2001 5079

Figure 5. Lateral diffusion coefficient D as a function of Alipo at different lipopolymer molar concentrations of 0, 5, 10, 20, and 30 mol % at the air-water interface. Alipid ) 65 Å2. Sections I and II indicate the situations of noninteracting and interacting lipopolymers, respectively.

values of MSD. The latter point is supported by the corresponding plots of the averaged MSD over time (Figure 4F-J), which in general show a decreasing slope with increasing lipopolymer molar concentration. Interestingly, MSD vs time retains for all lipopopolymer concentrations investigated (0, 5, 10, 20, and 30 mol % lipopolymer) a linear relationship, thus indicating a random distribution of obstacles (phospholipid moiety of lipopolymers) within the polymer-tethered phospholipid monolayer. More direct evidence for the effect of the lipopolymer concentration on the lateral diffusion of phospholipids can be obtained if the diffusion coefficient, D, is plotted as a function of the area per lipopolymer, Alipo, as shown in Figure 5. At large area per lipopolymer of Alipo g 650 Å2 (0, 5, 10 mol % lipopolymer), we observe a small decrease of D with decreasing Alipo, from D ) 2.45 µm2/s at 0 mol % lipopolymer to D ) 2.34 µm2/s at 10 mol % lipopolymer, which is within the range of the experimental error of 5%. At these relatively low lipopolymer molar concentrations, lipopolymers do not interact with each other (section I in Figure 5). This is verified from fluorescence microscopy studies that provide no indication of macroscopic phase separation between phospholipids and lipopolymers (data not shown). Since lipopolymers and phospholipids are likely to show similar diffusion coefficients, no significant decrease in phospholipid diffusion is expected. As recently discussed,2 polymer moieties of adjacent DSPE-EO45 molecules start to interact with each other at around Alipo ) 600 Å2. Because interacting lipopolymers are less mobile, they now act as obstacles for phospholipid diffusion, thus leading to decreased diffusion coefficients of phospholipids (section II in Figure 5). This can be seen in Figure 5 for Alipo < 600 Å2 (20 and 30 mol % lipopolymer), which is characterized by a significant drop of D with increasing lipopolymer molar concentration from D ) 2.34 µm2/s at 10 mol % lipopolymer to D ) 1.97 µm2/s at 20 mol % and to D ) 1.24 µm2/s at 30 mol %. Since the area per phospholipid, Alipid, was kept constant in this experiment, the observed change in phospholipid diffusion can be directly related to the lipopolymer molar concentration. Changing Area Per Molecule at 30 mol % Lipopolymer. Recent FRAP experiments on DSPE-EO45/ DMPC mixtures showed not only that diffusion coefficients of phospholipids change significantly if the strength of lipopolymer-lipopolymer interactions is modified but also that the immobile fraction (percentage of immobile phospholipids) is changing. Since single molecule detection techniques are able to resolve molecules in their different

Figure 6. (A-D) Distribution histograms of MSD obtained from tracks based on 11 subsequent frames (t ) 1 s). (E-H) The corresponding averaged MSD(t) for Alipo ) 146, 180, 196, and 230 Å2. The lipopolymer molar concentration is maintained at 30 mol %. Approximately 105 tracks were counted in each case. Since the left column of the histogram of parts C and D of Figure 6 represents MSD values of 0-1 µm2, it also represents data from immobile molecules (MSD ) 0 µm2). Consequently, immobile molecules are considered in the case of the averaged MSD shown in parts G and H of Figure 6.

dynamic states (other than the ensemble-averaging FRAP approach), we also performed single molecule fluorescence imaging experiments on DSPE-EO45/DMPC mixtures for a fixed lipopolymer molar concentration of 30 mol % lipopolymer at different areas per lipopolymer molecule, Alipo. The chosen area values represent the phospholipidlipopolymer monolayer above and below its rheological transition at Arheo ∼ 170 Å2. Such a transition was recently found in the case of poly(ethylene glycol) and poly(oxazoline) lipopolymers 25,26 and poly(ethylene glycol) lipopolymer-phospholipid mixtures 27 at the air-water interface. Figure 6A-D shows the histograms of MSD for 30 mol % lipopolymer at Alipo of 230, 196, 180, and 146 Å2. While the shapes of histograms for 230 and 196 Å2 exhibit no significant deviation from that of pure DMPC (see Figure 4A), those for 180 and 146 Å2 are clearly different. In the latter cases, an increasing number of immobile or very slowly moving phospholipids can be found with decreasing area per lipopolymer (parts C and D of Figure 6). This can be seen from the left column of the histogram in parts C (25) Naumann, C. A.; Brooks, C. F.; Fuller, G. G.; Knoll, W.; Frank, C. W. Langmuir 1999, 15, 7752. (26) Naumann, C. A.; Brooks, C. F.; Fuller, G. G.; Lehmann, T.; Ruehe, J.; Knoll, W.; Kuhn, P.; Nuyken, O.; Frank, C. W. Langmuir 2001, 17, 2801. (27) Naumann, C. A.; Brooks, C. F.; Wiyatno, W.; Knoll, W.; Fuller, G. G.; Frank, C. W. Macromolecules 2001, 34, 3024.

5080

Langmuir, Vol. 17, No. 16, 2001

Ke and Naumann

Figure 8. Lateral diffusion coefficient D as a function of Alipo at the air-water interface recorded using FRAP and SMD, respectively.

Figure 7. (A) Lateral diffusion coefficient D as a function of Alipo at the air-water interface. The lipopolymer molar concentration is 30 mol %. DSPE-EO45 and DMPC/DSPE-EO45 mixtures show a rheological transition at an area per lipopolymer of Arheo ) 170 Å2.23,25 Sections II and III indicate the regions below and above the rheological transition. The letters a, b, c, and d correspond to Alipo ) 230, 196, 180, and 146 Å2, respectively. (B) The dependence of lateral diffusion coefficient D on free area for DMPC phospholipid (squared markers) and DMPC/DSPE-EO45 phospholipid-lipopolymer mixtures (diamond markers) at the air-water interface, respectively. af ) Alipid - 44 Å2. The letters a, b, c, and d correspond to Alipo ) 230, 196, 180, and 146 Å2, respectively.

and D of Figure 6, which shows MSD in the range of 0-1 µm2. While this column is exclusively represented by immobile molecules (MSD ) 0 µm2) in the case of Figure 6C, it includes about 30% slowly diffusing molecules (0 < MSD e 1 µm2) in the case of Figure 6D. Interestingly, the averaged MSD over time shows in all four cases a rather linear relationship (Figure 6E-H). This is somewhat surprising if we keep in mind that lipopolymers can interact with each other via their hydrophobic chains for Alipo e 250 Å2, thereby forming microclusters.27,28 The observed linear MSD-time relationship indicates that the existing obstacles are randomly distributed before and after microphase separation. Figure 7A illustrates a plot of diffusion coefficients vs Alipo for 30 mol % lipopolymer (a, 230; b, 196; c, 180; d, 146 Å2), as derived from the results in Figure 6. In this case, a significant decrease of D can be found from D ) 1.53 µm2/s at 230 Å2 to D ) 1.24 µm2/s at 196 Å2 to D ) 1.08 (28) Wiesenthal, T.; Baekmark, T. R.; Merkel, R. Langmuir 1999, 15, 6837.

µm2/s at 180 Å2 to D ) 0.81 µm2/s at 146 Å2 with decreasing values of Alipo, exhibiting a linear D-Alipo relationship. There is no indication from Figure 7A of a qualitative change of the phospholipid diffusion around the rheological transition (below the transition, Figure 7A, section II; above the transition, Figure 7A, section III), which has been verified for DSPE-EO45 at Arheo ∼ 170 Å2. Since the area per phospholipid, Alipid, was changed in the case of our measurements shown in Figure 7A, the free area model introduced by Sackmann and co-workers 5,6 can be applied to our data. This model describes the diffusion behavior of a rigid cylinder with the crosssectional area, a0, in a two-dimensional layer of a specific viscosity. It predicts a linear relationship between ln D and the inverse of the free area per cylinder, af ) A - a0 via ln D ∼ (1/af). Figure 7B compares the corresponding plots of ln D vs (1/af) for 30 mol % lipopolymer (diamonds) and pure DMPC (squares), which was recently determined.1 Interestingly, the ln D-(1/af) plot shows a linear relationship for areas below the rheological transition of Arheo ) 170 Å2 (a, 230; b, 196; c, 180 Å2), but a significant deviation from linearity for areas above the rheological transition (d, 146 Å2). This can be seen as an indication of the existence of the rheological transition based on diffusion data of phospholipids. Another interesting result from Figure 7B is the similarity of the slopes of fitting curves for 30 mol % lipopolymer at areas per lipopolymer of 230, 196, and 180 Å2 and for pure DMPC. It suggests that the change in diffusion observed in Figure 7A is caused mainly by a change of the free area among phospholipids. Comparison of FRAP and Single Molecule Imaging Data. One aspect of our study was to compare diffusion data on DSPE-EO45/DMPC mixed monolayers at the airwater interface obtained by FRAP and single molecule fluorescence imaging. Not surprisingly, very similar D-Alipo relationships are found for both techniques, as shown in Figure 8, because the diffusion coefficients in both cases represent ensemble-averaged magnitudes even though the size of the ensemble is different (single molecule imaging, ∼105 molecules; FRAP, ∼1010 molecules). One possible reason for the observed difference between absolute values of D for a given Alipo, obtained from FRAP and single molecule fluorescence imaging in Figure 8, should be seen in deviations of temporal and spatial resolutions for both techniques. While FRAP conditions (mercury bulb of the microscope used as bleaching source; long bleaching time of 10 s could result in thermal excitation of the sample; large diameter of bleaching spot

Hindered Diffusion in Monolayers

of 150 µm made setup relatively prone to surface flow) do not allow the measurement of absolute values of diffusion coefficients with high precision,2 the absolute diffusion values from single molecule imaging experiments are very reliable under the experimental conditions used (see also Materials and Methods). Another reason for the observed deviation could be related to differences in the fluorescence dyes used. While the FRAP experiments were performed using phospholipids labeled with the rather hydrophilic N-(7-mitrobenz-2-oxa-1,3-diazol-4-yl) dye (NBD) to their hydrophilic headgroups, single molecule experiments were conducted on headgroup-labeled phospholipids labeled with the photostable but rather hydrophobic TRITC dye. Deviating lateral diffusion values could, therefore, be affected by the different dye-phospholipid interactions. Single molecule fluorescence imaging provides a more subtle picture, however, when one compares the histograms in Figures 4 and 6 with the immobile fraction, IF, obtained from FRAP studies. While IF provides a single number for the amount of immobile molecules, the histograms show a specific distribution. This becomes especially obvious when the histograms in Figure 6 for different Alipo at 30 mol % lipopolymer are considered. Thus, histograms in parts C and D of Figure 6 exhibit not only a specific percentage of immobile molecules but also a significant amount of slowly moving ones. Interestingly, there is still a significant amount of mobile phospholipids even above the rheological transition at 170 Å2 (Figure 6D). Conclusion We have extended our recent single molecule fluorescence imaging experiments on Langmuir films of phospholipids at the air-water interface1 to those of phospholipid-lipopolymer mixtures. Our studies on polymertethered phospholipid monolayers at the air-water interface show that single molecule fluorescence imaging

Langmuir, Vol. 17, No. 16, 2001 5081

allows for a more detailed analysis than FRAP regarding diffusion properties in heterogeneous monolayers, thus making it a powerful tool to study problems of obstructed diffusion in monolayers of amphiphiles at the air-water interface. The experiments presented are qualitatively in good agreement with recent FRAP experiments on analogue samples.2 Three different regions could be distinguished. As long as adjacent lipopolymers do not interact with each other (Alipo > 650 Å2), there is no relevant change in phospholipid diffusion for different lipopolymer molar concentrations. This verifies that mobile lipopolymers do not affect the phospholipid diffusion when adjacent lipopolymers interact solely via their polymer moieties (300 Å2 < Alipo < 650 Å2), a moderate decrease in phospholipid diffusion is observed with increasing lipopolymer concentrations. Since the free area within the lipid moiety is kept constant, the measured drop can be directly linked to a decreased lateral mobility among lipopolymers. At Alipo < 300 Å2, this decrease in diffusion becomes even more pronounced when lipopolymers form small clusters via acyl chain condensation.28 Our studies support theoretical predictions that the size, the density, and the mobility of obstacles (tethered lipids of lipopolymers in our case) affect the lateral mobility of phospholipids.29,30 The experiments presented are complementary to the studies on solid-supported, polymer-tethered phospholipid bilayers, which are currently in progress in our laboratory. Acknowledgment. The authors thank John Coffman for his support with the film balance experiments and Miranda Stanley for her help with the preparation of the manuscript. Funding for this work was provided by the Purdue School of Science at IUPUI. LA010408P (29) Saxton, M. J. Biophys. J. 1987, 52, 989. (30) Saxton, M. J. Biophys. J. 1989, 56, 615.