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Single Molecule Fluorescence Imaging of Phospholipid Monolayers at the Air-Water Interface Pu Chun Ke and Christoph A. Naumann* Department of Chemistry, Indiana University-Purdue University Indianapolis, 402 N. Blackford St., Indianapolis, Indiana 46202 Received January 22, 2001. In Final Form: April 2, 2001
Langmuir films of amphiphilic biomolecules represent promising systems to study diffusional properties in model membranes under controlled area conditions. Here, we present for the first time single molecule fluorescence imaging experiments on phospholipid monolayers at the air-water interface. The technique is used to track the lateral diffusion of single molecules of N-(6-tetramethylrhodaminethiocarbamoyl)1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (TRITC-DHPE), in phospholipid monolayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero3-[phospho-rac-(1-glycerol)] (sodium salt) (DMPG) at different areas per phospholipid molecule. Our tracking data of the averaged mean-square displacement indicate for both phospholipids that surface flow could be suppressed significantly. The diffusion behavior of TRITC-DHPE in DMPC and DMPG monolayers is characterized by unobstructed diffusion and can be described well by a free area model. Our experiments show that single molecule fluorescence imaging can be successfully applied to monolayers of amphiphiles at the air-water interface. This opens the door to future studies of hindered diffusion in monolayers of specific heterogeneity.
Introduction The lateral diffusion of phospholipids and membrane proteins in plasma and model membranes has fascinated numerous research groups over the past two decades because a fundamental understanding of this membrane property is expected to explain a broad variety of membrane-located biological processes (e.g., intramembrane signaling of proteins). Several excellent reviews have been published in this area.1-4 Lateral diffusion in model membranes, which is now well understood, shows two different size regimes. If the solute (e.g., protein) is large in comparison to the solvent (lipid), the lateral diffusion is well described by the Saffman-Delbrueck model, which treats the lipid bilayer as a continuum.5,6 In contrast, if the solvent and the solute are of the same size (phospholipid diffusion), a twodimensional version of the free volume model of Cohen and Turnbull seems to fit the data best.7-9 Still not quite understood is the situation of solutes that are only slightly larger than solvent molecules. The only systematic study, the investigation of the lateral diffusion of macrocyclic polyamides (size range: 30-300 Å2) in phospholipid bilayers (area per molecule: 65 Å2), showed that the threshold value for the transition from the free area * To whom correspondence should be addressed. E-mail
[email protected]; Tel 317-2782512. (1) Vaz, W. L. C.; Goodsaid-Zalduondo, F.; Jacobson, K. FEBS Lett. 1984, 174, 199. (2) Zhang, F.; Lee, G. M.; Jacobson, K. BioEssays 1993, 15, 579. (3) Almeida, P. F. F.; Vaz, W. L. C. In Handbook of Biological Physics; Sackmann, E., Lipowsky, R., Eds.; Elsevier: Amsterdam, 1995; Vol. 1, p 305. (4) Saxton, M. Curr. Top. Membr. 1999, 48, 229. (5) Saffman, P. G.; Delbrueck, M. Proc. Natl. Acad. Sci. U.S.A. 1975, 73, 3111. (6) Saffman, P. G. J. Fluid Mech. 1976, 73, 593. (7) Cohen, M. H.; Turnbull, D. J. Chem. Phys. 1959, 31, 1164. (8) Traeuble, H.; Sackmann, E. J. Am. Chem. Soc. 1972, 94, 4499. (9) Galla, H. J.; Hartmann, W.; Theilen, U.; Sackmann, E. J. Membr. Biol. 1979, 48, 215.
Figure 1. Molecular structures of the unlabeled phospholipid molecules DMPC and DMPG and of the fluorescence-labeled phospholipid TRITC-DHPE, which have been used in this study.
model to the continuum model is reached if the solute occupies a 1.5-fold higher area value than the solvent does.10 (10) Liu, C.; Paprica, A.; Petersen, N. O. Biophys. J. 1997, 73, 2580.
10.1021/la010116o CCC: $20.00 © 2001 American Chemical Society Published on Web 05/15/2001
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Figure 2. Schematic diagram of the experimental setup of single molecule fluorescence microscopy. O1, O2, O3, O5: lenses; O4: microscope objective (40×, NA ) 1.15, water immersion); M1 and M2: mirrors; D1: diaphragm.
The lateral diffusion of phospholipids and proteins in plasma membranes, which is characterized by diffusion coefficients 1-2 orders of magnitude smaller than those for similar molecules in model membranes, is, so far, much less understood.11-13 This remarkable discrepancy is based on the complexity of the plasma membrane which can be seen in (1) the parallel existence of several diffusioncontrolling mechanisms, (2) the heterogeneous character of the membrane, and (3) the complex composition of the membrane (e.g., existence of glycocalix and cytoskeleton) and is further complicated by uncertainty about the area fraction of obstacles and the molecular areas of biomolecules in the membrane.4 The latter aspect would especially benefit from model studies under controlled molecular area or area fraction conditions. A model system that allows such controlled-area studies is the Langmuir film at the air-water interface. Fluorescence recovery after photobleaching experiments (FRAP) on Langmuir films of phospholipids verified, for example, the free area model for the diffusion of phospholipids.14,15 A complicating problem of air-water in-
terface experiments is, however, the surface flow that can result in overestimated diffusion values. FRAP is especially prone to flow because it monitors the fluorescence recovery of a large ensemble of molecules over a relatively large area (diameter of bleaching spot g 0.5 µm vs diameter of single phospholipid molecule ∼ 1 nm). Most molecules of biological relevance exist in different dynamic states, which are not distinguishable by the ensemble-averaging FRAP technique. Different dynamic states can be resolved, however, if corresponding studies are done at the single molecule level. Schuetz et al. reported, for example, anomalous diffusion within solid supported phospholipid bilayers and polymer-supported phospholipid monolayers using single molecule fluorescence imaging.16 Single molecule detection (SMD) has, furthermore, a much better spatial resolution (∼10 nm) than FRAP (g0.5 µm).17 Kaes and co-workers recently performed diffusion studies on a phospholipid (DMPC) Langmuir film at the single molecule level using gold-labeled phospholipids as probe molecules.18 Here, we present for the first time single molecule fluorescence imaging studies on Langmuir films.
(11) Abney, J. R.; Scalettar, B. A. Biophys. J. 1995, 55, 817. (12) Webb, W. W.; Barak, L. S.; Tank, D. W.; Wu, E.-S. Biochem. Soc. Symp. 1981, 46, 191. (13) Edidin, M. Curr. Top. Membr. 1996, 43, 1. (14) Peters, R.; Beck, K. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 7183.
(15) Kim, S.; Yu, H. J. Phys. Chem. 1992, 96, 4034. (16) Schuetz, G. J.; Schindler, H.; Schmidt, T. Biophys. J. 1997, 73, 1073. (17) Qian, H.; Sheetz, M. P.; Elson, L. Biophys. J. 1991, 60, 910. (18) Forstner, M. B.; Kaes, J.; Martin, D. Langmuir 2001, 17, 567.
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Figure 4. Fluorescence image of a single TRITC-DHPE molecule embedded into a phospholipid monolayer of DMPC at the air-water interface (up) and its corresponding fluorescence intensity cross section (bottom). Exposure time: 15 ms; Alipid ) 90 Å2.
Figure 3. Pressure-area isotherms of DMPC (A) and DMPG monolayers (B) doped with TRITC-DHPE at the air-water interface (molar ratio of TRITC-DHPE over DMPC or DMPG: 10-5 mol/mol). Points a, b, c, d, and e correspond to areas per phospholipid molecule, Alipid, of Alipid ) 50, 57, 65, 90, and 115 Å2. The corresponding film pressures are (A) π ) 30.3, 19.5, 10.6, 1.2, and 0.08 mN/m and (B) π ) 16.1, 9.6, 4.8, 0.3, and 0.04 mN/m, respectively.
We report our experiments on monolayers of zwitterionic and negatively charged phospholipids at different area per molecule using a TRITC-labeled phospholipid as probe molecule. The following problems will be addressed: (1) Are single molecule imaging experiments on Langmuir films possible under conditions in which the surface flow can be suppressed significantly? (2) Can lateral diffusion of phospholipids in Langmuir films at the single molecule level be described by the free area model as found in the case of corresponding FRAP experiments? (3) What are the differences in diffusion behavior between Langmuir films of zwitterionic and negatively charged phospholipids? Materials and Methods Sample Preparation. The chemical structures of DMPC and DMPG phospholipid molecules and a TRITC-DHPE fluorescence molecule are shown in Figure 1. The molar ratio of TRITCDHPE over DMPC (or DMPG) was set at 10-5 mol/mol. The cover glasses used for sealing the Teflon sample cell, as shown in the inset of Figure 2, were immersed in Micro-90 cleaning solution (Cole-Parmer Instrument Co.) and then rinsed with Milli-Q water thoroughly before being baked in a furnace at 420 °C for 4 h. Chloroform (Fisher, HPLC grade) was used as the spreading solvent for phospholipid monolayers at the air-water interface. Milli-Q water (pH ) 5.5, 18 MΩ resistivity) was used as the subphase material. To match the short working distance of the microscope objective (see Figure 2), the thickness of the water layer above the cover glass was maintained at approximately 200 µm. The temperature of the sample cell was maintained at 23.8 °C via a Peltier cooling system (TE Technology). A Teflonencapsulated silicone O-ring of inner diameter 12 mm was placed
on top of the cover glass to suppress direct water flow. The surface of the sample cell was mechanically sealed to avoid the perturbation of environmental air flow. Optical Setup. Our experiment was performed on an inverted microscope (Zeiss Axiovert S100TV) as illustrated in Figure 2. A frequency-doubled Nd:YAG laser at a wavelength of 532 nm (CrystaLaser) was employed as the excitation source. The laser beam was expanded and collimated by lenses O1 and O2 and spatially filtered using a diaphragm to attain a Gaussian intensity profile. To reduce photobleaching of the sample irradiated by the laser beam, a Uniblitz shutter (VMM-D1) of 3 mm open aperture was utilized. To suppress the elongation artifact induced by the interaction between the electric field of the laser beam and single phospholipid molecules, a quarter wave plate was inserted in the beam pass to convert the beam from a linearly to a circularly polarized state, thus ensuring an accurate particle tracking at the later stage. The beam was then delivered to the EPI port of the microscope by mirrors M1 and M2 and focused by lens O3 and microscope objective O4 (Olympus, water immersion, NA ) 1.15) to a diffraction spot of 20 µm in diameter at the air-water interface. For all the measurements, the optical power after mirror M2 was fixed at 100 mW. As a result, the light intensity at the focus of the microscope objective was approximately 19.1 kW/cm2. The fluorescence signal, centered at 566 nm, was focused by lens O5 and collected with an intensified CCD camera (iPentaMAX 512EFT, Princteon Instruments). 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. A dichroic mirror (Omega XF1051) used in conjunction with a Raman filter (Omega 540AELP) ensured optically a high signalto-noise ratio. Data Acquisition and Processing. For image recording and single molecule tracking, Isee imaging software (Inovision Corp) installed in a Dell workstation (750 MHz) was employed. Utilizing the nanotrack program in Isee, we obtained the mean-squaredisplacement (MSD) for individual phospholipid molecules based upon
MSD(t) )
∑
|r(t) - r(t - t0)|2
0et-t0e0.9
where t0 ) 0.1 s is the time lag between two consequent frames,
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Figure 5. Twenty MSDs from individual tracks with a total time of 1 s for each track for DMPC monolayers at the air-water interface (left). The MSD averaged over 50 individual tracks (right). Alipid ) 65 Å2. and r(t) is a position vector at time t. The lateral diffusion coefficient D can be derived then using formula
D ) MSD(t)/4t In our experiment, the accuracy of single molecule tracking is assured by choosing each single track of 11 steps with a total time of 1 s. Approximately 600 tracks were obtained separately for each value of area per phospholipid molecule. About 90% percent of the tracks were discarded because of incomplete (less than 11 steps caused by photobleaching) or unreasonable (e.g., starlike or recycling tracks caused by on-off blinking of fluorophores) positional trajectories. By including the shorter tracks (represent approximately 50% of the rejected ones), we only observed a variation in D of no more than 5% without qualitative change. On the basis of our selection criteria, the diffusion data obtained for 105 and 50 tracks show discrepancies of less than 10% approximately. The histograms of MSD over time for 105 and 50 tracks follow asymmetric profiles similar to that predicted by Monte Carlo simulations,19 thus indicating that the MSD data are not affected significantly by our selection criteria. The induced error in determining the value of D is less than 12% according to the theoretical model.17 The observed on-off blinking of single fluorophores ensured that single molecules were imaged. (19) Sonnleitner, A.; Schu¨tz, G. J.; Schmidt, Th. Biophys. J. 1999, 77, 2638.
Results and Discussion DMPC. Figure 3A shows the pressure-area isotherm of a DMPC monolayer at a temperature of 23.8 °C. As the gradual slope of the isotherm curve indicates, the area per molecule of DMPG can be varied over a large area range without leading to phase transitions. The letters a, b, c, d, and e represent the areas per phospholipid molecule of Alipid ) 50, 57, 65, 90, and 115 Å2, respectively, at which single molecule imaging experiments have been performed. Figure 4 exemplifies imaging of a DMPC monolayer doped with TRITC-DHPE. It shows the backgroundcorrected image (32 µm × 32 µm, 0.25 µm/pixel) for an area per DMPC molecule of Alipid ) 90 Å2. The on-off blinking of single molecules and permanent photobleaching were observed clearly in the experiment. Figure 5 (left) shows the distribution of the mean-square displacement, MSD, over time for single tracks of TRITCDHPE in a DMPC monolayer (molar ratio of TRITCDHPE over DMPC: 10-5mol/mol) at three different areas per phospholipid of Alipid ) 57, 65, and 90 Å2. It is well established, however, that single molecule tracking experiments do not provide meaningful results based on the analysis of single tracks.3,17,20 Since a stochastic process (20) Saxton, M. J. Biophys. J. 1997, 72, 1744.
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Figure 6. Twenty MSDs from individual tracks with a total time of 1 s for each track for DMPG monolayers at the air-water interface (left). The MSD averaged over 50 individual tracks (right). Alipid ) 65 Å2.
is observed, sufficient statistics is necessary to interpret the data properly. Rudnick and Gaspari showed, for example, that random walks reveal a surprisingly broad spectrum of different single tracks.21 Thus, a meaningful diffusion coefficient can only be derived after averaging over a sufficient number of single tracks. Such a diffusion coefficient is, furthermore, only well-defined if long-range diffusion is considered with MSD being several orders of magnitude larger than the size of a single molecule. This is different from short-range diffusion in which MSD does not extend beyond a few times the molecular size, which is better described by local fluctuations and molecular movements in regions of momentarily high free volume.3 Since the maximum values of MSD (5-25 µm2) in our experiments are 3-4 orders of magnitude larger than the size of a single phospholipid molecule (∼1 nm2), we can consider long-range diffusion with a well-defined diffusion coefficient. The corresponding averaged data from Figure 5 (right), which represent the average over 50 single tracks (each of 11 frames), indeed, provide a different picture (21) Rudnick, J.; Gaspari, G. Science 1987, 237, 384.
than the distribution of single tracks suggests. In this case, MSD shows a linear relationship as a function of time. This interesting result indicates that the lateral mobility of TRITC-DHPE in a DMPC monolayer is based only on Brownian diffusion without a measurable surface flow. The derived diffusion coefficients are clearly dependent on the area per molecule, Alipid, as the values of D ) 1.61 µm2/s at Alipid ) 57 Å2, D ) 2.61 µm2/s at Alipid ) 65 Å2, and D ) 4.26 µm2/s at Alipid ) 90 Å2 show. Interestingly, Kaes and co-workers could not verify such a dependence from their single molecule imaging experiments on DMPC monolayers using gold-labeled probe molecules (size of the gold labels: 30 and 100 nm).18 While their determined diffusion coefficients are with ∼1 µm2/s in the range of those we obtained (0.4-4.5 µm2/s), our results differ qualitatively. The statistical differences between both experiments may not be responsible for the discrepancy since our data (in contrast to those from Kaes’s group) are qualitatively in agreement to FRAP experiments on phospholipid monolayers at the air-water interface,14,15 even though FRAP averages over a large ensemble of molecules. In addition, recent experiments
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Figure 7. Lateral diffusion coefficient D as a function of area per molecule for DMPC and DMPG monolayers at the airwater interface (A). The dependence of lateral diffusion coefficient D on free area for DMPC and DMPG monolayers at the air-water interface (B). a0 ) 44 Å2.
on phospholipid-lipopolymer monolayers at the airwater interface based on very similar experimental conditions22 agreed qualitatively well with corresponding FRAP experiments on the same system.23 We attribute the discrepancy between Kaes’s data and our results mainly to the different labels used. While the on-off blinking of single fluorophores ensures the employment of single probe molecules, it is much more difficult to confirm that a single gold bead is not conjugated to multiple phospholipid molecules. In the later case, the measured diffusion properties might be distorted. Figure 7 presents the diffusion coefficients of DMPC monolayers measured for different values of Alipid. Each point represents the average of 50 tracks, each of 11 frames. As shown in Figure 7A, the D-Alipid curve derived from DMPC exhibits primarily monotonic increases of diffusion coefficients with increase of Alipid. On the basis of the result in Figure 7A, the diffusion coefficient was evaluated as a function of the area per molecule using the two-dimensional free area model introduced by Sackmann and co-workers (Figure 7B).8,9 The model considers the diffusion of a rigid cylinder with the cross-sectional area, a0, in a two-dimensional layer of a specific viscosity. In this case, the diffusion coefficient is dependent on the free area per cylinder, af ) Alipid - a0, via ln D ∼ 1/af. Following (22) Ke, P. C.; Naumann, C. A. Hindered diffusion in polymer-tethered phospholipid monolayers at the air-water interface. Submitted to Langmuir. (23) Naumann, C. A.; Knoll, W.; Frank, C. W. Hindered diffusion in polymer-tethered membranes: a monolayer study at the air-water interface. Submitted to Biomacromolecules.
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Almeida et al., a0 corresponds to the cross-sectional molecular area of a phospholipid molecule in its solid phase.24,25 The corresponding plot of ln D over 1/af for DMPC in Figure 7B shows that our experimental data for DMPC can be well described by the free-area model, in good agreement with previous FRAP experiments.14,15 DMPG. DMPG, another phospholipid with a similar pressure-area isotherm as DMPC, also shows no phase transition within the observed area range (Figure 3B). The letters A, B, C, D, and E in Figure 3B represent the areas per phospholipid molecule of Alipid ) 50, 57, 65, 90, and 115 Å2, respectively, at which single molecule imaging experiments have been performed. In contrast to the zwitterionic DMPC, DMPG is characterized by a negative net charge. Figure 6 shows the distribution of the mean-square displacement, MSD, over time for single tracks of TRITCDHPE in a DMPG monolayer (molar ratio of TRITCDHPE over DMPG: 10-5mol/mol) at three different areas per phospholipid of Alipid ) 57, 65, and 90 Å2 (left). Qualitatively, the results are identical to our findings on DMPC. While a significant distribution of single tracks can be observed for a given Alipid (left), the averaged values of MSD (averaged over 50 single tracks each of 11 frames) show a linear relationship with respect to the observation time (right), thus showing again Brownian motion without measurable surface flow. The derived diffusion coefficients are again clearly dependent on the area per molecule, Alipid, as the values of D ) 1.75 µm2/s at Alipid ) 57 Å2, D ) 2.1 µm2/s at Alipid ) 65 Å2, and D ) 3.1 µm2/s at Alipid ) 90 Å2 show, thus supporting our results on DMPC. Figure 7A,B reveals interesting differences between the diffusion behavior of DMPC and DMPG. D is smaller for DMPG than for DMPC at large Alipid but larger for DMPC at small Alipid, the values coinciding around Alipid ) 60 Å2. As shown in Figure 7B, the diffusion data obtained for DMPG also fit the free area model. Figure 7B is based on the assumption that the cross-sectional areas of both phospholipids in the solid state, a0, are very similar. The different slopes in Figure 7B are, however, an indication for different effective areas per DMPC and DMPG molecule. In addition, it should be considered that the diffusion of charged TRITC-DHPE probe molecules is likely to depend on the electrostatic properties of the corresponding phospholipid monolayer. To understand the observed differences between DMPC and DMPG, single molecule experiments at different ionic strengths would be necessary. Such measurements are, however, beyond the scope of this publication. Conclusion The technique of single molecule fluorescence imaging has for the first time been used to track the lateral diffusion of TRITC-DHPE probe molecules in phospholipid monolayers of DMPC and DMPG at the air-water interface. Our data indicate that surface flow can be suppressed successfully, and the diffusion behavior of both phospholipids can be described well by the two-dimensional free-area model. The observed qualitative differences between these results and those of Kaes and coworkers indicate that the selection of the probe molecule can be crucial.18 The next step is to apply the technique of single molecule fluorescence imaging to Langmuir films, which show hindered diffusion based on specific membrane (24) Almeida, P. F. F.; Vaz, W. L. C.; Thompson, T. E. Biochemistry 1992, 31, 6739. (25) Almeida, P. F. F.; Vaz, W. L. C.; Thompson, T. E. Biochemistry 1992, 31, 7198.
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heterogeneities. Corresponding experiments on polymertethered phospholipid monolayers are currently in progress. Acknowledgment. The authors thank Mr. John Coffman for assistance with the Langmuir trough experiments. Discussions with Dr. Peter To¨ro¨k (on suppressing the spherical aberration of a water-immersion objective)
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and Dr. Michael Saxton (about the proper interpretation of random walks) are acknowledged. The authors also thank Dr. Josef Kaes for providing their single molecule tracking results on gold-labeled Langmuir films prior to publication. Funding for this work was provided by the Purdue School of Science Indianapolis. LA010116O
