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Direct Observation of Structural Evolution in Palmitic Acid Monolayers following Langmuir-Blodgett Deposition Sarah A. Vickery and Robert C. Dunn* Department of Chemistry, University of Kansas, Malott Hall, Lawrence, Kansas 66045 Received June 18, 2001. In Final Form: August 31, 2001 Confocal microscopy, atomic force microscopy, and near-field scanning optical microscopy are utilized to study the evolution in structure of palmitic acid monolayers transferred onto mica using the LangmuirBlodgett technique. Monolayers transferred at a surface pressure of 1.5 mN/m contain condensed islands surrounded by expanded regions of the film. The densed-branch morphology of the condensed domains indicates a nonequilibrium growth mechanism driven by diffusion limited aggregation. Immediately following film transfer, significant evolution in the film structure is observed as palmitic acid in the expanded regions of the film diffuses toward and combines with the condensed domains. The data from the complementary high-resolution techniques suggest a mechanism for domain growth dominated by dewetting following film transfer.
Introduction Langmuir-Blodgett (LB) films of lipids or fatty acids have long been utilized as simple models for biological membranes.1-4 These studies have focused on the twodimensional structures formed under various external influences or the effects that additives such as cholesterol or small peptides have on film properties.5,6 Mounting evidence, however, suggests that the deposition process itself can play a significant role in influencing the final film structure.7-11 Understanding these processes at the molecular level is of interest not only for interpreting the results from LB studies but in gaining new insights into the forces that drive domain formation in these supported two-dimensional films. Several studies have shown that surface-mediated condensation can influence film structure in LB films transferred onto substrates.8,9,12 Direct observations at the three-phase line where the substrate is pulled through the film layer on the subphase have unambiguously revealed a phase transition in monolayers of DPPC and DMPE during the dipping process.10,13 Following transfer, other processes can also affect the final film structure and phase partitioning observed in LB films. For example, subphase drainage from the monolayer following transfer, dehydration, and changes in the relative humidity have all been shown to influence the final film structure in many LB films.14-16 * Corresponding author. (1) Gennis, R. B. Biomembranes: Molecular Structure and Function; Springer-Verlag: New York, 1989. (2) McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171-195. (3) Mo¨hwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441-476. (4) Gaines, G. L. Insoluble Monolayers at Gas Liquid Interfaces; Interscience: New York, 1966. (5) Lipp, M. M.; Lee, K. Y. C.; Waring, A.; Zasadzinski, J. A. Biophys. J. 1997, 72, 2783-2804. (6) Hwang, J.; Tamm, L. K.; Bohm, C.; Ramalingam, T.; Betzig, E.; Edidin, M. Science 1995, 270, 610-614. (7) Rana, F. R.; Widayati, S.; Gregory, B. W.; Dluhy, R. A. Appl. Spectrosc. 1994, 48, 1196-1203. (8) Sikes, H. D.; Schwartz, D. K. Langmuir 1997, 13, 4704-4709. (9) Sikes, H. D.; Woodward, J. T.; Schwartz, D. K. J. Phys. Chem. 1996, 100, 9093-9097. (10) Riegler, H.; Spratte, K. Thin Solid Films 1992, 210-211, 9-12. (11) Riegler, J. E.; LeGrange, J. D. Phys. Rev. Lett. 1988, 61, 24922495. (12) Shiku, H.; Dunn, R. C. J. Microsc. 1999, 194, 455-460. (13) Spratte, K.; Riegler, H. Langmuir 1994, 10, 3161-3173.
In the present study, several high-resolution techniques are utilized to directly follow changes in film structure following transfer. By direct observation of the structural evolution, this work complements previous studies that have found indirect evidence for such changes.8-10,12-16 While several mechanisms can be envisioned that would lead to the structural changes, we suggest that the data is most consistent with a mechanism driven by dewetting. Research over the past decade has significantly advanced our understanding of the mechanisms involved in dewetting, although it remains less understood than the more studied process of substrate wetting. Theoretical and experimental investigations have shown that thin films can dewet through the formation and nucleation of holes or as a result of instabilities arising from thermally activated surface waves. The latter, known as spinodal decomposition, is characterized by the generation of surface waves with a wavelength proportional to the square of the film thickness. These surface waves grow exponentially until the amplitude is such that they contact the substrate, presumably starting the dewetting process.14,15,17 In this study we follow the evolution in structure of palmitic acid monolayers immediately following LB deposition onto mica substrates. These structural changes appear to be associated with a dewetting process that drives diffusion-limited aggregation of PA to condensed regions of the film, thus depleting the expanded regions initially present following film transfer and increasing the size of the condensed PA domains. Several high-resolution techniques are utilized to follow the structural evolution. Progressive confocal fluorescence microscopy measurements show a large migration over time in the location of the fluorescent marker added into the film. Complementary high-resolution AFM measurements of the sample topography confirm that at least some of this motion arises from the bulk diffusion of PA on the mica surface. Immediately following transfer of PA onto mica, lipid in the expanded regions of the film can diffuse on the surface until nucleating at the condensed islands. (14) Elbaum, M.; Lipson, S. G. Phys. Rev. Lett. 1994, 72, 3562-3565. (15) Reiter, G. Phys. Rev. Lett. 1992, 68, 75-78. (16) Shiku, H.; Dunn, R. C. J. Phys. Chem. B 1998, 102, 3791-3797. (17) Bischof, J.; Scherer, S.; Herminghaus, S.; Leiderer, P. Phys. Rev. Lett. 1996, 77, 1536-1539.
10.1021/la010906y CCC: $20.00 © 2001 American Chemical Society Published on Web 11/09/2001
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These processes result in the self-similar growth of fractallike domains with dimensions of 1.45. Finally, simultaneous near-field fluorescence and topography images reveal that an evolution in the fluorescence signal continues even after domain growth terminates. Experimental Section Palmitic acid (Sigma Chemical Co., St. Louis, MO; >99% pure)and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (diIC18, Molecular Probes, Inc., Eugene, OR) were used without further purification. Working solutions were prepared by adding concentrated diIC18 in methanol to a stock solution of PA dissolved in spectral grade chloroform (1 mg/mL) to a final concentration of 0.25 mol %. A small amount of the PA/0.25 mol % diIC18 mixture was dispersed onto a buffered subphase composed of 0.15 M NaCl and 2 mM NaHCO3 (pH ) 6.9). Lipid monolayers were transferred at room temperature onto cleaved mica surfaces using a computer-controlled Langmuir-Blodgett trough (Nima Technology, model 611). Surface pressure during lipid compression and transfer was monitored with a Wilhelmy sensor. The films were compressed at a rate of 100 cm2/min and transferred at a dipper velocity of 24 mm/min while maintaining constant surface pressure. A custom-designed, high-resolution microscope capable of confocal microscopy, atomic force microscopy (AFM), and nearfield scanning optical microscopy (NSOM) was utilized to perform all measurements of PA films. The microscope is mounted on a commercial inverted fluorescence microscope (Zeiss, Axiovert 135TV) equipped with a fluar 40×, 1.3 NA oil immersion objective lens. All the NSOM data were collected in active feedback while the tip was maintained at a constant force. Confocal measurements were carried out by attaching samples to a closed-loop x-y piezo scanner (Mad City Labs) mounted on the inverted microscope. The sample was excited by epi-illumination from the 514 nm line of an argon ion laser (Liconix 5000), and sample fluorescence was collected from below with the objective lens. After residual excitation light was removed with filters, sample fluorescence was imaged onto an avalanche photodiode detector (EG&G, SPCM-200). AFM measurements were performed in contact mode with a Dimension AFM head (Digital Instruments) that is mounted onto the microscope and used in a configuration similar to the Bioscope AFM (Digital Instruments). For NSOM measurements, a cantilevered near-field fiber-optic tip is mounted into a Dimension AFM head that positions the tip above the microscope objective lens. As the sample is raster scanned by the piezo scanner, it is excited by 514 nm light coupled into the NSOM probe. All sample scanning and data collection is controlled using a Digital Instruments Nanoscope IIIIa controller.
Results Figure 1 shows a series of 50 µm × 50 µm confocal fluorescence images of the same area of a PA monolayer doped with 0.25 mol % of the fluorescent membrane probe diIC18. The film was transferred onto a freshly cleaved mica surface from the buffered subphase at a surface pressure of 1.5 mN/m. At this pressure, the PA monolayer contains a mixture of less ordered liquid expanded (LE) phase and more ordered liquid condensed (LC) phase domains. These domains clearly can be seen in the confocal fluorescence image shown in Figure 1A. The fluorescent diIC18 partitions into the less ordered LE domains which appear bright in the confocal image while the more ordered LC domains exclude the dye and remain dark. Interestingly, fluorescence images taken as a function of time, shown in Figure 1, exhibit an evolution in the location of the fluorescent marker added into the PA monolayers. The irregular, dendritic LC domains present in these images are characteristic of the majority of films transferred under these conditions. The fluorescent dye is initially present in the LE phase and over time moves
Figure 1. 50 µm × 50 µm confocal fluorescence images of the same area of a PA monolayer doped with 0.25 mol % diIC18. The film was transferred from a buffered subphase onto mica at a pressure of 1.5 mN/m. The series of images taken (A) 7, (B) 18, and (C) 29 min, respectively, following LB deposition onto mica reveals an evolution in the location of the fluorescent dopant. The migration of the fluorescent dye toward the dark, dendritic condensed domains in the film is observed. Films transferred under these conditions demonstrated complete evolution in film properties within approximately 30 min.
into the LC domains. Images A, B, and C of Figure 1 were collected at times of 7, 18, and 29 min, respectively, following transfer onto the substrate. The times reported are the times at which the data acquisition was initiated.
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For all of the high-resolution techniques employed, each image took approximately 2 min to acquire. The complete evolution in film properties within approximately 30 min was observed in the majority of the samples studied. The images displayed in panels A, B, and C of Figure 2 demonstrate the effect of small amounts of cholesterol added into the PA monolayers. These images were recorded on the same area of a PA/0.025 mol % cholesterol monolayer at times of 12, 43, and 80 min, respectively, following transfer of the monolayer onto the mica substrate. In Figure 2A, the dye is completely excluded from the LC regions of the PA monolayer which appear as dark semicircular domains. Over time, however, an evolution in the dye marker location is observed as illustrated in panels B and C of Figure 2. Figure 2B, taken 43 min following film transfer, shows the initial stages of dye migration where the diIC18 has accumulated around the edges of the LC domains. By 80 min, Figure 2C shows that the migration process is complete and the dye has incorporated itself into the LC regions of the film. To gain further insight into the possible structural changes taking place in the PA monolayers, high-resolution AFM measurements were carried out as a function of time. These measurements are sensitive to the small height difference between the less ordered LE phase and the higher topography LC domains present in the PA monolayers. Figure 3 shows a series of AFM images taken in the same region of a PA monolayer transferred onto mica at a pressure of 1.5 mN/m. The images displayed in Figure 3A-D were collected 5, 7, 16, and 33 min, respectively, following film transfer. Highly branched LC domains of higher topography are evident in the AFM images which are consistent with the initially dark, dye excluding regions seen in Figure 1A. The height differences measured in Figure 3 between the LE and LC domains do not vary significantly from the initial image. Close examination of the AFM images, however, does establish that the area of the LC domains increases over time. Qualitatively, this suggests that the evolution seen in both the confocal and AFM images reflects the bulk diffusion of PA from the expanded regions to the condensed domains upon transfer of the film onto the mica surface. To investigate this process quantitatively, the AFM images were subjected to a height threshold condition to calculate the area occupied by the LC domains in these two level films. The resulting images, shown in Figure 3E-H, are easily analyzed to calculate the small changes in LC area as a function of time. The total increase in LC area as a function of time is plotted as a log-log plot in Figure 4, with a fit showing the trend in the data. The results from this analysis indicate that the area occupied by the LC domains in the initial AFM image taken 5 min following film transfer corresponds to 36.7% of the film area. This increases significantly to 49.8% of the film area in the final AFM image after which no further evolution in the film was detected. This percentage change in LC coverage corresponds to a total increase in LC area of 9.7 µm2. The results presented in Figures 1 and 3 illustrate the utility of both the optical and topographical signatures to track the evolution in the PA phase structure. To provide a direct comparison between these markers of film structure, near-field scanning optical microscopy (NSOM) was utilized.18,19 Panels A and B of Figure 5 display the simultaneously collected near-field fluorescence and (18) Dunn, R. C. Chem. Rev. 1999, 99, 2891-2927. (19) Hollars, C. W.; Dunn, R. C. Biophys. J. 1998, 75, 342-353.
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Figure 2. 45 µm × 45 µm confocal fluorescence images demonstrating the effects of a small amount of cholesterol added to PA monolayers doped with 0.25 mol % diIC18. The film was transferred from a buffered subphase onto mica at a pressure of 1.5 mN/m. With the addition of 0.025 mol % cholesterol, the condensed domains appear less branched than the pure PA films. Again, an evolution in the location of the dye marker can be observed clearly in this series of images taken (A) 12, (B) 43, and (C) 80 min, following LB deposition.
topography images from a PA/0.25 mol % diIC18 film transferred at a surface pressure of 1.5 mN/m. The 30 µm × 30 µm images shown in panels A and B of Figure 5 were measured 7 min following film transfer. Due to the more time-consuming alignment needed for NSOM and the
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Figure 4. log-lot plot of LC area as a function of time for the binary images displayed in panels E-H.
near-field fluorescence images reveal that the fluorescent probe location remains mobile inside the condensed domains. This suggests that the dye can continue to diffuse within the condensed domains even once the bulk flow of material toward the domains terminates. Discussion
Figure 3. Time series of AFM measurements taken in the same 37 µm × 37 µm region of a PA monolayer transferred onto mica from a buffered subphase at a pressure of 1.5 mN/m. The images were collected (A) 5, (B) 7, (C) 16, and (D) 33 min following LB deposition. The condensed domains, which appear bright in the height image (5-8 Å), grow as a function of time as PA molecules in the surrounding expanded regions of the film are transported to the condensed islands. To quantify this process, images A-D were subjected to a height threshold condition resulting in the binary images displayed in E-H, respectively. The resulting images were analyzed to quantify the increase in LC area over time.
rapid nature of the lipid migration, it was not possible to capture an image while the dye was still completely dispersed within the LE phase. Panels C and D of Figure 5 show the results from near-field measurements taken in the same area of the film 15 min following film transfer. Analysis of the topography images reveals no discernible changes in the area of the LC domains. In contrast, the
Many studies have found evidence for structural evolution in LB films during and following film transfer onto substrates.8-11,16 The measurements presented here using several complementary high-resolution microscopy techniques provide direct evidence for a large time-dependent structural change in PA monolayers transferred onto mica near lift-off on the pressure isotherm. These changes are evidenced by the striking migration of the fluorescent marker, diIC18, into the condensed PA domains observed with both confocal and NSOM measurements and through the direct observation with AFM of domain growth as a function of time following deposition. The confocal images displayed in Figures 1 and 2 clearly show the structural evolution in the fatty acid film immediately after deposition. Previous reports have shown that PA films transferred in the LE region often condense to form close-packed islands.8,9 This process is intimately linked to substrate and subphase conditions which affect the surface charge.16,20-22 The fluorescent marker, which partitions into the less-ordered LE phase, originally surrounds the condensed islands formed upon transfer. The series of confocal images shown in Figures 1 and 2 presumably reflects the movement of PA from the LE regions toward the condensed LC islands, thus depleting the expanded regions of the monolayer. This process continues until the expanded regions are either depleted of PA or the process is quenched through other mechanisms such as loss of the hydration layer. The AFM measurements shown in Figure 3 confirm that the evolution observed in the confocal measurements involves the diffusion of both the dye marker and the PA molecules. A significant increase in the area of the condensed domains is directly observed as a function of time in these measurements. Moreover, the binary nature of the height difference between the LE phase and more (20) Israelachvili, J. Intermolecular and Surface Forces, 2nd ed.; Academic Press Inc.: San Diego, CA, 1992. (21) Pashley, R. M. J. Colloid Interface Sci. 1981, 80, 153-162. (22) Pashley, R. M.; Israelachvili, J. N. J. Colloid Interface Sci. 1984, 97, 446-455.
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Figure 5. 30 µm × 30 µm NSOM images of a PA film doped with 0.25 mol % diIC18 and transferred onto a mica substrate from a buffered subphase at a pressure of 1.5 mN/m. Simultaneously collected (A) fluorescence and (B) topography images were measured 7 min following LB deposition. Near-field fluorescence and topography images of the film 15 min after LB deposition are shown in (C) and (D), respectively. As in the AFM data, the condensed domains in the force images, (B) and (D), are 5-8 Å taller than the surroundings and appear light in the gray scale images. Changes in the location of the fluorescent marker are observed in the fluorescence images, while the simultaneously collected near-field topography images reveal little or no changes in the size or shape of the LC domains. This indicates that the fluorescent dye and probably the PA molecules remain mobile in the LC islands even after the bulk flow of material to the islands terminates.
upright LC phase observed in the AFM data provides a straightforward route for calculating the changes in area of these domains. With the images subjected to a thresholding condition, they can be converted to truly binary height images that are easily analyzed to calculate the LC area. The results from this analysis indicate that for the 37 µm × 37 µm images shown in Figure 3, the area of the condensed islands increased 9.7 µm2 over the time period studied. The molecular area for PA decreases from 40 Å2/molecule to 20 Å2/molecule when transitioning from the less-ordered LE phase to the more ordered LC phase, respectively. Assuming that the areas surrounding the LC islands observed in Figure 3 are initially homogeneously covered with PA in the LE phase and that the evolution observed in Figure 3 goes to completion, the calculated total increase in LC area should be 11.6 µm2. The lower measured value of 9.7 µm2 undoubtedly reflects the loss of initial migration information due to the experimental time constraints associated with transferring the sample to the AFM. The
lower value may also indicate a quenching of the diffusion by other mechanisms at later times. Regardless, it is clear from these measurements that there is a substantial change in the domain structure following film deposition. The condensed PA domains exhibit a dendritic or densed-branch morphology that is reflective of nonequilibrium growth processes. This can be seen most clearly in the higher resolution AFM measurements shown in Figure 3. Significant branching and self-similar growth is observed in the domains, which is consistent with a diffusion limited aggregation mechanism for domain growth. Interestingly, analysis of the fractal dimension of the domains using the dilation method indicates a dimension of approximately 1.45, which is somewhat lower than the 1.67 expected for diffusion limited aggregation. However, slightly lower fractal dimensions than those expected for diffusion limited aggregation have been found previously in similar studies on lipid monolayers of
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DMPE.23 We also find that the fractal dimension remains approximately constant throughout the domain growth process. We have previously shown that monolayers of DPPC can become mobile on mica substrates above a certain critical relative humidity threshold.16 This was attributed to a hydration force that weakens the interactions between the lipid molecules and the mica substrate.21,22 The result was the homogeneous coalescence of smaller condensed domains to form the energetically more favorable larger domains, without any noticeable change in the relative areas occupied by the expanded and condensed phases.16 In contrast, the results reported here suggest that rather than a simple diffusive random walk, the PA molecules loosely oriented in areas surrounding the condensed islands are driven to coalesce by factors such as line tension gradients and dewetting. Previous reports have shown that LB films transferred in the LE phase can condense on the surface to form densely packed islands. The resulting phase coexistence can create a surface tension gradient at the phase domain boundaries that pulls the remaining expanded phase molecules toward the condensed islands, leaving the surrounding substrate bare.8,9 While this gradient may contribute to the lipid diffusion seen here, dewetting is likely to play the dominate role in the evolution in film structure observed. Dewetting or drainage of the aqueous layer trapped between the lipid film and solid substrate following LB deposition has been observed in many studies to affect the final film structure. The bulk hydrodynamics involved in drainage of the subphase often lead to characteristic stripe structures in the resulting film.10,12 These structures are not observed in our data, which suggests that this is not the dominant mechanism involved. Moreover, the perturbation to film structure caused by drainage of the subphase occurs during film dipping and not over the long time periods observed here. Dewetting, however, would be expected to occur over extended periods of time and is consistent with the island growth observed in the AFM data. Our data suggest that holes exposing the mica substrate nucleate in the expanded regions of the film following film transfer. On the basis of previous studies, these dry regions are expected to grow radially carrying the lipid material on the rims of the expanding hole, leaving the remaining substrate bare.8 In studies of thin water films absorbed on mica, these holes grow homogeneously until instabilities in the expanding rim of the ring cause breakup and the formation of a succession of droplets on the surface.14 In our system, the expanding holes carrying the lipid material on the toroidal rim encounter the condensed domains before the instabilities initiate the collapse of the rim. The condensed domains, being less compressible and more stationary on the mica substrate, act as an effective barrier to the growth of the dry domains. These condensed lipid domains, therefore, grow as PA molecules are transported to them by the growing dry regions. The homogeneous formation of nucleation sites (23) Miller, A.; Knoll, W.; Mo¨hwald, H. Phys. Rev. Lett. 1986, 56, 2633-2636.
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for holes in the expanded regions of the film results in the uniform growth of the condensed islands observed in the AFM data. All the domains observed in the AFM measurements grow homogeneously approximately 10% during the course of the aggregation process. It is interesting to note that the NSOM measurements reveal continued diffusion of the dye marker, and presumably PA molecules, within the condensed domains even after the bulk diffusion from the expanded regions is quenched. This is evidenced in the NSOM data by changes in the fluorescence distribution within the condensed domains that persist well beyond the time required for domain growth as measured by the simultaneously collected topography data. From this we can conclude that the lipids, at least in the condensed regions, retain diffusional freedom on the surface for time periods longer than that associated with domain growth. This probably reflects the retention of the hydration layer under the condensed islands, which has been shown previously to strongly effect the mobility of the lipids on mica substrates.16,21 Conclusion Confocal microscopy, atomic force microscopy, and nearfield microscopy are utilized to study the evolution in structure of PA monolayers transferred onto mica substrates using the LB technique. Time-resolved confocal microscopy measurements reveal a significant migration of the fluorescent membrane probe diIC18 from the expanded regions of the monolayers to the condensed islands. High-resolution AFM measurements confirm that this diffusion involves the bulk movement of both the dye marker and the PA lipids toward the condensed islands where the molecules fuse and incorporate into the domains. Analysis of time-resolved AFM images indicates that most of the condensed island growth can be accounted for by assuming a homogeneous coverage of PA in the expanded regions. The densed-branch morphology of the condensed domains indicates a nonequilibrium growth mechanism driven by diffusion-limited aggregation. The fractal dimension of the condensed domains, calculated using the dilation method, is found to be 1.45 and is invariant with domain size or stage of domain growth. Simultaneously collected near-field fluorescence and topography images reveal that the dye marker, and presumably the PA molecules, retain diffusional freedom within the condensed islands even once the bulk diffusion of PA toward the islands ceases. Although a number of mechanisms may be active in this system, the data from the complementary high-resolution techniques suggest a mechanism for domain growth dominated by dewetting following film transfer. Within this mechanism, holes exposing the mica substrate nucleate in the expanded regions of the film and expand carrying lipid to the condensed islands. Acknowledgment. S.A.V. gratefully acknowledges support from the University of Kansas Madison and Lila Self Graduate Fellowship. Support for this work was generously provided by NSF (CHE-9982052) and the Alfred P. Sloan Foundation. LA010906Y
