J. Phys. Chem. B 1998, 102, 3791-3797
3791
Direct Observation of DPPC Phase Domain Motion on Mica Surfaces under Conditions of High Relative Humidity Hitoshi Shiku and Robert C. Dunn* Department of Chemistry, UniVersity of Kansas, Lawrence, Kansas 66045 ReceiVed: January 22, 1998; In Final Form: March 11, 1998
Langmuir-Blodgett monolayers of L-R-dipalmitoylphosphatidylcholine (DPPC) doped with 1,1′-dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine perchlorate (diIC18) are studied under conditions of high relative humidity (RH). Monolayers transferred to a freshly cleaved mica surface in the liquid expanded (LE)-liquid condensed (LC) phase coexistence region of the pressure isotherm are studied with tapping-mode atomic force microscopy (TM-AFM), near-field scanning optical microscopy (NSOM), and confocal microscopy. As the RH is increased above 65%, the small LE and LC domains become mobile on the surface and aggregate to form larger domains of like phase. Analysis of the AFM images confirms that this process does not reflect phase transitions in the monolayer at increased RH but is driven by the energetically favorable decrease in the interfacial line energy of the lipid domains upon aggregation. NSOM measurements of the monolayer confirm that the evolution in the monolayer structure at high RH does not involve defect formation, collapse of the film, or removal of lipid. The evolution in monolayer properties at increasing RH is also found to be very sensitive to the composition of the subphase used in the film transfer process, which may be important for practical applications of these films.
Introduction Highly ordered organic films can be transferred from the air/ water interface onto solid supports using the Langmuir-Blodgett (LB) technique.1,2 This process is useful in modifying the surface properties of materials and creating novel new structures at interfaces. As such, supported thin organic films have received a great deal of interest in areas such as optics and microelectronics.1,3 For the majority of these applications, strict requirements are placed on film properties such as stability and the density of defect sites. Therefore, much of the LB film research to date has concentrated on understanding and controlling the microscopic structures present in these films. LB monolayers formed from phospholipids, such as L-Rdipalmitoylphosphatidylcholine (DPPC), are prototypical systems for investigating the structural properties of thin films. Upon compression, DPPC monolayers confined at the air/water interface undergo two-dimensional phase transitions, essentially free from the effects of gravity.4,5 Distinct changes are observed in the pressure isotherm which reflect a reorientation of the lipid film structure on the subphase surface. From the Gibbs’ rule and the degrees of freedom for a pure DPPC film, regions in the pressure isotherm exist where multiple lipid phases are present. These regions are particularly interesting for studying the forces that govern the equilibrium size and shape of lipid phase domains, as well as probing modifications to the phase structure introduced by changes in the environment. The interplay of electrostatic interactions and interfacial line tension energies between the phase domains leads to distinct structural patterns in the monolayer4 that can be monitored using several methods. Fluorescence microscopy studies have proven very informative in these studies and have arguably provided the greatest * Corresponding author.
amount of information concerning the two-dimensional phase partitioning in lipid films.4-7 Small mole percentages of a fluorescent probe molecule are doped into the lipid film, and the resulting regions of high and low fluorescence intensity are used to map the distribution of the lipid phases present. These studies, however, are normally limited in spatial resolution to the micron level and provide little information on the submicron structure present in the film. Interest in film properties and structures on the nanometric level motivated the recent application of high-resolution techniques such as atomic force microscopy (AFM) for LB film research.8-11 Early AFM studies investigating the domain structures in the LC-LE phase coexistence region of stearic acid8 and dipalmitoylphosphatidic acid9 found that over distances greater than several microns the AFM data were in general agreement with previous fluorescence studies. However, at the submicron level, AFM images of the monolayers revealed the presence of small lipid domains less than 100 nm in diameter. Similar small domains have since been found in many LB lipid films,12-15 including those composed of DPPC.10,11 AFM studies have been useful in probing LB film stability, structure, and mechanical properties at the nanometric level, an understanding of which is essential before these films can become incorporated into useful devices.3,16,17 The influence of external conditions on film properties and structures is an important part of this effort. The effects of humidity on the monolayer can be directly measured using AFM, and several reports have appeared investigating LB films under controlled humidity environments.9,8,18 These studies, however, have been mainly concerned with the collapse of lipid monolayers stored under arid conditions or in modifying the lipid layers at high RH using the AFM tip. In this report, we discuss results from confocal, AFM, and NSOM measurements of DPPC monolayers exposed to a controlled relative humidity (RH) environment. These measure-
S1089-5647(98)00833-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/17/1998
3792 J. Phys. Chem. B, Vol. 102, No. 19, 1998 ments provide a dynamical view of the lipid monolayer by directly probing the motions of the lipid phase domains under conditions of increasing RH. The results show that the small LC and LE domains become mobile on the mica surface above a certain critical humidity level and coalesce into larger domains. The relative fraction of LC to LE lipid phase only drops slightly during this process, indicating that the domains are not simply undergoing a phase transition at high RH. The aggregation of like phase domains at high RH effectively reduces the number of boundary lipids between the coexisting LC and LE phases and lowers the energy of the film. The observed aggregation of the small lipid domains at high RH, which mimics the airwater interface, suggests that these small structures are not present in Langmuir films but are introduced into the film by the transfer process. The presence of ions in the subphase during LB film preparation is also found to greatly influence the mobility of the small lipid domains. Both the presence and identity of the ions are important factors in determining the stability of the final supported DPPC film at high RH, which may become important considerations for future practical applications. Experimental Section L-R-Dipalmitoylphosphatidylcholine (DPPC, Sigma) and 1,1′dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (diIC18, Molecular Probes) were used without further purification. Lipid monolayers were transferred onto a freshly cleaved mica surface at room temperature using a computer-controlled Langmuir-Blodgett trough (Nima Technology, Model 611). The LB trough incorporates a Wilhelmy sensor to monitor the surface pressure during the lipid compression and transfer processes. DPPC was dissolved in spectral grade chloroform (1 mg/mL) into which small volumes of a concentrated solution of diIC18 in methanol were added to a concentration of 0.25 mol %. Approximately 50 µL of the DPPC or DPPC/0.25 mol % diIC18 mixture was dispersed onto the subphase and compressed at rate of 100 cm2/min. The films were transferred onto a mica surface at a dipper velocity of 25 mm/min while maintaining constant surface pressure. High-resolution measurements of DPPC films were performed using a custom-designed microscope capable of atomic force microscopy (AFM), near-field scanning optical microscopy (NSOM), and confocal microscopy. The microscope is built on a commercial inverted fluorescence microscope (Zeiss, Axiovert 135TV) equipped with a fluar 40×, 1.3 NA oil immersion objective lens. For NSOM measurements, a cantilevered near-field fiber-optic tip is mounted onto a Dimension AFM head (Digital Instruments) that positions the tip above the microscope objective lens.19,20 A separate x-y closed-loop piezo scanner (Physik Instrumente) raster scans the sample under the NSOM tip during imaging. Excitation from the 514 nm line of an argon ion laser (Liconix 5000) is coupled into the NSOM tip, and sample fluorescence is collected from below with the objective lens. Once the residual excitation light is removed with filters, the sample fluorescence is imaged onto an avalanche photodiode detector (EG&G, SPCM-200). For NSOM compliance measurements, a lock-in amplifier (Stanford Research Systems, SR830) is used to monitor the phase of the NSOM tip resonance.20 For the AFM measurements, a Dimension AFM head (Digital Instruments) is mounted onto the microscope and used in a similar configuration to the Bioscope AFM (Digital Instruments). For imaging LB samples at elevated humidity, tappingmode AFM was utilized. For the controlled humidity studies,
Shiku and Dunn
Figure 1. Pressure isotherm of DPPC/0.25 mol % diIC18 on a subphase of 10 mM MgCl2. Pressure isotherms for monolayers of pure DPPC and with subphases of pure (18 MΩ) water are quantitatively similar. The arrow, located in the liquid expanded (LE)-liquid condensed (LC) coexistence region, marks the pressure at which the monolayers were transferred onto a mica surface.
a custom-designed environmental chamber was used to house the sample. Relative humidity was monitored using a digital hygrometer/thermometer (Fisher Scientific) with a useful operating range of 10-95% ( 0.1% RH. The humidity in the chamber was controlled with a water-saturated nitrogen gas flow, capable of holding the humidity constant (within 1%) for more than 30 min. Results Figure 1 shows the pressure isotherm for DPPC/0.25 mol % diIC18, along with an arrow marking the position on the curve where the DPPC monolayers were transferred onto a freshly cleaved mica surface. Fluorescence images of DPPC monolayers transferred at this pressure are shown in Figure 2 under conditions of normal (A and C, 50% RH) and high relative humidity (RH) (B and D, 95% RH). The subphase used in film formation was 10 mM MgCl2 in (A) and (B) and pure water in (C) and (D). The confocal fluorescence images exhibit the distinctive patterns indicative of the coexisting LC and LE lipid phases.5 The fluorescent diIC18 molecule preferentially partitions into the LE regions, providing a marker for the LE (bright) and LC (dark) lipid phases. Under these conditions, the circular dark islands of LC phase are dispersed in the fluorescently doped LE phase region. With a resolution less than 1 µm, the fluorescence profiles in the LE phase appear homogeneous. Comparing fluorescence images from the same film region under low (Figure 2A) and high (Figure 2B) RH reveals that the large LC islands do not change position or shape but become blurred around the rim of the structure at high RH. The fluorescently doped LE regions, on the other hand, become distinctly more heterogeneous at high RH. Similar experiments, carried out on mica supported LB films transferred from a pure water subphase, however, show different behavior. For these films, the fluorescence distribution present in the LE phase is relatively inhomogeneous at low RH (Figure 2C) and does not visibly evolve at high RH, even when exposed for more than 120 min (Figure 2D). For DPPC monolayers transferred from the 10 mM MgCl2 subphase, the increase in structure observed with confocal microscopy in the LE regions at high RH can be precisely measured with TM-AFM measurements. Figure 3 shows a selected series of high-resolution TM-AFM images, measured on the same region of a DPPC monolayer while the RH was
DPPC Phase Domain Motion on Mica
J. Phys. Chem. B, Vol. 102, No. 19, 1998 3793
Figure 2. Confocal fluorescence images of DPPC/0.25 mol % diIC18 monolayers on mica transferred from a subphase of 10 mM MgCl2 (A, B) and 18 MΩ water (C, D). Patterns indicative of the LE (fluorescent) and LC (dark) coexistence are visible at normal (A and C, 50% RH) and elevated (B and D, 95% RH) relative humidity. For DPPC monolayers transferred from the MgCl2 subphase, a change in film structure is visible at high humdity. For the film transferred from water, no evolution in the film structure is observable.
gently raised. The RH was slowly increased at a rate of 0.35%/ min while the sample was continuously scanned with TM-AFM. Each image takes approximately 2 min to complete, resulting in a RH change during the capture time of less than 1%. The initial frame (Figure 3A, RH ) 55%) of the series shows that the DPPC monolayer consists of large LC islands (>5 µm) surrounded by a coexistence region of smaller LC and LE islands. The small LC and LE domains observed in the “LE” regions of the monolayer, which appear homogeneous in the lower resolution confocal fluorescence measurements, have been seen previously in both AFM8-11 and NSOM21 studies. A height difference of 1.2 ( 0.5 nm is observed in Figure 3, between the upright LC phase and tilted LE. As the RH value is increased above 65%, the small domains in the monolayer become mobile. The motion of the small domains is random, but clearly each LC or LE grain aggregates to their own phase, presumably to decrease the interfacial line energy. At a RH greater than 85% (Figure 3G,H), the domain mobility on the mica surface decreases as the domains reach a critical size. As evident in Figure 3, while the smaller domains are very dynamic, the larger LC domains remain stable, except
for small changes that occur near the domain boarders. These experiments were carried out on pure DPPC monolayers, though we find no evidence for perturbation of the film structure by incorporation of 0.25 mol % diIC18 dye. Experiments on dyedoped films show that the structures present and dynamic behavior of the film at high RH remain the same as that shown in Figure 3. To confirm that the evolution in the film structure is due to the aggregation of lipid domains and not simply a result of defect formation, high-resolution NSOM measurements were made at low and high RH. Figure 4 shows the NSOM (A) fluorescence, (B) height, and (C) compliance images of a DPPC/0.25 mol % diIC18 monolayer exposed to a humid atmosphere (KBr-saturated solution, 84% RH) for 30 min. The high resolution in the NSOM fluorescence image is able to resolve the small structures present in the “LE” regions of the monolayer. These structures can be compared with the simultaneous topographic image shown in Figure 4B, which is sensitive to the small height differences associated with the LC and LE domains, similar to the AFM results. The measured height difference in Figure 4B between the lipid phases is 0.8 ( 0.3 nm, comparable to that
3794 J. Phys. Chem. B, Vol. 102, No. 19, 1998
Shiku and Dunn
Figure 3. Sequential 10 µm × 10 µm tapping-mode AFM images of a DPPC (no diIC18) monolayer on mica observed while the relative humidity is gently increased. The film was transferred from a 10 mM MgCl2 subphase, and the RH was increased from 50 to 95% at a rate of 0.35%/min. The images correspond to RH values of (A) 55%, (B) 62%, (C) 70% (D) 73%, (E) 75%, (F) 78%, (G) 86%, and (H) 91%. Each image takes approximately 2 min to complete, resulting in a RH change during the capture time of less than 1%.
seen in the AFM results. Thus, comparing parts A and B of Figure 4, the brighter LE regions in the fluorescence image correlate directly with the lower height LE regions in the topo-
graphic image. The compliance image shown if Figure 4C is measured by monitoring the phase of the NSOM tip resonance.20 This imaging mode responds to the compliance differences
DPPC Phase Domain Motion on Mica
J. Phys. Chem. B, Vol. 102, No. 19, 1998 3795 Discussion
Figure 4. The 18 µm × 18 µm tapping-mode NSOM (top) fluorescence, (middle) height, and (bottom) compliance images of a DPPC/ 0.25 mol % diIC18 monolayer exposed to a humid atmosphere (KBrsaturated solution, 84% RH) for 30 min.
between the two lipid phases and is sensitive to defects or contamination in the film which may not be visible in the height image. The simultaneous collection and comparison of all three of these contrast modes allows for the unambiguous identification of the LC and LE phase domains in the film. As in the AFM experiments, the small LE and LC domains seen in Figure 4 are formed from the aggregation of smaller domains present at low RH. Comparison of images at low and high RH shows that the monolayers are not simply forming defects in the film or collapsing into multilayer structures but are, instead, evolving in response to changes in the RH.
The series of AFM images shown in Figure 3 clearly show the increased mobility of the small lipid domains in the DPPC monolayer under conditions of high humidity. Both the AFM and NSOM results show that this motion is not due to a degradation in the film integrity, removal of lipid from the monolayer, or collapse into multilayer structures. This is evidenced by the conservation in the
