Langmuir 1992,8, 2509-2514
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Fluorescence Microscopy Study of Langmuir Monolayers of Stearylamine Keith J. Stine' and David T. Stratmann Department of Chemistry, University of Missouri-St. Louis, St. Louis, Missouri 63121 Received February 26, 1992. In Final Form: July 6, 1992
The liquid-expanded (LE) to liquid-condensed (LC) coexistence region of monolayers of stearylamine deposited on water acidified to pH 2.5 with HC1 has been observed using fluorescence microscopy and surface pressure isotherm measurements. Protonation of the amine headgroup on acidificationwith HC1 shifts the LE-LC region down more than 30 OC in temperature, widens the coexistence region, and leads to the observation of numerous shape transitions of the LC phase domains. The LC domain shapes become progressively less compact on compressions at a seriesof increasing temperatures. Image analysis of the domain shapes at two temperatures is used to obtain a critical domain radius for transition to a noncircular shape. The LEkLC line tension is estimated from the observed critical radius. Introduction In recent years, the technique of fluorescence micro~ c o p y l -has ~ enabled studies of the morphologies of coexisting phases in Langmuir monolayers. Domain shapes have been generally understood in terms of the opposing tendencies of the line tension between coexisting surface phases favoring compact shapes, and repulsive electrostatic forces favoring extended Coupling between domain size andamphiphile tilt has been observed by fluorescence anisotropy e~periments.~ Amphiphile chain tilt is favored by a mismatch between headgroup size and chain profile.8 The experimental work of McConnell and co-workers4and of Mohwald and co-workersg has focused on the liquid-expanded (LE) to liquidcondensed (LC) phase transition in phospholipids. The recent work of Seul and co-workers on phospholipid + cholesterol mixtures has focused on analysis of domain shape transitions and dynamics in a system with monolayer liquid-liquid Monolayers of long-chain amines have long been known to undergo substantial expansion on acidification of the subphase and the resultant protonation of the headgroup, with the effect dependent upon the particular anion in the subphase.13J4 The large shifts in the phase diagram and the substantial increase in surface potential15 on acidification of the subphase suggested that monolayers of amine salts would be interesting systems in which to observe LC domains using fluorescence microscopy. We
report observations of LC domain structure in the LE-LC coexistence region of stearylamine deposited on water acidified to pH 2.5 with hydrochloric acid. In choosing to study monolayers of long-chain amines on acidified subphases, our goal was to use a simple single-chain surfactant salt as a model system to study the dependence of domain structure on subphase conditions. Stearylamiie monolayers on an HC1-acidified subphase are found to exhibit a variety of LC domain shapes dependent on temperature, and this paper reports our basic findings on this system.
2186. (9) MBhwald, H. In Annual Reviews of Physical Chemistry; Straws,
Experimental Section 1. Isotherm Measurements. Isotherms were measured in a home-built Langmuirtrough constructed of Teflon with a water surface of 10.2 cm X 29.7 cm enclosed in a Plexiglas housing with access holes for aspiration of the water surface and deposition of the spreading solution. Temperature was controlled by circulatingwater through coppertubing welded to a '/kin. copper plate beneath the Teflon and was measured by a fine thermocouple in a glass sheath inserted into the side of the trough positioned 3 mm beneath the water surface. Thesurfacepressure was measured by a Cahn Model 25 electrobalance using filter paper as a Wilhelmy plate. The BCD output from the Cahn electrobalance was read by a PCL-720 32-bit digital I/O and counter card (B&CMicrosystems)in a 386 computer. A Teflon barrier on a threaded rod attached to a universal joint was driven by a stepping motor using a home-built circuit such that the area covered by the monolayer could be varied at rates of 2.0-50 cm2 min-1 in 0.01 cm* steps. The force on the Wilhelmy plate was recorded every 0.25 s by a BASIC program, and the average of every 15 readings was saved. The force vs time data were converted to surface pressure vs area per molecule after the completion of each timed run. The initial surface pressure built up on deposition of the film molecules was measured immediately prior to a run. 2. Materials. Stearylaminewas purchased from Sigma (9996 pure) and used without further purification. Solutions in 9 1 benzene/methanol, the solvent system used by Hoffman," were prepared of concentration (3-6) X lo1' moleculeslpl, and monolayers were formed by deposition onto the water surface from a 25-pL microsyringe. The solutions for the microscopy observations contained 0.3 mol % of the fluorescent probe
62,784. (13) Adam, N. K. Roc. R. SOC.London, A 1930,126, 526. (14) Hoffman, E. J.; Boyd, G.E.; Ralston, A. W. J. Am. Chem. SOC. 1942,64,498. (16) Betta, J. J.; Pethica, B. A. Trans.Faraday SOC.1956,52, 1581.
4-(hexadecylamino)-7-nitrobenz-2-oxa-1,3-diazole (Molecular Probes). Millipore water was acidified to pH 2.5 using highpurity HCl from Aldrich. 3. Fluorescence Microscopy. A home-built Langmuir trough with a water surface of 7.8 cm X 13.9 cm was fitted onto the stage of an Olympus BH-2 microscopewith an epifluorescence attachment. Temperature could be controlled over the range 5-65 "C and was measured by a fine thermocouple in a Teflon
(1) von TschaPner, V.;
McConnell, H. M. Biophys. J. 1981, 36, 409.
(2) I.&che, M.; Sackmann, E.; MBhwald, H. Ber. Bunsen-Ges. Phys.
Chem. 1983,87,848. (3) Peters, R.; Beck, K. Proc. Natl. Acad. Sci. U.S.A. 1983,80,7183. (4) McConnell, H. M. In Annual Reuiews of Physical Chemistry; Straw, H. L., Babcock, G. T., Leone, S.R., Eds.; Annual Reviews Inc: Palo Alto,CA, 1991; Vol. 42, p 171. (6) Andelman, D.; Brochard,F.; Joanny, J. F. J. Chem. Phys. 1987,86, 3673. (6) Vanderlick, T. K.; MBhwald, H. J. Phys. Chem. 1990, 94, 886.
(7) Moy, V. T.; Keller, D. J.; Gaub, H. E.; McConnell, H. M. J. Phys. Chem. 1986,90,3198. (8)Satran, S. A.; Robbins, M. O., Garoff, S. Phys. Reo. A 1986, 33,
H. L., Babcock, G. T., Moore, C. B., Eds.;Annual Reviews Inc.: Palo Alto, CA, 1990, Vol. 41, p 441. (10) Seul, M. Physica A 1990,168,198. (11) Sed, M.; Sammon, M. J. Phys. Reu. Lett. 1990, 64, 1903. (12) Seul, M.; Sammon, M. J.; Monar, L. R. Rev. Sci. Instrum. 1991,
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Stine and Stratmann
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Figure 1. Gradient image of image D from Figure 5 formed by the method used in the treatment of individual domains for boundary length determination. sheath positioned just below the water surface. Compression was achieved by manually moving a Teflon barrier with a notch which held a glass cover in place over the monolayer. The top of the trough coverglass (Delta Technologies)had a transparent conductive coating and could be slightly heated by a Variac to burn off condensation. The monolayer was observed through a 20X or 40X ultralong working distance objective using a DageMTI SIT-68 camera and a Dage-MTI HR-1000 monitor. Fluorescence excitation was provided by a mercury lamp or by a Omnichrome 150-mW multiline multimode argon ion laser. 4. Image Analysis. Image analysis was carried out using a DT2851 frame grabber board, DT2858 auxilliaryframe processor board, and DT-IRIS subroutine library (Data Translation) in a 386 computer. Image analysis programs were written in Fortran. The image analysis work of Seul12on phospholipid + cholesterol mixtures has utilized these two boards. Binary images were constructed from each captured image. The image was first convertedto a checkerboard pattern of 24 X 25 pixel boxes where the 600 pixels in each box were set equal to the average pixel intensity in the box. The resulting checkerboard pattern was then convolved three times with a 15 X 15 low-pass filter to form the backgroundframe. The background file was then subtracted from the original image file offset by the average intensity of the background. The binary image was then created by choosing the value for the cutoff pixel intensity which gave the best binary representation of the original image; pixels above the cutoff were set to 50, and those beneath the cutoff were set to 0. This procedure gave binary images much superior to those obtained by using the original image smoothed with a 30 X 30 low-pass fiiter as the background. The pixels comprising a selecteddomain were identified by using an initial point inside the domain as input to a homemade region growing algorithm. The number of pixels in the region was converted to domain area. The boundary pixels of the domainwere determined by applyinga 3 X 3 gradient filter to the image and deleting those pixels from the gradient image which by their value were identified as noise along the boundary. As an example, the gradient image obtained from image D of Figure 5 is shown in Figure 1. The remaining pixels were counted and converted to boundary length. For some of the binary images, the total number of edge pixels and total number of pixels in domains were measured. The number of domains in the image was then manually counted on the screen.
Results 1. Isotherms. In Figure 2,surface pressure vs area per molecule isotherms of stearylamine on water acidified to pH 2.5 with HC1 are presented. The isotherms on the acidified water show kinks signifyingthe LE-LC transition at areas similar to those observed by Hoffman.14 The isotherm at 25.0 "C on unacidified water in Figure 3 shows no LE-LC transition. The lack of an LE-LC transition
Figure 2. Surface pressure vs area per molecule isotherms measured for stearylamine monolayers on water acidified to pH 2.5 with HC1 at T = 19.8, 23.0, 26.0, 29.0, and 32.1 "C. The average compression rate used was 6 A2 molecule-' mi+. At 32.1 "C, the compression rate was 10 A2 molecule-' mi+.
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agrees with our observations by fluorescence microscopy that on the unacidified subphase the monolayer is below the triple point temperature. 2. FluorescenceMicroscopeImages. The monolayer films were formed on the acidified subphase at a series of temperatures between 14.0 and 32.0 "C and at areas of 120-90 A2 molecule-l in the uniformly fluorescent LE phase. After allowingabout 10min for solvent evaporation (5 min for temperatures above 25 "C), the films were observed under compression from the LE phase into the LE-LC coexistenceregion. On compression,the LC phase domains nucleate, grow, and assume the shape they are observed to have while the trough is scanned and images are recorded. As expected, additional growth results from further compression. The dark LC phase is always contained within the bright LE phase background. The LC domains are not observed to undergo dynamic shape fluctuations, as observed for the denser darker liquid domains in the liquid-liquid coexistence region of phospholipid + cholesterolmixtures.l*12 With the microscope trough covered by the coverslip, a very slow drift (approximately 1pm/s) of monolayer domains, probably due to small lateral temperature gradients, occurs which does not influence the domain shapes. The monolayers were examined at constant temperatures for time periods ranging from 10 to 50 min. At temperatures below 20 "C, a decrease in the fraction of the LC phase was not visually discernable in films observed at a constant surface density over times up to 50 min. In observations of monolayers above about 23 "C, we observe discernable and then
Langmuir, Vol. 8, No. 10,1992 2511
Langmuir Monolayers of Stearylamine
Figure 4. Fluorescence microscope images of stearylamine on unacidified water at 48 "C in the LE-LC coexistence region at approximately30 A2/molecule. The monolayercontains0.3 mol 76 of the fluorescentprobe NBD-hexadecylamine. Image B was recorded 4 min after image A. The gray block in the lower left corner is 250 pm in length.
obvious dissolution at 30 "C. We have not measured the dissolution rate, but at 30 "C the fraction of the monolayer covered by the LC phase after compression can be clearly seen to decrease with time. Rapid compressions to 20 A2 molecule-l or less at 35 "C failed to show the nucleation of any LC phase. We also observed the monolayer films on cooling from the LE phase into the LE-LC region. The monolayer on the unacidified subphase was formed near room temperature and then heated into the LE phase within 10 min. Observations under mercury lamp illumination are reported, as no anisotropywas detected under oblique illumination by a p-polarized laser beam at 488 nm. On an unacidified subphase up to 45 "C, the monolayer exhibited domain patterns like those observed for pentadecanoic acid below the triple point,16but the domains were smaller. LE-LC coexistence was seen in the temperature range 45-50 "C. On cooling from the LE phase, the LC domains grew as fractal shapes which then healed into compact shapes in a few minutes as shown in Figure 4. Fractal growth was never observed on the acidified subphase. Studies at these high temperatures are difficult due to dissolution into the subphase. Observations on the acidified subphase indicate that the triple point ~
(16) Moore, B. G.; Knobler, C. M.; Akamatau, S.; Rondelez, F. A. J. Phys. Chem. 1990,94,4588.
temperature is about 13 "C, as compared with 45 OC on the unacidified subphase. Figure 5 shows images of the monolayer on compression a t different temperatures. Compression at 14.0 "C from the LE phase into the LE-LC region showed that the smallest LC domains were round, with larger ones being elongated and slightly curved, resembling beans. Compression at 16.2 "C showed elongated LC domains which curved back and forth in a "serpentine" manner and beanshaped LC domains. The smallest LC domains observed first on compression at 16.2 "C are small and slightly curved. Compression at 18.5 "C showed serpentine LC domain shapes more elongated than those at 16.2 "C. If the monolayer was further compressed at 16.2 "C, the LC domains did not become as elongated as those at 18.5 "C; they were observed to become both longer and thicker. Compression at 21.0 "C initially showed very elongated branched domains and shorter almost straight strip domains at a low LC phase area fraction. Compression to lower areas resulted in lamellar-like patterns. It is interestingto note that, at 21.0 "C and higher temperatures, the serpentine distortion observed at 16.2 and 18.5 "C appears to be absent. Images F-H were taken from the same monolayer. Images F and G were taken from different regions of the monolayer 5min after compression from 75 to 52 A2 molecule-l. Image H was taken after compression to 40 A2 molecule-' 15 min later. The pair of images G and H show that the lamellar pattern at lower area per molecule is comprised of structures like those of image G packed closer together. The branching observed for many domains, as readily observed in image G, occurs during the rapid initial growth by splitting at the ends. Compression at 23.5 "C and higher temperatures showed only lamellar patterns; the temperature dependence of the lamellae thickness is currently under study. The lamellar images are similar to those observed in monolayers of racemic dipalmitoylphosphatidylcholinewith 2 mol % ch01esterol.l~The observation of increasinglyless compact structures on increasingtemperature is the reverse of that observed18 for dimyristoylphosphatidic acid with 1 % cholesterol at pH 11. The inhomogeneity of domain sizes and shapes is controlled by the variation in the number of domains initially nucleated in a given region of the monolayer. After the initial domain nucleation, there is very little additional nucleation on further compression. In regions of greater nucleation density, the domain sizes are smaller and the shapes are more compact. The initial nucleation density is not a readily controllable variable and most likely depends upon how much the water surface is locally agitated by the motion of the barrier when nucleation occurs. Over our periods of observation, homogenization of the monolayer patterns was not observed. Inhomogeneity of monolayer films has been observed in many other fluorescence microscopy experiments. When a lamellar pattern formed on compression at 23.5 "C was cooled to 14 "C over 25 min, the pattern changed slightly. Some of the strips split into two or three shorter strips which began to evolve toward a more compact shape. This process was slower in regions of the film where the lamellar pattern was denser. Domains which were elongated in the less dense regions of the film at higher temperature became more compact on cooling. Attempts were made to observe fluorescence images on subphases acidified to pH 2.5 with hydrobromic acid and (17) Weiss, R. M.; McConnell, H. M. J. Phys. Chem. 1985,89,4453. (18) Heckl, W. M.; Mijhwald, H. Ber. Bunsen-Ges. Phys. Chem. 1986, 90,1159.
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Figure 5. Fluorescence images of stearylamine monolayers compressed into the LE-LC coexistence region on water acidified to pH 2.5 with HC1. The monolayer contains 0.3 mol % of the fluorescent probe NBD-hexadecylamine. Images were taken at 14.0 "C (A, B); 16.2 "C (C, D); 18.5 "C (E); 21.0 "C (F-H). The area per molecule values and times after compression for the images are as follows: (A) 105 A2/molecule, 5 min; (B) 95 A2/molecule, 7 min; (C) 88 A2/molecule, 8 min; (D) 78 A2/molecule, 20 min; (E) 70 A2/molecule, 16 min; (F) 52 A2/molecule,5 min; (G) 52 A2/molecule, 5 min; (H) 40 A2/molecule, 20 min. In images A-E, the gray block in the lower left corner is 125 pm in length; in images F-H, it is 250 pm long.
hydroiodic acid. On the HBr subphase, the probe phase segregated from the monolayer in many places. On an HI
subphase, the probe fluorescence was substantially quenched, as might be expected for a highly polarizable
Langmuir, Vol. 8, No. 10,1992 2513
Langmuir Monolayers of Stearylamine 2.5 I
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decreases with increasing temperature. The upper branch of the roughly linear S vs log A dependence at 14.0 "C becomes 1 at log A close to 1.6. At 21.0 "C, it appears that the S vs log A dependence is nonlinear and appears to extrapolate to 1 at S less than 1.6 and most likely around 1.3. These are estimated by visually examining the plots; the functional dependence of S on log A is not known. In any case, the critical radius does not change much between these two temperatures. Determination of SG for four images at 14.0 "C gave values between 0.7 and 1.0, while values between 0.2 and 0.8 were calculated for 4 images at 16 "C. In the lamellar patterns, it is difficult to visually count the number of domains. Using the expression for &hape, the domain radius above which the domain becomes non~ircular,~ estimates of the LE-LC line tension may be made at 14.0 and 21.0 OC. The expression for line tension A is
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counterion. On a pH 2.5 nitric acid subphase, preliminary studies show similar images as found on the HC1subphase, but with the phase diagram apparently shifted downward by about 5 "C as evidenced by observation of lamellar patterns near 15 "C. Observation of anequimolar mixture of stearyl alcohol and stearylamine on the pH 2.5 HC1 subphase showed LE-LC coexistence at 25 "C with round LC domains. 3. Image Analysis Results. The image analysis procedure described above has been used to analyze domain shapes from images of the compressions at 14.0 and 21.0 "C. About 10 images were captured at each temperature and a few domains analyzed from each one. A shape factor S = P/C for individual domains is defined where P is the perimeter of the domain and C is the circumference of the domain calculated from the area assuming a circular shape. The shape factor S will be 1 for circular domains and will increase for less compact shapes. There is a critical domain area above which the shape becomes non~ompact.~ For entire images, we have also calculated SG,a global generalization of S, defined as (P)/ (C) where (P)is the average perimeter per domain in the image and (C) is the circumference calculated from an average domain area obtained by dividing the total area of the LC phase by the number of domains counted in the image. In Figure 6, results are shown for S at 14.0 and 21.0 "C. The value of S follows a dual dependence on area at 14.0 "C. S remains close to 1 for the "bean-shaped" domains, but increases for the other domains. At 21.0 "C, where only elongated shapes are observed, the value S increases consistently with domain area. From the calculations of McConnell,4 the domain area at which S deviates from 1 willdecrease with increasingtemperature if the line tension
(1)
where D is the dielectric constant, Ap = ~ L -C LE is the density difference between the LC and LE phases, p is the dipole moment per molecule assumed equal in the two phases, €0is the permitivitty, 6 is the dipole spacing taken here as ( A L C ) ' ~and ~ , Rsbw is the critical domain radius determined from the measured critical domain area. The pKa of alkylammonium salts in water19 is reported as 1011. The surface PKa of the stearylammonium cation will be decreased by 3-4 pH units due to the positive Guoy potential of the interface.20 If the surface PKa of the stearylammonium cation was 6, as a lower estimate, then the fraction of unprotected amine headgroups at a bulk pH of 2.5 will be negligible and the amine monolayer can be taken to be fully protonated. A crude estimate of the line tension can then be made for the charged monolayer using the approximation of treating the charged headgroup and counterion as an effective dipole p of strength (e/K)(l D - W , where e is the electronic charge and K is the Debye-Hiickel screening length of the subphase.6 Given the lack of information concerning the variation of D near the interface, D will be taken as the dielectric constant of bulk water. For the pH 2.5 HCl subphase, K = 0.40 k1 at these temperatures. We take ALE= 90 A2/molecule at 14.0 "C, on the basis of the compression observations, ALE = 60 A2/molecule a t 21.0 "C, and ALC= 30 A2/molecule a t both temperatures. From the estimated critical domain areas we will use RShape= 3.5 pm at 14.0 "C and Rshaw = 2.6 pm at 21.0 "C, giving X = 9.8 X lo-" J m-l at 14.0 "C and X = 5.4 X lo-" J m-l a t 21.0 "C.
+
Discussion These results indicate that protonated amine monolayers exhibit many of the shape transitions possible for LC phase domains. The shift of the phase diagram and qualitative change in the domain patterns confirm the expected important effect of protonation of the headgroup. The temperature dependence of the patterns observed indicates a decrease in the LE-LC line tension with temperature, as would be expected on approaching the top of the LE-LC coexistenceregion. There is considerable similarity between the domain shapes observed here and the shapes of plane drops of magnetic fluids in a perpendicular magnetic field, as described by Tsebers and (19) March, N. Advanced Organic Chemistry; McGraw-Hill: New York, 1977; p 228. (20) Davies, J. T.; Rideal, E. K. Interfacial Phenomena; Academic Press: New York, 1963; p 94.
2514 Langmuir, Vol. 8, No. 10, 1992
Maiorov.21 The elongation from circular shapes is similar to the "elliptic" instability of the magnetic fluid drops, the oscillatory distortions observed in images C and E of Figure 5 resemble the serpentine instabilityof the magnetic fluid drops, and the elongated and branched shapes of image G resemble the structure of magnetic fluid drops in large fields. The ammonium cation headgroup does not have an asymmetric shape as do the headgroups of phospholipids; thus, the serpentine nature of the shapes in images C and E and the curving of the fatter domains cannot be explained as due to asymmetric molecular packing.22 Although no fluorescence anisotropy was observed, the existence of a tilted LC phase, perhaps at lower temperatures, cannot be ruled out. It is not at all guaranteed that probe orientation is coupled to the orientation of the neighboring amphiphiles. Our estimate of line tension is admittedly crude, as we have assumed the dielectric constant D to be that of water. This may be a more reasonable assumption for small charged headgroups than for small polar headgroups if the charged headgroup is immersed deeper and given that the 'effective dipole" involves the diffuse part of the electric double layer. Since h depends weakly on R,bp, and R s b p decreases slightly between 14.0 and 21.0"C, the difference in X between the two temperatures is caused almost exclusively by the change in density difference between the LE and LC phases. Prior estimates of 1.4 X J m-l for the LE-LC line tension of a pho~pholipid~~ and of DX = 3.4 X 10-l2J m-l for the LE-G line tension of ethyl heptadecanoate24 have been reported. Recently, the LELC line tension has been determined for a fluorescent (21) Tsebere, A. 0.; Maiorov, M. M. Magnetohydrodynamics (Engl. Trawl.) 1980, 16, 21. (22) Heckl, W. M.; Lbche, M.; Cadenhead, D. A,; Mdhwald, H. Eur. Biophys. J. 1986, 14, 11. (23) Helm, C. A.PbD. Thesis, Technical University of Munich, 1988, unpublished.
Stine and Stratmann
bipolar amphiphile26 for which the formation of an anisotropic condensed phase involves the dynamics of lifting a large fluorescent group attached to the 12th carbon of a stearic acid chain up from the surface. Using surface pressure relaxation data and domain nucleation rates determined by fluorescence microscopy, a value of (5.0 f 0.5) X 10-l2J m-l at 20 OC was obtained. Slow and readily measurable surface pressure relaxation upon compression just into the LE-LC coexistence region occurs for other bipolar amphiphiles.2* Our values for h are about 10times larger than the two reported values of LE-LC line tension. The measurement of surface potential isotherms on this system would enable better estimation of the electrostatic forces. The charged amine monolayer system appears to be a promising candidate for image analysis studies. Study of the temperature and surface density dependence of the spacing of the lamellar patterns, and of the behavior of mixtures with a compound with an uncharged headgroup such as stearyl alcohol, should provide further interesting results. Acknowledgment. D.T.S. is thankful for a Monsanto/ UMSL undergraduate research fellowship for the summer of 1991. Technical assistance from T.Windsor and J. Kramer is acknowledged. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. Registry No. NBD-hexadecylamine, 94102-47-3; stearylamine, 124-30-1. (24) Stine, K. J.; Knobler, C. M.; Desai, R. C. Phys. Reo. Lett. 1990, 65,1064. (25) Muller, P.;Gallet, F. Phys. Reo. Lett. 1991,67, 1106. (26) Bois, A. G.; Baret, J. F.; Kulkami, V. S.;Panaiotov, I. I.; Ivanova, M. G.Langmuir 1988,4, 1358.
