Scanning Force and Scanning Near-Field Optical Microscopy of

Jun 16, 1998 - With scanning force microscopy (SFM) we found domains with diameters in the micrometer range protruding several angstroms from the inte...
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Langmuir 1998, 14, 3895-3900

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Scanning Force and Scanning Near-Field Optical Microscopy of Organized Monolayers Incorporating a Nonamphiphilic Metal Dyad Achim K. Kirsch,† Achim Schaper,† Heinz Huesmann,† Maria A. Rampi,‡ Dietmar Mo¨bius,† and Thomas M. Jovin*,† Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Go¨ ttingen, Germany, and Department of Chemistry, University of Ferrara, Via L. Borsari, 46, I-44100 Ferrara, Italy Received January 5, 1998. In Final Form: April 24, 1998 A water-soluble metal complex dyad, Rh(III)-X-Ru(II), was incorporated in monolayers at the air/ water interface by cospreading with a positively (eicosylamine) or negatively (stearic acid) charged amphiphile. After transfer of the monolayer onto a mica substrate via the Langmuir-Blodgett technique, a domain structure similar to that at the air/water interface was observed by scanning probe microscopy (SPM). With scanning force microscopy (SFM) we found domains with diameters in the micrometer range protruding several angstroms from the interstitial areas. The same structure was imaged with scanning near-field optical microscopy (SNOM) with joint registration of topographic features and ruthenium fluorescence. We present a model for the complex structure of the molecular film compatible with the different microscopy results.

Introduction Organized monolayers offer a versatile range of applications including the investigation of photoinduced electron-transfer processes and the conversion and storage of light energy. Monolayers formed and organized at the air/water interface can be transferred by the LangmuirBlodgett (LB) technique onto solid substrates in order to construct monolayer assemblies with a well-defined orientational order in all three dimensions. Amphiphiles with moieties suited for electron-transfer processes have been successfully incorporated into mixed monolayer assemblies, and photoinduced charge separation has been investigated in such systems.1-3 The water-soluble metal complex dyad Rh(III)-XRu(II) is particularly suited for the construction of devices for the conversion and storage of light energy due to the vectorial electron transfer from the Ru(II) to the Rh(III) subunit.4,5 Monolayers of amphiphilic molecules spread at the air/water interface are capable of binding nonamphiphilic molecules [e.g. the Rh(III)-X-Ru(II) dyad] by either adsorption from the subphase or cospreading the nonamphiphilic molecule with the lipid.6 In previous studies, the dyad was incorporated in welldefined monolayer assemblies at the air/water interface.7 * To whom correspondence should be addressed. † Max Planck Institute for Biophysical Chemistry. ‡ University of Ferrara. (1) Mo¨bius, D. In Kinetics of Nonhomogeneous Processes; Freeman, G. R., Ed.; John Wiley & Sons: New York, 1987; p 533. (2) Mo¨bius, D.; Ahuja, R. C.; Caminati, G.; Chi, L. F.; Cordroch, W.; Li, Z.-M.; Matsumoto, M. In Photoinduced Electron Transfer: Fundamental Differences between Homogeneous Phase and Organized Monolayers; Mataga, N., Okada, T., Masuhara, H., Eds.; Dynamics and Mechanisms of Photoinduced Transfer and Related Phenomena; Elsevier: Amsterdam, 1992. (3) Huesmann, H.; Striker, G.; Mo¨bius, H. Langmuir 1997, 13, 4929. (4) Balzani, V.; Scandola, F. Supramolecular Photochemistry; Ellis Horwood: New York, 1991. (5) Indelli, M. T.; Bignozzi, C. A.; Harriman, A.; Schoonover, R. J.; Scandola, F. J. Am. Chem. Soc. 1994, 116, 3768. (6) Hada, H.; Hanawa, R.; Haraguchi, A.; Yonezawa, Y. J. Phys. Chem. 1985, 89, 560.

Spectroscopic and thermodynamic characterization of the dyad in mixed monolayers provided evidence for an interaction of the dyad with both negatively and positively charged matrices, which determined the orientation of the dyad underneath the lipid monolayer.7 For mixed monolayers of dyad/stearic acid (molar ratio 1:3) at the air/water interface two coexisting phases, both incorporating the dyad, have been detected in a broad range of surface pressures by Brewster angle microscopy (BAM). From the geometric parameters of the dyad it was concluded that the dyad forms multilayers upon compression.8 After transfer of these monolayers onto mica substrates, two phases were again found by SPM.9 The round domains exhibited less luminescence of the metal-to-ligand chargetransfer (MLCT) transition of the Ru(II) subunit than the separating interstices. From quantitative photobleaching analysis it was deduced that the dyad density in the interstices was twice that of the domains but that the luminescence quantum efficiency was reduced. In the simultaneously recorded shear-force topographic data the mixed monolayer in the round domains appeared thicker than that in the interstice. Experimental Section Monolayer Preparation. The dyad (Figure 1) [Rh(III)(dcbipy)2-(Mebipy-CH2-CH2-Mebipy)-Ru(II)(phen)2]5+ (PF6)-5 (dc-bipy ) 4,4′-dicarboxy-2,2′-bipyridine; Mebipy ) 4-methyl2,2′-bipyridine; phen ) 4,7-dimethyl-1,10-phenanthroline) was synthesized and isolated by M. T. Indelli (University of Ferrara, Italy). Eicosylamine and stearic acid were purchased from Sigma Chemicals. Dyad and amphiphiles were dissolved in a mixture of methanol and chloroform (1:3 v/v). The stock solutions were mixed in the appropriate ratio and spread at the air/water interface of a Fromherz type circular trough.10 Subphase water (7) Huesmann, H.; Bignozzi, C. A.; Indelli, M. T.; Pavanin, L.; Rampi, M. A.; Mo¨bius, D. Thin Solid Films 1996, 284-285, 62. (8) Huesmann, H.; Spohn, D. B.; Indelli, M. T.; Rampi, M. A.; Mo¨bius, D. Langmuir 1997, 13, 4877. (9) Kirsch, A. K.; Meyer, C. K.; Huesmann, H.; Mo¨bius, D.; Jovin, T. M. Ultramicroscopy 1998, 71, 295. (10) Fromherz, P. Rev. Sci. Instrum. 1975, 46, 1380.

S0743-7463(98)00049-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/16/1998

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Kirsch et al. Sutter Instruments, Novato, CA) were mounted in a shear-force sensor head, replacing the SFM head of the NanoScope III SPM system. The shear-force detection system regulated the distance between the fiber tip and the sample surface to 5-10 nm.13 The lateral vibration amplitude of the tip for the shear-force detection was on the order of 5 nm at frequencies in the range 20-80 kHz. The fiber tip was used for both illumination of the sample and detection of the emitted light (shared aperture mode).12,14-17 The 488 nm line of an Ar-Kr mixed gas laser (Spectra Physics, Mountain View, CA), stabilized with an LS-PRO HP Noise Eater (Cambridge Research & Instrumentation, Cambridge, MA), was used for the excitation of the metal-to-ligand charge-transfer (MLCT) transition of the Ru(II) subunit of the dyad. The luminescence of this transition, which is strongly quenched by the Rh(III) subunit,5 was isolated from the excitation light by means of a dichroic mirror and a band-pass filter (505DRLP02 and 590DF35, Omega Optical, Brattleboro, VT). The luminescence was detected with a single photon counting avalanche photodiode (SPCM-AQ 131, EG&G Optoelectronics, Canada) and recorded by the pulse counting board of the NanoScope electronics. The counting time per pixel was 4.1 ms, and no further image processing was applied. The images (128 pixels × 128 pixels) were obtained at a 0.2 Hz line scan frequency dictated by the response time of the shear-force feedback system. Measurements were performed under ambient conditions at room temperature.

Figure 1. Structure of the metal complex dyad Ru(II)Rh(III). The dimensions of the dyad are ∼1.5 nm × 2.8 nm.

Results

was from a Milli-Q water purification system (Millipore Corp., Bedford, MA). The surface pressure was measured by a Wilhelmy balance. Monolayers were transferred onto freshly cleaved mica by the LB technique. A 15 mm circular piece of mica was immersed vertically in the subphase prior to spreading and compression of the monolayer. After evaporation of the solvent the monolayer was compressed to a surface pressure π of either 5 or 20 mN/m and left to relax for 10 min at the air/water interface. Finally, the mica plate was pulled out at constant surface pressure, leaving a single monolayer deposited on the substrate surface. Scanning Force Microscopy (SFM). The samples were scanned with a NanoScope III multimode SFM (Digital Instruments, DI, Santa Barbara, CA) operated in the permanent contact or tapping mode using a J-scanner with a 135 × 135 (x,y) × 5 (z) µm scan range. Calibration of the scanner was with a pitch crosshatch standard [10 × 10 × 0.2 (x,y,z) µm, STS 1800, DI] and with holes in multilayer assemblies of saturated fatty acids (for z).11 Microfabricated Si tips integrated into triangular cantilevers with a sensitivity (dF/dz) of ∼0.1 N/m and a resonance frequency of ∼160 kHz (Ultralevers, Park Scientific Instruments, PSI, Sunnyvale, CA) were used. Images were obtained in the topographic (isoforce) mode. Measurements were performed in air (relative humidity 15-30%) at room temperature (18-24 °C). The tip load was minimized by adjusting the damping amplitude and the cantilever bending to a minimum value. Images (512 pixels × 512 pixels) were taken at a 5 Hz line scan frequency. Standard plane-fit and flattening corrections were applied to the data. Cross-sectional, roughness, and bearing analyses were performed with the NanoScope software. The root-mean-square (rms) surface roughness was computed for 4 µm2 surface areas. Scanning Near-Field Optical Microscopy (SNOM). The scanning near-field microscope used for collecting near-field luminescence and shear-force data was a slightly modified version of the system described in previous reports.9,12,13 Uncoated fiber tips produced from an optical fiber (SMF 1528 CPC6, Siecor, Neustadt, Germany; radius of curvature ∼ 70 nm) in a heating and pulling process with a commercial pipette puller (P-2000,

Mixed Monolayers of Dyad/Stearic Acid Transferred onto Mica. SFM images of the monolayer topography were acquired after transfer of the mixed dyad/ stearic acid monolayer (molar dyad fraction fD ) 0.25) onto the mica surface at π ) 5 mN/m (Figure 2a). For convenience (see reference 8) the term monolayer is used for these films of complex morphology in which lipid and dyad molecules might be arranged in different planes. Circular domains of different size were homogeneously distributed. Domains in contact exhibited distinct borders (arrows in Figure 2a). Occasional small defects (holes of a few square micrometers) were restricted to the interdomain areas and were absent on the bare mica surface. Figure 2d shows a near-field luminescence image of a similar mixed monolayer transferred under identical conditions to those for the monolayer in Figure 2a. The domains as well as the defects in the interdomain areas (arrows in Figure 2d) exhibited less luminescence than the interstices. The average domain size was approximately the same as that in Figure 2a. The SFM image (Figure 2b) depicts a surface area in which a defect appears in the interdomain region. From the cross-section along the dashed line the height of the interdomain layer was ∼4 Å over the basal plane while the domains contributed an additional thickness of ∼9 Å (inset in Figure 2b). The three-level monolayer architecture is clearly perceptible from the depth histogram in Figure 2c. An SFM micrograph of the LB film structure after transfer to the mica at higher surface pressure (π ) 20 mN/m) is shown in Figure 3a. Large, more amorphous domains were visible. Features of the monolayer architecture are given in Figure 3b. The domain surface had a granular morphology, the roughness of which increased from 1 Å at low surface pressure (domain surface in Figure 2a) to 7 Å at the higher surface pressure. In the interdomain region defects were obvious, similar to those discerned at lower surface pressure (cf. Figure 2a). From

(11) Wolthaus, L.; Schaper, A.; Mo¨bius, D. J. Phys. Chem. 1994, 98, 10809. (12) Kirsch, A.; Meyer, C.; Jovin, T. M. In NATO Advanced Research Workshop: Analytical use of fluorescent probes in oncology, Miami, Fl; Kohen, E., Hirschberg, J. G., Eds.; Plenum Press: New York, 1996; p 317. (13) Kirsch, A. K.; Meyer, C. K.; Jovin, T. M. J. Microsc. 1997, 185, 396.

(14) Jalocha, A.; van Hulst, N. F. Opt. Commun. 1995, 119, 17. (15) Spajer, M.; Courjon, D.; Sarayeddine, K.; Jalocha, A.; Vigoureux, J. M. J. Phys. III 1991, 1, 1. (16) Bielefeldt, H.; Ho¨rsch, I.; Krausch, G.; Lux-Steiner, M.; Mlynek, J.; Marti, O. Appl. Phys. A 1994, 59, 103. (17) Bozhevolnyi, S. I.; Vohnsen, B. J. Opt. Soc. Am. B 1997, 14, 1656.

Monolayers Incorporating a Nonamphiphilic Metal Dyad

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Figure 2. SFM and SNOM imaging of the dyad/stearic acid monolayer structure after transfer to mica. fD ) 0.25 and π ) 5 mN/m. (a) Tapping mode SFM. The horizontal color bar codes for topography in a and b. (b) Zoom to a surface region exhibiting a hole in the basal film structure. (Inset) Cross-section along the dashed line drawn in b. Step heights are indicated by the markers. (c) Height histogram of the area marked by the box indicated in b. (d) SNOM image of a similar sample. Excitation intensity: 2.4 µW. The vertical color bar codes for fluorescence intensity (kcps ) 1000 counts per second).

the surface profile along the dashed line in Figure 3b a three-level architecture similar to that in Figure 2c was revealed. However, the histogram in the bearing analysis (Figure 3c) was smoothed out due to the increased surface granularity. The near-field luminescence of the mixed monolayers transferred at the high (Figure 3d) and low (Figure 2d) surface pressures had similar distributions. The highest lateral resolution achieved in the luminescence imaging mode was ∼600 nm. The fine structures in Figures 2d and 3d were caused by noise in the optical detection system. For example, the shot noise of the background in Figure 2d was ∼5000 cps, leading to a small signal-to-noise ratio. Inasmuch as the observed resolution in an image depends fundamentally on this parameter,18 the lateral resolution was probably limited by the signal intensity. In the corresponding images obtained in a confocal laser scanning microscope, the domain structure of these films was barely visible19 due to the inherently lower contrast generated in such systems. The LB film consisting of dyad and stearic acid exhibited time-dependent morphological changes. While the samples (18) den Dekker, A. J.; van den Bos, A. J. Opt. Soc. Am. A 1997, 14, 547. (19) Arndt-Jovin, D. J. Private communication, 1996.

prepared at low surface pressure were stable over months, the interstitial area of samples transferred at higher surface pressures over time developed protuberances exceeding the domain level by several angstroms. However, these were not reflected in perceptible changes in the luminescence distribution (data not shown). Mixed Monolayers of Dyad/Eicosylamine Transferred onto Mica. SFM micrographs were taken of a mixed metal complex/eicosylamine monolayer (π ) 5 mN/ m, fD ) 0.25) after transfer onto mica (Figure 4). The monolayer was imaged in tapping mode and showed domains surrounded by a granular interstice; these features were stable during repeated scanning (Figure 4a). The tops of these granules were well above the domain level, and their morphologies varied in size and shape. The domains were smaller than those of films consisting of dyad and stearic acid (Figures 2 and 3). A typical area is presented at higher magnification in Figure 4b. From the cross-section along the dashed line, the domain was ∼7 Å in height, measured from the basal smooth interdomain surface from which the granules extended for several nanometers normal to the surface (see surface profile in Figure 4e). Within the domains a subpattern was observed (patches on the domain in the lower right

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Figure 3. SFM and SNOM imaging of the dyad/stearic acid monolayer structure after transfer to mica. fD ) 0.25 and π ) 20 mN/m. (a) Tapping mode SFM. (b, c) Same analysis as in Figure 2 (for details see text). (d) SNOM image of a similar sample. Excitation intensity: 150 nW. The horizontal color bar codes for topography in a and b. The vertical color bar codes for fluorescence intensity.

corner of Figure 4b), indicating some kind of structural heterogeneity. In Figure 4c and d the same region was imaged in permanent contact mode. A porous domain structure was perceived, which was stable during repeated scanning and differed significantly from the smoothly contoured domain surface in Figure 4b. The latter was recovered after switching back to the tapping mode. From the surface profile along the dashed line drawn in Figure 4d the apparent depth of the pores within a domain was ∼13 Å (surface profile Figure 4f). In contrast to the case of mixed monolayers of dyad and stearic acid, it was not possible to resolve the domains on these films optically with the near-field optical microscope, whereas the simultaneously recorded shear-force image confirmed the presence of the domains (data not shown). Discussion Dyad/Stearic Acid Mixed Monolayers. In previous work8 it was found that, in the complex monolayer of stearic acid and dyad, the latter was completely bound at the air/water interface with no loss into the subphase upon compression. At surface pressure onset (π ) 0 mN/m) a two-phase model for the complex monolayer was proposed with one phase consisting of “supramolecular clusters” with a ratio of dyad/lipid of nLD ) 1:2 to 1:2.25 coexisting with free stearic acid. Upon compression the average area

per dyad molecule decreased below the cross-sectional area deduced from a three-dimensional structural model of the dyad, leading to a different two-phase model, with one phase consisting of a multilayer of dyad underneath a monolayer of stearic acid (XD-phase) and the second having only a monolayer of dyad beneath a monolayer of stearic acid (MD-phase). These two-phase systems, previously visualized by BAM8 at the air/water interface, were probably not affected by the transfer onto a solid substrate, as judged from a series of findings: (i) No dyad was lost during the transfer. This could be seen by comparing the absorptions of the MLCT transition on the solid substrate and at the air/ water interface (data not shown). (ii) The lateral patterns revealed by SFM and SNOM were similar to those seen by BAM. (iii) The persistence of visible boundaries between adjacent domains confirmed that the latter consisted of differently oriented molecules, corresponding to the optically anisotropic domains seen by BAM. The anisotropy reflects different orientations of the molecules with long-range order.20 The boundary can be attributed to different tilt or azimuthal angles of the molecules preventing a coalescence of the domains. It is thus reasonable to infer that the domains visible in the SFM (20) Overbeck, G. A.; Ho¨nig, D.; Mo¨bius, D. Thin Solid Films 1994, 242, 213.

Monolayers Incorporating a Nonamphiphilic Metal Dyad

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Figure 4. SFM images of a monolayer of metal complex/eicosylamine after transfer to mica. fD ) 0.25; π ) 5 mN/m. (a) Overview, tapping mode. A smooth domain structure is evident. (b) Zoom to a surface region of the monolayer shown in a. (c) Same region as in a imaged in contact mode. (d) Zoom to a surface region of the monolayer shown in c. The color bar codes for topography in a-d. (e, f) Cross-section analysis of the monolayer architecture along the lines drawn in b, d.

and SNOM images corresponded to the anisotropic round domains at the air/water interface seen by BAM. The SNOM measurements revealed a higher luminescence emission of the interstitial areas. The fluorescence quantum yield in these dyads depends on the intra- and intermolecular photoinduced electron-transfer efficiency, which is in turn a function of the local environment. The environment of the dyads was different in the domains and the interstitial areas, thus resulting in a different quantum yield. The latter quantity can be measured either by luminescence lifetime or by quantitative photobleaching.9 From the latter technique, the fluorescence quantum yield of the dyad molecules in the domains was found to be higher than that in the interstitial areas.9 The relative luminescence distribution and quantum yields led to the conclusion that the dyad density in the

interstitial area was greater, implying a complex (multilayered) film architecture. We have to reconcile this observation with the SFM results suggesting that the interstitial surface was lower than the domain level, and we do so with the model illustrated in Figure 5. The interstitial phase consists of a dense layer of dyad to which the excess of dyad is attached in the form of subsequent layers (XD-phase). The lipid molecules on top of the interstitial phase are less densely packed, as in a gasanalogue state. This phase coexists with the domain phase of a monolayer of the dyad underneath a more densely packed monolayer of stearic acid (MD-phase). Due to the weaker lateral interactions between the single lipid molecules in the interstitial phase, they constitute a layer that is thinner and/or more susceptible to penetration by the scanning tips, and therefore contribute less to the

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Figure 5. Model of the film architecture for LB films consisting of dyad and stearic acid transferred onto mica. The arrow chain depicts a possible path of a probe scanning the sample surface.

topographic SPM signal compared to the more densely packed lipid molecules in the domain phase. The approximate thickness of the two phases can be estimated from the model parameters by replacing the dyad with two adjacent spheres of 14 Å diameter. This value was deduced from 3D modeling of the molecule. The length of a fully extended stearic acid aliphatic chain is 22 Å. According to our model, the interdomain region consists of two layers of closely packed spheres, corresponding to one layer of dyad molecules underlying one layer of stearic acid, yielding a combined thickness of hdom ) 25 Å + 22 Å ) 47 Å. The influence of the chain tilt angle on the thickness is neglected in this simplified approach. For the interdomain region a thickness of hint ) 37 Å was derived according to an arrangement of three layers of closely packed spheres, that is, 1.5 layers of dyad molecules depicted in Figure 5. This value was calculated from the average area per dyad molecule (1.25 nm2) in a monolayer transferred at π ) 5 mN/m8 and the SFM result that at this surface pressure ∼70% of the surface was covered by domains. The stearic acid molecules are disregarded because they are assumed to be in a gas- or liquid-analogue state and thus do not contribute to the monolayer architecture revealed by SFM. Strikingly, the theoretical value for the domain-interdomain height difference, ∆h ) 10 Å, is in good agreement with the experimental height difference between domains and interstices of ∼9 Å. Dyad/Eicosylamine Mixed Monolayers. In mixed monolayers of dyad cospread with eicosylamine, much less dyad was retained at the interface than in mixed monolayers of dyad and stearic acid.7 In LB films of dyad and eicosylamine the complex morphology shown in the SFM images was more difficult to interpret, since the

Kirsch et al.

topology depended on the SFM mode of operation. In tapping mode a smooth domain surface structure was apparent (Figure 4a), but the same surface area appeared in a porous morphology during imaging in the permanent contact mode (Figure 4c). Thus, we have to assume that within round domains an inherent structural heterogeneity was responsible for the observed changes in the topographic contrast. We attribute the heterogeneity to the coexistence of fluid- and solid-like regions exhibiting different resistance to the force applied by the tip. One has to assume that in general surface profiles registered by both SFM modes reflect the tip load as well as the viscoelastic properties of the scanned surface. The frequency-dependent elastic response of our mixed monolayers to an external force reflects the viscoelastic behavior of the hydrocarbon chains.21 In the tapping mode a cyclic force is applied with a driving frequency of ∼160 kHz, a frequency at which all hydrocarbon chains are probably coupled rigidly. In contrast, the point scan rate is much lower (kHz), such that the locally applied force is modulated accordingly. We expect that the extent of monolayer deformation depends strongly on the molecular structure and alignment, accounting for the porous domain structure observed in the contact mode. Conclusion The nonamphiphilic Rh(III)-X-Ru(II) dyad has been incorporated in organized monolayers at the air/water interface by interaction with two different lipid anchors, stearic acid and eicosylamine. The morphologies of the two resulting systems after transfer onto mica determined by two scanning probe techniques differed strongly due to the different extent of dyad incorporation. No dyad was lost with stearic acid upon compression and transfer of the monolayer, giving rise to the formation of a phase with a bi- or multilayer of dyad underneath the lipid anchor. In contrast, much less dyad remained at the air/ water interface and was transferred to the solid with eicosylamine as the lipid anchor. We have used different scanning probe microscopy techniques to visualize these systems inasmuch as neither technique alone is capable of revealing all pertinent features of the films. SNOM and SFM yield complementary topographic and spectroscopic information, which together reveal the complexity of these mixed monolayers. LA9800497 (21) Sheiko, S. S.; Eckert, G.; Ignat’eva, G.; Muzafarov, A. M.; Spickermann, J.; Ra¨der, H. J.; Mo¨ller, M. Macromol. Rapid Commun. 1996, 17, 283.