Contrast Studies on Organic Monolayers of Different Molecular

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Contrast Studies on Organic Monolayers of Different Molecular Packing in FESEM and Their Correlation with SFM Data A. G. Bittermann,† S. Jacobi,‡ L. F. Chi,‡ H. Fuchs,‡ and R. Reichelt*,† Institut fu¨ r Medizinische Physik und Biophysik, Robert-Koch-Strasse 31, Westfa¨ lische Wilhelms-Universita¨ t, D-48149 Mu¨ nster, Germany, and Physikalisches Institut, Wilhelm Klemm-Strasse 10, Westfa¨ lische Wilhelms-Universita¨ t, D-48149 Mu¨ nster, Germany Received April 3, 2000. In Final Form: December 14, 2000 Organic monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol or dipalmitoyl-phosphatitylcholine, transferred by the Langmuir-Blodgett technique in phase-coexistence regions onto a solid support, show domains differing in their molecular packing. Domains with dense molecular packing and quite upright orientation of the molecules are neighbored by a matrix with lower packing density and variable molecular orientation. In the scanning force microscope, these domains can be distinguished by a small difference in their layer heights. In the field emission scanning electron microscope (FESEM), these small height differences were not detectable. Coated monolayers show no topographic features of different domains in secondary electron (SE) micrographs. In opposition to that, the domains are easily recognizable in SE micrographs of the uncoated monolayer by a very strong contrast which presents almost a black/white pattern. Thus, the FESEM may serve as a sensitive tool for mapping areas of different molecular packing in chemically homogeneous organic monolayers.

Introduction Thin organic films on solid supports are becoming increasingly important, especially in medicine and sensor technology.1-6 Reliable methods for the characterization of these films are necessary in development and product control. In the past few years, scanning force microscopy (SFM)7 has been established as a powerful and frequently used tool to characterize organic films on solids at high spatial resolution.8,9 Field emission scanning electron microscopy (FESEM) allows a fast routine screening of large specimen areas at high lateral resolution and therefore may also serve as an excellent tool for characterization of thin organic films on solid supports in the future. FESEM is well-known as an imaging technique to characterize specimens with regard to their topographic features and mean atomic number at high resolution (e.g., see refs 10-12). Recent evaluations show that it is also * To whom correspondence should be addressed. † Institut fu ¨ r Medizinische Physik und Biophysik. ‡ Physikalisches Institut. (1) Roberts, G. Langmuir-Blodgett Films; Plenum Press: New York, 1990. (2) Gregory, P.; Go¨litz, P. Organic thin films. Adv. Mater. 1991, 3, 8. (3) Ulman, A. An introduction to ultrathin organic films: from Langmuir-Blodgett to Self-Assembly; Academic Press: Boston, 1991. (4) Schwartz, D. K. Langmuir-Blodgett film structure. Surf. Sci. Rep. 1997, 27, 242. (5) Hui, S. W.; Rao, N. M. Scanning 1995, 17, Suppl. 5, V-7. (6) Gooding, J. J.; Erokhin, P.; Hibbert, D. B. Biosens. Bioelectron. 2000, 15, 229. (7) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 56, 930. (8) Chi, L. F.; Anders, M.; Fuchs, H.; Johnston, R. R.; Ringsdorf, H. Science 1993, 259, 213. (9) Garnaes, J.; Schwartz, D. K.; Viswanathan, R.; Zasadzinski, J. A. N. Nature 1992, 357, 54. (10) Reimer, L. Scanning electron microscopy; Springer Press: New York, 1999. (11) Reimer, L. Image formation in low voltage scanning electron microscopy; SPIE Optical Engineering Press: Bellingham, WA, 1993; Vol. TT12.

suitable for the study of thin organic layers on flat solid supports, that is, specimens possessing almost no topographic details. Contrast phenomena, other than the wellknown topographic and atomic number contrast, allow for the characterization of thin organic layers (e.g., see refs 13-16). Properties contributing to these contrasts are, for example, chemical differences in the monolayer, differences in the local film thickness, or differences in the orientation and packing of molecules in the monolayer. Here, we will present some results concerning the “molecular packing contrast”. As model systems, we used chemically homogeneous monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) and dipalmitoyl-phosphatityl-choline (DPPC). DPPTE and DPPC are both lipids, consisting of a hydrophilic headgroup adhering to the silicon wafer and two hydrophobic carbohydrate chains on the opposite site of the molecule. Monolayers of DPPTE and DPPC were transferred from the liquid subphase onto silicon wafers by the Langmuir-Blodgett technique under controlled conditions (see Figure 1), resulting in a distinct patterning of the monolayer. In the liquid condensed (LC) phase, the molecules are densely packed, whereas in the liquid expanded (LE) phase the molecules are just loosely arranged and have random orientations. As shown by SFM, there is a small difference in height between these domains. Although the height differences are not detectable in FESEM, the differences in the molecular packing can be visualized in the secondary electron (SE) mode by strong contrast. (12) Nakayama, T.; Gemma, N.; Miura, A.; Azuma, M. Thin Solid Films 1989, 178, 477. (13) Lopez, G. P.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1993, 9, 1513. (14) Wollman, E. W.; Frisbie, C. D.; Wrighton, M. S. Langmuir 1993, 9, 1517. (15) Reichelt, R. Optik 1997, 106, Suppl. 7, 15. (16) Reichelt, R.; von Nahmen, M.; Bittermann, A. G. Proc. 14th Intern. Congr. Electron Microscopy; Benavides, H. A. C., Yacaman, M. J., Eds.; Inst. Phys. Publ.: Bristol & Philadelphia, 1998; Vol. 1, p 449.

10.1021/la0004956 CCC: $20.00 © 2001 American Chemical Society Published on Web 02/24/2001

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Figure 1. Isotherm for DPPTE at 25 °C. The two-phasecoexistence region lies between the LE phase region and the LC phase region. The LB transfer was done within this twophase-coexistence range. The chemical structure of DPPTE is depicted in the upper right.

Figure 2. Hypothetic model of molecular arrangements in a LB film on a solid support, transferred under two-phasecoexistence conditions. The film consists of densely packed domains of quite upright molecular orientation (LC phase) as well as of areas where the packing density of the molecules is lower and the molecules have various orientations (LE phase).

Materials and Methods A piece of RCA-cleaned silicon (ammonium-peroxide cleaning procedure17) was coated with a monolayer of DPPTE (kindly provided by Dr. K. Reihs, Bayer AG, Leverkusen/Germany, MG ) 730.7 g/mol, 2mM in chloroform) by Langmuir-Blodgett (LB) transfer at a pressure of 9-10 mN/m, a transfer speed of 2 mm/ min, and a temperature of about 25 °C with a Lauda FW2 (Lauda, Germany) film balance. The film transfer is carried out in the two-phase-coexistence region (Figure 1), where the resulting monolayer will consist of a LC phase (domain of densely packed molecules with almost identical tilt orientation) and a LE phase (loosely arranged molecules with various orientations; Figure 2). The LB transfer of DPPC (Avanti Polar Lipids, Inc.) was performed in the two-phase-coexistence region as well. For correlative studies in SFM and FESEM, the siliconsupported DPPTE film was finder-coated perpendicularly with 1.2 nm Pt/C using a Balzers evaporation device (BAF 300, Balzers, Principality Liechtenstein; equipped with a turbo molecular pump). The coating was applied through a finder mask; an H2 finder grid was placed on a spacer over the DPPTE-coated silicon wafer. These finder coordinates allow an easy orientation on the sample in both types of microscopes. In the areas covered by the grid bars during the coating process, the DPPTE film remains uncoated. After this preparation, the specimens are very well suited for contrast studies in the FESEM. Scanning force micrographs were taken with a Park Autoprobe microscope (Park Scientific Instruments, U.S.). The sample was scanned in contact mode, using a force of 2 nN. Scanning electron microscopy was performed with a S-5000 “in-lens” FESEM (Hitachi Ltd., Tokyo, Japan) mostly at an accelerating voltage of 6 kV in the SE and backscattered electron (BSE) modes. (17) Brzezinski, V.; Peterson, I. R. J. Phys. Chem. 1995, 99, 12545.

Langmuir, Vol. 17, No. 6, 2001 1873 Secondary ion mass spectrometry was done with a ToF-SIMS (time-of-flight secondary ion mass spectrometer) device (reflector type of ToF-SIMS, built at the University of Mu¨nster, Germany), equipped with a gallium source for primary ions.18 Positive and negative ion mass spectra were recorded under static conditions. Specimen areas were imaged for preselected negative ion masses. For quantitative studies, a DPPTE film was transferred by the LB technique at 65 Å2/molecule onto a silicon wafer. SE micrographs were taken by FESEM and digitally analyzed using Scion Image Software (Soft-Imaging Software GmbH, Mu¨nster, Germany). Gray values were thresholded, resulting in a black/ white-pattern. Both black and white areas were quantified in terms of their size. Results were processed using Microsoft Excel software. Brewster angle microscopy19 was done with a BAM 2 plus (Nanofilm Technologies, Go¨ttingen, Germany), working with an analyzer angle of 45°. The DPPTE film was for that purpose floating on the water surface of the LB trough. Ellipsometry of the DPPTE film on a solid support was performed on an I-Elli 2000 (Imaging Ellipsometer, Nanofilm Technologies, Go¨ttingen, Germany).

Results and Discussion DPPTE monolayers on silicon wafers, consisting of two different phases, were correlatively imaged by FESEM and SFM. In the SE mode of the FESEM, the two phases of the uncoated DPPTE and DPPC layers can be easily distinguished by a strong contrast (Figure 3A,B and Figure 10). The bright areas correspond to the LC phase, where neighbored molecules are identically arranged and densely packed. Dark regions correspond to the LE phase, where the molecules are loosely packed and can possess various orientations (Figure 2). After Pt/C coating, the contrast between the two phases in the SE micrographs disappears (Figures 3A and 5A). No topographic features can be recognized in the two-phase monolayer by FESEM after the coating (Figure 3A). This fact suggests that the strong contrast between the two phases of the uncoated monolayer has no significant topographic component. Instead of topography, the observed contrast indicates differences in the packing density and orientation of molecules in the uncoated monolayer. SFM images (Figure 3C,D) show a mean difference in height between the two phases of the DPPTE film of about 1 nm (Figure 4). The liquid condensed phase is elevated by this amount against the liquid expanded phase, which correlates with a more upright position of the molecules in the LC phase (Figure 2). For the interpretation of this rather small height difference, it has to be taken into account that the SFM tip might enter the organic layer to a certain extent and therefore does not measure absolute values. Also, there will be no upright-versus-flat difference in the molecular orientations between the two phases. More likely, the tilt angles will vary to a smaller degree, resulting in height differences of far less than molecular length. The measured height difference could not be detected even at high magnification in FESEM. Characterization of thin organic films on solid supports in the FESEM can best be done in the SE mode. The contrast between the two phases, depending on differences in the molecular packing, is visible in the SE mode of the FESEM only (Figure 5A). In the BSE mode, however, no contrast between the phases can be detected (Figure 5B). It is well-known that the BSE contrast of flat samples depends mainly on differences in atomic number (e.g., see (18) Hagenhoff, B. Sekunda¨ rionenmassenspektroskopie an molekularen Oberfla¨ chen; Deutscher Universita¨tsverlag: Wiesbaden, 1994; p 179. (19) Ho¨nig, D.; Mo¨bius, D. Thin Solid Films 1992, 210/211, 64.

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Figure 3. FESEM-SFM correlation of images of the same area of the sample. The DPPTE monolayer on the silicon wafer was finder-coated with 1.2 nm Pt/C and analyzed with SFM (contact mode, micrographs C and D). Subsequently, the very same region was depicted with FESEM (SE mode, micrographs A and B). The brighter regions of the SE micrograph correlate with the elevated domains in the SFM topograph (compare B and D). In contrast to the SE micrograph, the height differences in the SFM topograph are visible also in areas of Pt/C coating (micrograph A and C).

Figure 4. Example for height measurements on DPPTE in the SFM. Even taking into consideration that the molecular layers might be deformed by the force of the SFM tip during the scan, there is still a small but significant difference in height between the two phases.

ref 10). At an acceleration voltage of g5 kV, a majority of BSEs are generated in the sample at a greater depth than the emitted SEs (e.g., see ref 10). Thus, the lack of contrast in chemically homogeneous ultrathin organic layers on a likewise chemically homogeneous support in the BSE mode is not surprising. The SE signal on the contrary is well suited for characterization of organic monolayers, because it carries information from the very surface of the sample as a result of direct interaction of primary electrons with atoms in the monolayer. The higher the packing density in the

monolayer, that is, the more molecules per surface area, the higher the amount of SEs. An orientation of the molecules more or less perpendicular to the surface results in a higher yield of SEs than free orientation at a low angle to the surface. Furthermore, the molecular orientation determines the number of accessible atoms at each point. Thus, molecular orientation and density are expected to have a direct influence on the SE output. However, the tilting of the whole sample relative to the primary electron beam in the FESEM by (40° had no significant effect on contrast distribution (data not shown).

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Figure 7. High-resolution SFM topograph of the DPPTE film in the two-phase-coexistence region. In addition to the large LC domains, numerous microdomains are visible. These LC microdomains are embedded in the LE phase.

Figure 5. FESEM micrograph of a Pt/C-finder-coated DPPTE film on a silicon wafer. Micrograph A was recorded in the SE mode, and micrograph B was recorded in the BSE mode. In both detection modes, the finder-coating is easily visible. Whereas the SE image allows a clear distinction between the two phases in the uncoated area, no differences are visible in the BSE mode.

Figure 6. ToF-SIMS maps visualizing the chemical composition of the LC and LE phases. (A) CH--fragment signal. The signal is most prominent in the LC phase and is less strong in the LE phase. CH- fragments originate from DPPTE molecules, especially from the hydrophobic carbohydrate chains of the molecule. (B) SiO2--fragment signal. There is nearly no signal in LC areas; LE regions yield a moderate SiO2- signal. SiO2fragments are derived from the silicon substrate.

One also has to be aware that other properties of the monolayer related to the molecular packing such as, for example, surface energy can affect the secondary electron yield. ToF-SIMS data show a higher molecule-specific signal in the LC phase. In the LE phase, the molecule-specific signal is lower and the signal from the silicon support is visible (Figure 6). The observed signal distribution correlates with the distribution of DPPTE molecules; there

are more molecules per unit area in the LC phase than in the LE phase, where fewer molecules are just loosely arranged on top of the solid silicon support. When the DPPTE-coated wafer is softly scratched, bare areas of the wafer get exposed. In SE micrographs, these bare regions of the silicon wafer and the LE phase show very similar contrast in respect to the LC phase (data not shown). Measurements of the microroughness of both phases reveal a higher roughness of the LE phase (data not shown). This phenomenon might be related to a solidification process during the LB transfer of the DPPTE monolayer from the water surface onto the silicon wafer. On the solid substrate, the molecules of the LE phase, formerly lying at different flat angles homogeneously distributed onto the water surface, aggregate preferentially to microclusters of up to 100-200 nm in diameter; just a few single molecules stay unaggregated. These microclusters are quite uniformly arranged all over the area of the LE phase and contribute to the higher roughness of this phase, whereas the densely packed molecules in the LC phase provide a smoother surface. These microclusters are occasionally visible in SFM topographs at higher magnifications (Figure 7). Reflection microscopic techniques (Brewster angle microscopy, BAM, and ellipsometry; Figure 8) before and after the LB transfer of the lipid monolayer from the water surface onto the silicon wafer show distinctly different situations. After the transfer onto the solid support, in addition to the LC/LE phases observed in the floating film of the LB trough there are numerous microdomains in the area of the former homogeneous LE phase. Depending on the applied surface pressure in the LB trough, the LC areas vary strongly in shape, density, and size. The Brewster angle micrograph (Figure 8A) reveals some further details of the LC areas of the DPPTE film on top of the water surface. The LC areas show different image intensities which correspond to various shades of gray in the micrograph. Every domain itself is radially structured into different regions of brighter to darker tones of gray. According to the principle of BAM, various gray values indicate different molecular orientations. Groups of neighbored molecules have the same tilt angle, which is just a few degrees different from the common tilt angle

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Figure 8. Reflection microscopy of DPPTE monolayers in the LE/LC-coexistence region before and after the LB transfer. (A) Brewster angle micrograph of the DPPTE monolayer on top of the water surface in the LB trough. Bright areas of densely packed molecules are surrounded by a dark field of loosely arranged molecules. The bright areas show various shades of gray, indicating different molecular orientations. (B) The DPPTE monolayer on the silicon substrate after the LB transfer, depicted by ellipsometry. Bright large areas of densely packed molecules are visible. The darker areas in between are speckled with tiny bright spots, indicating microdomains of crystalline molecular arrangements between few loosely packed molecules.

of the next group. The molecules of each LC domain therefore exhibit a small number of slightly different, more or less upright orientations. The LE phase on the other hand appears homogeneous. This means that DPPTE molecules of this phase do not have any tilt angle in common but show a large variety of orientations instead. Furthermore, the strong contrast between the LE and LC phases in the Brewster angle micrograph corresponds to a large difference in molecular orientation and molecular packing density between phases, assigning the DPPTE molecules of the LE phase quite flat tilt angles. The picture of the supported DPPTE film, recorded by ellipsometry (Figure 8B), shows large bright LC areas as well as microdomains of the same brightness, both surrounded by the darker area of the LE phase. This bright/dark pattern reflects differences in the index of refraction because of material differences or differences in film thickness. The appearance of microdomains after the transfer process of the two-phase-coexistence lipid is a strong hint for a solidification process, described above.

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Figure 9. SE micrographs, demonstrating the effect of electron beam irradiation on uncoated DPPTE. The darker squares in the middle of the SE micrographs were previously irradiated with almost identical electron doses. (A) Beam damage at room temperature versus (B) beam damage at low temperature (-170 °C).

A quantitative evaluation of the DPPTE film in the twophase-coexistence region indicates an artificial drifting of molecules during the LB transfer. Molecules of the LE phase in the DPPTE film on the water surface get incorporated into the LC phase during the transfer of the film from the water onto the silicon wafer. Less than a quarter of the molecules in the LE phase gets translocated during this crystallization process, whereas the majority stays in the LE phase. As a consequence, the LE phase decreases in area. Beyond that, each molecule in this new LE phase has even more space available for various orientations. The molecules in the LC phase, on the other hand, stay densely packed side by side in a more upright position with a minimal spatial need of about 50 Å2/ molecule. Thus, the observed contrast between the LC and LE phases in FESEM is obviously due to local differences in packing densities and molecular orientations in between the structured DPPTE monolayer. Organic monolayers on solids are sensitive to electron beam damage. Even during the first scan at a low electron dose and at room temperature, the monolayer gets irreversibly modified by the electron beam. As a result of damage, a darkening of the previously scanned central area in the SE micrograph is detectable (Figure 9A), which may be due to adsorption of contaminants and/or formation of carbonaceous material under the influence of the electron bombardment. Imaging at low temperature (LT) indicates clearly that the total effect of electron irradiation is strongly reduced (Figure 9B). The beam electrons affect the sample on one hand directly (e.g., structural damage) and on the other hand indirectly by inducing contamina-

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vation that the darkening of preirradiated sample areas originates to a large extent from contamination due to surface diffusion. The contamination will have an even stronger effect on the contrast in SE micrographs of conventional microscopes which have by far a worse pressure and a less clean vacuum in the specimen chamber.

Figure 10. SE micrograph of DPPC on silicon wafer. The micrograph was recorded at 4 kV. Two phases are recognizable. Dark regions correspond to the LE phase, and bright areas to the LC phase.

tion by molecular debris which can readily diffuse at room temperature at the sample surface (e.g., see ref 20). The surface diffusion is strongly reduced at low temperature, and thus the contamination of the irradiated sample area is reduced correspondingly. We conclude from our obser(20) Engel, A. Microscopie E Ä lectronique en Science des Mate´ riaux Bombannes 1981; Editions du CNRS; Centre Nationale de la Recherche Scientifique: Paris, 1983; p 185.

Conclusions A new type of contrast was demonstrated in the secondary electron mode for field emission SEM at low acceleration voltage which correlates with differences in the molecular packing within a chemically homogeneous organic layer supported by a solid. Domains of the layer comparable in their thickness but different in their molecular packing density and their molecular order reveal a strong contrast in the SE mode which indicates a high sensitivity on this film property. This molecular packing contrast will raise further applications for field emission SEM to study thin organic films on solids. Acknowledgment. This project was supported by Grant Re 782/3-1, awarded by the Deutsche Forschungsgemeinschaft. The authors appreciate the expert photographic work of Mrs. G. Kiefermann and thank Mr. C. Hennesthal and Mr. M. Schenk for SFM work. The ToFSIMS measurements were done by Mr. R. Kamischke and Mr. F. Kollmer. Brewster angle microscopy and ellipsometry were performed by Mr. M. Gleiche. LA0004956