An Atomic Force Microscopy Study of Ordered Molecular Assemblies

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Langmuir 1999, 15, 8505-8513

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An Atomic Force Microscopy Study of Ordered Molecular Assemblies and Concentric Ring Patterns from Evaporating Droplets of Collagen Solutions Hideatsu Maeda JRCAT/National Institute for Advanced Interdisciplinary Research and National Institute of Bioscience and Human-Technology 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Received December 17, 1998. In Final Form: July 19, 1999

Ordered molecular assemblies spontaneously appeared from evaporating droplets of collagen solutions on glass substrates. Nanoscopic structures of the assemblies were examined using an atomic force microscope; well aligned collagen molecules oriented their ends toward the assembly surface and formed layered structures for concentrations >0.05 mg/mL, while the molecules, mostly curved, lay for concentrations 5 mg/mL, concentric ring patterns additionally appeared in the assemblies; each ring consisted of multilayers of well-aligned tilting collagen molecules. The color of the rings alternatingly changed from blue to orange when using a color-sensitive plate. The ring width increased from 3 to 120 µm with increasing collagen concentration and also increased toward the center of the patterns. The observations of temporal and spatial evolution of the concentric ring patterns with a polarizing-light microscope showed the repetitive appearance of the multilayered region which underwent a subsequent transition into a pair of blue and orange concentric rings near the droplet edge during evaporation. The formation time of one concentric ring was dependent on collagen concentration; it was 37-50 s at an early stage of the formation process.

Introduction In highly concentrated systems of rodlike molecules, the onset of various ordered phases (dependent on the concentration and ratio of particle length to diameter) has been suggested theoretically1,2 and by computer simulations3 and experimentally observed in a variety of concentrated rodlike colloidal systems.4 For example, concentrated suspensions of tobacco mosaic virus (TMV) particles (300 nm length and 15 nm diameter) have exhibited nematic, smectic, and colloidal crystalline phases,5 and monodisperse rodlike particles of β-FeOOH (typically 350 nm length and 60 nm width, although they are variable) have also exhibited smectic phases.6-8 Actually, their ordered assembly structures have been observed with an atomic force microscope (AFM) and electron microscope (EM). Zasadzinski et al.9 have visualized the distribution and orientation of TMV particles in nematic and crystalline phases by freeze-fracture electron microscopy. Using an AFM, Maeda10 has observed sideby-side intermeshed TMV particles in an ordered assembly to determine the minimum particle diameter and also found sharply bent TMV particles in zigzag and curved patterns from which he determined a critical bending angle of the individual particles. Using an EM, Watson et al.11 have examined structural assemblies of β-FeOOH par-

Figure 1. An AFM image of curved lying collagen molecules from a 0.005 mg/mL collagen solution assembling densely on the substrate.

(1) Onsager, L. Ann. N. Y. Acad. Sci. 1949, 51, 627. (2) Flory, P. J. Proc. R. Soc. London, Ser. A 1956, 234, 73. (3) Frenkel, D; Mulder, D. M. Mol. Phys. 1985, 55, 689. (4) Lekerkerkker, H. N W. NATO ASI Ser., Ser. C 1995, 460, 53. (5) Wen, X.; Meyer, R. B.; Casper, D. L. D. Phys. Rev. Lett. 1989, 63, 2760. (6) Maeda, Y.; Hachisu, S. Colloid Surf. 1983, 6, 1. (7) Maeda, Y.; Hachisu, S. Colloid Surf. 1983, 7, 357. (8) Maeda, H.; Maeda, Y. Langmuir 1996, 12, 1446. (9) Zasadzinski, J. A. N.; Sammon, M. J.; Meyer, R. B.; Cahoon, M.; Casper, D. L. D. Mol. Cryst. Liq. Cryst. 1986, 138, 211. (10) Maeda, H. Langmuir 1997, 13, 4150. (11) Watson, J. H. L.; Cardell, R. R. Jr.; Heller, W. J. Phys. Chem. 1962, 66, 1757.

ticles embedded in methacrylate and found that the individual particles had square cross sections and aligned to form an orthogonal array. Moreover, Maeda et al. have observed smectic structures of β-FeOOH using an EM7 and AFM8 which explained the origin of their iridescent colors and also observed surface structures of the individual particles therein.8 Collagen is the major protein constituent of diverse tissues such as bone, skin, tendon, and cornea, where the fibrils, consisting of collagen molecules arranged in a quasihexagonal lattice,12 frequently form a variety of macro-

10.1021/la981738l CCC: $18.00 © 1999 American Chemical Society Published on Web 09/29/1999

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Figure 2. (a) An AFM image of a molecular assembly from a 0.2 mg/mL collagen solution. (b) An enlarged AFM image of (a), where the molecular heads are seen to be closely packed forming layers (running mostly from the lower left to the upper right in this image). The arrows in (a) and (b) point out the directions of the layers. (Note that chains of the bright molecular ends, perpendicular to the arrow, are not the layers. This was confirmed under higher magnification.)

scopic ordered assemblies.13,14 Collagen is a rodlike macromolecule with a 280 nm length and a diameter of 1.2 nm15,16 and is composed of three amino acid chains wound into coaxial helices.17,18 In highly dense collagen solutions, Murthy19 has observed an isotropic to mesophase (12) Hulmes, D. J. S.; Miller, A. Nature 1979, 282, 878. (13) Fitton Jackson, S. In Treatise on Collagen; Gould, B. S., Ed.; Academic Press: London, 1968; Vol. 2. part B. (14) Harkness, R. D. In Treatise on Collagen; Gould, B. S., Ed.; Academic Press: London, 1968; Vol. 2. part A, p 247. (15) Hall, C. E.; Doty, P. J. Am. Chem. Soc. 1958, 80, 1269. (16) Hodge, A. J. In Treatise on Collagen; Ramachandran, G. N., Ed.; Academic Press: New York, vol. 1, 1967; 185, 205. (17) Ramachandran, G. N.; Kartha, G. Nature 1954, 174, 269. (18) Rich, A.; Crick, F. H. J. Mol. Biol. 1961, 3, 483. (19) Murthy, N. S. Biopolymers 1984, 23, 1261.

Figure 3. (a) A topographic AFM image for a molecular assembly from a 2 mg/mL collagen solution where many thin curved bandlike structures are visible. (b) An enlarged AFM image of (a), where the upward orienting collagen molecules were laterally connected to form curved layers, the layer edges corresponding to the bands in (a). (c) A height profile obtained by an AFM sectioning along the arrow indicated in (b) showed peaks corresponding to the layer edges.

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Figure 5. AFM images of molecular assemblies from 16 mg/ mL collagen solutions at slow evaporation: (a) the collagen molecules oriented their end toward the assembly surface to be regularly packed, and (b) the protruding (or bright in this image) molecules were separately visible in a closely packed molecular assembly (dark background).

Figure 4. AFM images of characteristic molecular assemblies formed at slow evaporation: (a) An assembly for a 5 mg/mL collagen solution where undulated and rolled layers are visible. (b) An enlarged image of the rolled layers for a 8 mg/mL collagen solution. (c) An enlarged image of the undulated (like a sine wave) layers for a 5 mg/mL collagen solution.

transition, and Giraud-Guille20,21 has observed cholesteric liquid crystalline domains. Using an EM, arced patterns characteristic of the cholesteric phases have been observed

in collagen fibrils of decalcified bone by Giraud-Guille22 and in collagen gels stabilized under ammonia vapor by Besseau et al.23 Baranauska et al.24 have obtained AFM images exhibiting the triple helical structure of collagen molecules (regularly arrayed in a native fibril) with main periods of 1.15 and 8.03 nm and a molecular diameter of 1.43 nm. In the present paper, we formed ordered molecular assemblies by evaporating collagen solutions on substrates (20) Giraud-Guille, M. M. J. Mol. Biol. 1992, 224, 861. (21) Giraud-Guille, M. M.; Besseau, L. Connect. Tissue Res. 1997, 37, 183. (22) Giraud-Guille, M. M. Mol. Cryst. Liq. Cryst. 1987, 153, 15. (23) Besseau, L.; Giraud-Guille, M. M. J. Mol. Biol. 1995, 251, 192. (24) Baranauskas, V.; Vidal, B. C.; Parizotto, N. A. Appl. Biochem. Biotechnol. 1998, 69, 91.

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Figure 6. Polarizing optical microscope (PLM) images (with a color-sensitive plate) for concentric ring patterns from (a) 19, (b) 16, and (c) 12 mg/mL collagen solutions (scale bars: (a) 0.5 mm, and (b) and (c) 100 µm).

and examined their structures using an AFM. We found ordered molecular arrangements for collagen concentrations >0.05 mg/mL and additionally found concentric ring patterns in the ordered assemblies for concentrations >5 mg/mL. We also briefly discuss a possible mechanism for the pattern generation.

Experimental Section Collagen solutions were prepared by dissolving purified proctase-solubilized collagen (consisting of 90% type I and 10% type III) from steer skin (Nippi Co. Ltd., Tokyo, Japan) into 0.20.5 M acetic acid solutions (about 5% of the collagen molecules

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are fragmented25). Collagen concentrations used were 0.005, 0.05, 0.1, 0.2, 0.5, 2, 5, 8, 12, 16, 19, and 23 mg/mL. To form ordered molecular assemblies, droplets of collagen solutions (30, 40, and 50 µL) were evaporated on slide glass substrates (manufactured by the Four Cault process, Matsunami Glass Ind. Ltd., Japan) in glass cells. The substrate surface was cleaned by extensively washing (mechanically shaking) first in a nonionic surfactant solution (which was three times exchanged for fresh solution) for 30-45 min and afterward in pure water (which was also exchanged similarly). The completion times of evaporation increased with increasing collagen concentration; they were 2-4 h when the cells were open, and 4-12 h when the cells were covered (called “slow evaporation” in this text). At slow evaporation rates, only the concentric rings near the innermost of the pattern were observed with polarizing light microscopy (PLM). In this paper, only the assembly structures of collagen shown in Figures 4 and 5 were formed during slow evaporation. Assembly structures of collagen molecules formed on substrates were examined using an AFM (Nanoscope IIIa, Digital Instruments Inc., Santa Barbara, CA), which was operated in the tapping mode. The microfabricated cantilevers of Si3N4 (Nanoprobes, Digital Instruments Inc.) used have a total length of 123 µm and a spring constant of 27-70 N/m. Calibration of the scanner was carried out using an optical calibration grating supplied with the system used. Macroscopic patterns appearing in the molecular assemblies were examined by a polarizing optical microscope (Olympus, BH2), and the pattern formation processes were analyzed using a videotape recording system (Sony, SVO260).

Results and Discussion We examined the assembly structures for collagen solutions on the substrates and found that the molecules were well aligned for concentrations >0.05 mg/mL. This suggests a critical concentration required for the ordered assembly formation, which is consistent with the onset of an isotropic to mesophase transition in concentrated collagen solutions found by Murthy.19 So far, AFM images for collagen molecules have been obtained to investigate the fibril formation mechanism,26 to compare with those of other biopolymers,27,28 and to examine the resolution of the AFMs developed.29 Figure 1 shows a molecular assembly from a 0.005 mg/mL collagen solution on the substrate, where many curved molecules lie and are overlapped with one another. The molecular (half-height) width observed was 6.8-7.4 nm, which is much larger than the nominal diameter of collagen, 1.2 nm. This results from a finite volume effect of the probe tip apex for the AFM imaging, because the apparent molecular (halfheight) width was easily estimated to be 7.03 nm from a height profile formula considering such a tip volume effect10 (using an apex radius of 10 nm for a probe tip used and the collagen diameter of 1.2 nm), which is in good agreement with the observed molecular width. Figure 2a shows a molecular assembly from a 0.2 mg/ mL collagen solution, where the molecules oriented their ends toward the assembly surface. The bright parts must be a group of the molecular ends sticking out slightly, as the molecular widths were significantly larger than the apparent molecular width shown in Figure 1. From its enlarged image (Figure 2b), the molecules were found to further form layers by laterally connecting themselves over 50-100 nm (the layers extended in the directions (25) Ebihara, T.; Iijima, K.; Sato, K.; Someki, I.; Kuwaba, K.; Hattori, T.; Irie, N. Connect. Tissue 1999, 31, 17. (26) Gale, M.; Pollanen, M. S.; Markiewicz, P.; Goh, M. C. Biophys. J. 1995, 68, 2124. (27) Mclntire, T. M.; Brant, D. A., Biopolymers 1997, 42, 133. (28) Anselmetti, D.; Dreier, M.; Luthi, R.; Richmond, T.; Meyer, E.; Frommer, J.; Guntherodt, H.-J. J. Vac. Sci. Technol. 1994, B12 (3), 1500. (29) Gustafsson, M. G. L.; Clarke, J. J. Appl. Phys. 1994, 76, 172.

Figure 7. Innermost (or maximum) ring width against collagen concentration for solution droplets of 30, 40, and 50 µL.

along the arrows indicated in Figure 2). In the molecular assembly for a 2 mg/mL collagen solution (Figure 3a), many undulated bandlike entities (which were bright and running approximately from the top to the bottom in the image) were observed. Its enlarged image (Figure 3b) shows that each band was the edge of an undulated layer consisting of collagen molecules which stuck out slightly; the lateral length of the layer was 300-700 nm. To further examine the band structure, we obtained a height profile (Figure 3c) along the arrow indicated in Figure 3b, which showed peaks with various widths corresponding to the bands in Figure 3b; the wide peaks further consisted of thinner ones, and the minimum peak separation resolvable with the probe tip was approximately 5.7 nm. Moreover, the profile further showed that the protruding lengths of the layers (or molecules) were approximately 1-1.5 nm, and the molecules were also packed among the protruding layers. The origin of the molecular end matching observed is not clear; it may arise on the surface of the concentrated edge region where the molecules are well oriented or arise at the edge line when discharged (where local forces act, as described later in Figure 12). It is of interest that TMV particles lay in ordered assemblies on substrates,10 unlike the collagen molecules. Figure 4a shows a molecular assembly for a 5 mg/mL collagen solution (formed at slow evaporation, ∼4 h); undulated and rolled layers were observed. The rolled layers were more clearly shown for a 8 mg/mL collagen solution (Figure 4b), where the layer width was 25-40 nm, and the hollow diameter was 0.1-0.3 µm. Furthermore, undulated layers (like a sine wave) were observed for a 5 mg/mL collagen solution (formed at slow evaporation, ∼4 h) as shown in Figure 4c; the spatial periodicity was approximately 5 µm. (More magnified AFM images showed the groups of the molecular ends in these layers.) Such a layer flexibility decreased with increasing collagen concentration; actually an assembly for a 16 mg/mL collagen solution (formed at slow evaporation, ∼7 h) showed that the molecular ends were regularly arranged

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Figure 8. (a) A PLM image (without a color-sensitive plate) for an intermeshing region of zigzag extinction lines appearing in the concentric ring pattern for a 16 mg/mL collagen solution; the parallel, opposite tapered ends come from the neighboring zigzag extinction lines, respectively; the bright (long arrow) and dark (short arrow) regions correspond to the blue and orange regions in the PLM image with a color-sensitive plate, respectively (scale bar: 100 µm). (b) A PLM image (with a color-sensitive plate) of dislocation in the concentric ring pattern for a 16 mg/mL collagen solution (scale bar: 100 µm).

as shown in Figure 5a. In addition, the individual molecular ends were observed in the assembly for a 16 mg/mL collagen solution (Figure 5b), as they happened to stick out with a separation of 10-40 nm, which is sufficient to be resolved by the probe tip used (having an apex radius of approximately 10 nm). The assembly structures of collagen (on the substrates) are the structures formed in the nonequilibrium state (or the evaporation process). The structural variation presented may couple with a moving

behavior of the droplet edge line (induced by evaporation), which may depend on the original collagen concentration. Concentric ring patterns appeared at collagen concentrations >5 mg/mL; panels a-c of Figure 6 are examples of the ring patterns for 19, 16, and 12 mg/mL collagen solutions, respectively. The alternating blue and orange rings were observed when a PLM with a color-sensitive plate was used. This indicates an alternating change in molecular orientations from one ring to the next, because

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Figure 9. A PLM image of the concentric ring pattern for a 16 mg/mL collagen solution. Approximately parallel grooves were visible in each ring; their average direction alternates at every ring (scale bar: 100 µm).

when introducing a color-sensitive plate at 45° between the polarizer and analyzer, the transmitted color from molecules oriented parallel to the plate is blue, and that from molecules oriented perpendicular to the plate is orange.20 The concentric rings were successively formed inward from the outer region of each pattern during evaporation (more details are shown in Figure 12), while no rings appeared in the central region. The number of the rings was frequently in the range of 100-300. The ring width increased toward the center of each pattern, e.g., from 20 (outer) to 55 µm (inner) for a 16 mg/mL collagen solution and from 7 (outer) to 15 µm (inner) for a 8 mg/mL collagen solution. The ring width also increased with increasing collagen concentration and with increasing droplet volume; e.g., as shown in Figure 7 the innermost (or maximum) ring widths (which are the average of the blue and orange ring widths) were determined against collagen concentration for 30, 40, and 50 µL droplets. These results suggest that the ring width is not directly dependent on the initial collagen concentration (but on the collagen concentration at the droplet edge presented later in Figure 12). Graud-Guille also observed concentric ring patterns in the textures which appeared from droplets (50 µL in volume) of acid solutions of collagen (10 mg/mL) on glass slides using a PLM.22 Figure 8a is a PLM image of a characteristic pattern (without a color-sensitive plate) appearing at every 90° in the concentric ring pattern for a 16 mg/mL collagen solution; the width of each ring gradually tapers and finally ends therein, although the orientation of the ends is alternately opposite from one ring to the next. The characteristic pattern is apparent, because it is independent of sample rotation in the PLM (indicating that the local molecular orientation with respect to the tangential line of the ring is almost constant). This apparent pattern corresponds to the color alteration regions observed in the left and right regions of the concentric ring pattern of Figure 6a, where the blue (bright in Figure 8a, long arrow) rings also taper and end to alternate with orange (dark in Figure 8a, short arrow). The opposite tapered ends are the branches of the neighboring “zig-zag extinction lines”,30 respectively (which extend in the concentric ring pattern); Keller also observed zigzag lines in a PLM image of a polymer spherulite and explained the origin of the zigzag shape using a math(30) Keller, A. J. Polym. Sci. 1955, 17, 291.

Figure 10. (a) An AFM image of a part of two successive concentric rings for an 8 mg/mL collagen solution; the bright regions indicate the ring ridges, and the dark regions between them indicate the valleys. (b) A radial height profile of the successive concentric rings of (a), where the peaks corresponding to the ring ridges were visible.

ematical model.30 The concentric ring patterns and zigzag extinction patterns found in this work are very similar to banded and zigzag extinction patterns, respectively, found by Giraud-Guille20,21 for collagen solutions placed between a slide and partially sealed cover glasses. She explained the banded and zigzag extinction patterns in terms of undulating molecular orientations and a small angular shift of the undulating structures, respectively.20,21 Also in the zigzag extinction pattern observed in this work, the ring ending behavior can be explained similarly by a gradual rotation of undulating molecular orientations occurring in the concentric ring patterns. In addition, dislocations were frequently observed in the concentric ring patterns as shown in Figure 8b, where a blue ring actually (not apparently) ended. The concentric ring structures were examined using a PLM and AFM. Figure 9 shows an enlarged PLM image (without the color-sensitive plate) of the concentric ring pattern for a 16 mg/mL collagen solution. Many, almost parallel, grooves were observed on each ring surface, whose directions alternatingly changed at every ring, approximately 15° with respect to the tangential lines of rings. The direction of the grooves was not coincident with that

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Figure 11. (a) A topographic AFM image of a ring in the concentric ring pattern for an 8 mg/mL collagen solution. The following features can be seen: (1) a grooved structure, consisting of alternating bright (that is, higher) and dark (lower) stripes running diagonally (as indicated by the arrow), and (2) thin undulated stringlike entities (running approximately parallel from top to bottom), corresponding to the layer edges of well-aligned collagen molecules. (b) An enlarged AFM image of (a), where the layers are observed to run diagonally (from upper left to lower right) with a tilting (from upper right to lower left). The direction of the arrow in this image corresponds to that shown in (a).

of the molecular orientations (as clearly seen from Figure 11). Figure 10a shows a part of the successive concentric rings for a 8 mg/mL collagen solution; the central region of each ring raised like a ridge, and the surface of each ring had grooves, corresponding to those in Figure 9. A radial height profile for the successive rings (Figure 10b) showed a curve like a sine wave; the separation between two peaks in the curve was 6.7 µm (approximately equal

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Figure 12. (a-c) PLM images (with a color-sensitive plate) of concentric ring patterns for a 16 mg/mL collagen solution at different advanced time stages in the formation process.

to the ring width). A topographic AFM image of a concentric ring for a 8 mg/mL collagen solution is shown in Figure 11a. In this image, the following characteristic structures were observed: (1) alternating bright and dark striations with a periodicity of approximately 0.6 µm (running diagonally as indicated by the arrow in this image), corresponding to the grooved structure shown in Figures 9 and 10a, and (2) thin undulated stringlike entities (running approximately parallel from top to bottom in this image). The angles between the directions of the strings and striations were approximately 40°. Figure 11b, which is an enlarged AFM image of Figure 11a, showed that the strings were the edges consisting of the tilting (from upper right to lower left) layers (running diagonally from upper left to lower right in this image). The direction of the arrow indicated in this image corresponds to that of the striations shown in Figure 11a.

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The ring color must be related to the layer tilting. The typical and resolvable minimum layer separations (or layer widths) were approximately 18 and 5 nm, respectively. Considering the formation process of the concentric ring patterns described below (Figure 12), the characteristic molecular assemblies of collagen presented can be formed in the following manner: the collagen solutions around the droplet edge were highly concentrated during evaporation, and the molecules were well aligned therein. With the inward movement of the edge line (induced by evaporation) the aligned molecules in the concentrated region were successively discharged onto the substrates to form a wet film which resulted in the observed molecular assemblies. The formation process of the ring patterns was observed for a 16 mg/mL collagen solution using the PLM with color-sensitive plate (Figure 12). A yellow band (ring as a whole) appeared near the droplet edge, from which a pair of blue and orange concentric rings were formed in the following manner: (a) The left half (purple red) region is a part of a collagen solution droplet, and the right half region is a part of the concentric ring pattern formed already. A yellow band (ring as a whole) appeared near the droplet edge (arrow). (b) When the width of the yellow ring increased up to a critical value (which is approximately 1-2 times as wide as those of the resulting concentric rings, dependent on collagen concentration), a small purple domain (arrow) abruptly appeared in the inner half of the yellow ring to grow along the ring (downward from the top of this image). (c) After a short lapse of time, the purple domain began to change its color into blue (long arrow) to grow (downward from the top of this image) with decreasing height. Simultaneously, the outer half of the yellow ring began to change its color to orange or red (short arrow) to grow with narrowing width, and the yellow ring appeared again at the droplet edge receding more inward with evaporation. The actions of (a)-(c) were repeated until evaporation was completed (it took about 2-4 h, dependent on the collagen concentration). The characteristic time required for the formation of one concentric ring decreased with decreasing collagen concentration and with proximity to the center of the pattern; the times were 13 (inner) to 37 s (outer) for a 8 mg/mL collagen solution and 28 (inner) to 50 s (outer) for a 16 mg/mL collagen solution. The appearance of the (yellow) ordered regions near the droplet edge and its subsequent transition into a pair of distinct (blue and orange) concentric rings were repeated during evaporation (Figure 12), suggesting the onset of a mechanical instability near the droplet edge region. The forces acting on the edge line (contact line31) receding with (31) Dussan, V. E. B. Annu. Rev. Fluid Mech. 1979, 11, 371.

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evaporation are mainly (inward) surface tension of the solution and the (outward) frictional force coming from the mechanical structures of the ordered edge region. The mechanical instability may be generated by the competition of such local opposing forces. So far, concentric ring patterns have been observed in colloidal precipitations of various inorganic salts (Liesegang rings)32,33 and in bacterial colonies.34,35 The concentric ring patterns observed for collagen solutions are new examples of spatially rhythmic patterns generated by a mechanical instability. (More details of the pattern formation will be published elsewhere.) Summary We observed characteristic molecular assemblies formed spontaneously from evaporating droplets of collagen solutions on glass substrates using an AFM and PLM. The results are summarized as follows: (1) For concentrations >0.05 mg/mL, collagen molecules oriented their ends toward the assembly surface to be regularly packed and to form layered structures, while they lay for concentrations 5 mg/mL, concentric ring patterns appeared in the molecular assemblies (the ring width increased with collagen concentration and with proximity to the center of each pattern). However, when the evaporation time was two or three times longer, only the concentric rings near the innermost of the pattern were observed using the PLM. These suggest the existence of minimum collagen concentration and minimum evaporation rates (dependent on collagen concentration) for the pattern generation. (4) Each concentric ring consisted of multilayers of well-aligned tilting collagen molecules; the direction of the layers alternatingly changed from one ring to the next, probably causing alternating blue and orange rings when using a color-sensitive plate. (5) The observations of the pattern formation process showed the repetitive appearance of an ordered layered region undergoing a subsequent transition to a pair of blue and orange concentric rings near the droplet edge. This action, connected with evaporation, suggests the onset of a mechanical instability near the droplet edge was responsible for the pattern generation. LA981738L (32) Liesegang, R. E. Naturwiss. Wochenschr. 1896, 11, 353. (33) Stern, K. H. Natl. Bur. Stand. (U.S.) Spec. Publ. 1967, No. 292. (34) Budrene, E. O.; Berg, H. C. Nature 1991, 349, 630. (35) Fujikawa, H. Physica A 1992, 189, 15.