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In Situ Stepwise Surface Analysis of Micropatterned Glass Substrates in Liquids Using Functional Near-Field Scanning Optical Microscopy Kristine E. Schmalenberg,†,| Deanna M. Thompson,‡,| Helen M. Buettner,‡ Kathryn E. Uhrich,*,† and Luis F. Garfias§ Department of Chemistry and Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, New Jersey 08854, and Bell Laboratories, Lucent Technologies, Murray Hill, New Jersey 07974 Received April 29, 2002 Imaging of two adsorbed proteins on micropatterned glass substrates in air and liquid was conducted using atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM). The micropatterned glass substrates were prepared in five steps and evaluated by NSOM at each processing step. The micropatterned samples consisted of alternating 10 µm wide laminin stripes and 20 µm wide bovine serum albumin stripes. The samples containing both protein stripes were imaged “dry” (i.e., in air) using conventional contact AFM, then imaged dry with NSOM, and finally imaged in the hydrated form (i.e., in liquid) using NSOM. When samples were imaged in air, similar topographical features were observed using both AFM and NSOM. However, after the sample was rehydrated and imaged with NSOM in solution, the topography changed significantly. Having confirmed that the “real” surface topography was obtained while the proteins were in the hydrated form, in situ NSOM was used to detect changes in surface topography with nanometer resolution as a function of the micropatterning process. Although lithographic micropatterning provides chemical cues, in situ NSOM demonstrates that topographical cues are also an important aspect of the surface that serves to regulate cellular attachment and migration.
Introduction Chemical cues have been used successfully to control cell attachment,1-4 spreading,5-11 and growth.12,13 Specifically, photolithographic patterning and sequential protein deposition have been shown to differentially enhance or repel attachment of peripheral nerve tissue components such as neurons and Schwann cells.11,14-16 Laminin, a protein found in the extracellular matrix, enhances neuronal and Schwann cell attachment and migration,17-20 whereas bovine serum albumin (BSA) discourages cellular * To whom correspondence should be addressed. † Department of Chemistry. ‡ Department of Chemical and Biochemical Engineering. § Lucent Technologies. | Authors contributed equally to this paper. (1) Britland, S.; Clark, P.; Connolly, P.; Moores, G. Exp. Cell Res. 1992, 198, 124-129. (2) Defife, K.; Colton, E.; Nakayama, Y.; Matsuda, T.; Anderson, J. J. Biomed. Mater. Res. 1999, 45, 148-154. (3) Folch, A.; Toner, M. Biotechnol. Prog. 1998, 14, 388-392. (4) Fraser, S. E. Dev. Biol. 1980, 79, 453-464. (5) Brunette, D. Exp. Cell Res. 1986, 164, 11-26. (6) Brunette, D.; Chehroudi, B. J. Biomech. Eng. 1999, 121, 49-57. (7) Chen, C.; Mrksich, M.; Huang, S.; Whitesides, G.; Ingber, D. Biotechnol. Prog. 1998, 14, 356-363. (8) Clark, P.; Connolly, P.; Curtis, A.; Dow, J.; Wilkinson, C. J. Cell Sci. 1991, 99, 73-77. (9) Craighead, H.; Turner, S.; Davis, R.; James, C.; Perez, A.; St. John, P.; Isaacson, M.; Kam, L.; Shain, W.; Turner, J.; Banker, G. J. Biomed. Microdevices 1998, 1, 49-64. (10) Curtis, A.; Clark, P. Crit. Rev. Biocomp. 1992, 5, 343-362. (11) Hammarback, J.; Letourneau, P. Dev. Biol. 1986, 117, 655662. (12) Carter, S. Nature 1965, 208, 1183-1187. (13) Chen, C.; Mrksich, M.; Huang, S.; Whitesides, G.; Ingber, D. Science 1997, 276, 1425-1428. (14) Kleinfeld, D.; Kahler, K.; Hockberger, P. J. Neuroscience 1988, 8, 4098-4120. (15) Matsuzawa, M.; Pitember, R.; Krauthamer, V. Brain Res. 1994, 667, 47-53. (16) Matsuzawa, M.; Liesi, P.; Knoll, W. J. Neurosci. Methods 1996, 69, 189-196.
attachment.21 By use of a combination of these two proteins, micropatterned substrates are created with alternating permissive (laminin) and less permissive (BSA) regions serving to spatially control cell behavior. The protein micropatterns were observed using both fluorescence and phase microscopy.22 Because the protein stripes are visualized using phase microscopy, regions which appear phase-dark and phase-light on the surface are due to density differences and are likely to be threedimensional features, suggesting that physical as well as chemical cues may influence cellular attachment and growth on these surfaces. Most research to date has focused on the importance of chemical cues on the protein micropatterns, while not assessing the impact of surface topography. Most methods for measuring surface topography require contact with the surface and a “dry” sample. Although atomic force microscopy (AFM) can measure surfaces in liquid, the method requires contact with the surface using tapping, or intermittent contact, mode. Any method that contacts the surface may change the conformation of the protein on the surface. The proteins patterned onto the surfaces are extremely sensitive to environmental changes in addition to being very pliable and therefore subject to deformation upon contact. Near-field scanning optical microscopy (NSOM)23 is a scanning probe microscopy technique that noninvasively (17) Manthorpe, M.; Engvall, E.; Ruoslahti, E.; Longo, F. M.; Davis, G. E.; Varon, S. J. Cell Biol. 1983, 97, 1882-1890. (18) Paulsson, M.; Saladin, K.; Engvall, E. J. Biol. Chem. 1991, 266, 17545-17551. (19) Son, Y.-J.; Thompson, W. J. Neuron 1995, 14, 133-141. (20) David, S.; Braun, P.; Jackson, D.; Kottis, V.; McKerracher, L. J. Neurosci. Res. 1995, 42, 594-602. (21) Schwab, M.; Kapfhammer, J.; Bandtlow, C. Annu. Rev. Neurosci. 1993, 16, 565-595. (22) Thompson, D.; Buettner, H. Tissue Eng. 2001, 247-265. (23) Betzig, E.; Trautman, J. Science 1992, 257, 189-195.
10.1021/la0204038 CCC: $22.00 © 2002 American Chemical Society Published on Web 10/03/2002
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visualizes surface topography.24 In conventional optical NSOM, shear-force feedback is commonly used to maintain a constant distance between the probe and the substrate surface.25 Alternately, NSOM can combine the feedback information from the tuning fork in noncontact mode to obtain both optical and topographical images of nanoscale solid surfaces in liquid.26,27 While both the conventional optical and tuning fork methods can be used to maintain feedback, the tuning fork method allows stable imaging in liquids for several hours28 by using an oscillatory damping feedback loop to maintain a constant distance from the surface. For noncontact imaging in liquids or gases, NSOM topographical analysis offers advantages over AFM and is a comparable technique to evaluate surface topography that can be operated in several modes depending on the surface. Noncontact mode in AFM produces topographic images of delicate dry surfaces29 but cannot reproducibly maintain stable feedback for long periods of time in liquids. Two other modes used by AFM in liquids, contact and tapping (or intermittent contact), require minimal contact between the probe and the substrate.30-33 Images of bacteria, for example, have been obtained using tapping, or intermittent contact, mode AFM.34,35 A significant advantage of NSOM imaging is the maintenance of stable noncontact feedback for several hours27,28 while the probe is immersed in liquids. Especially for biological systems, reliable imaging techniques are needed to observe cells or proteins in aqueous media with high resolution and high reproducibility. To paraphrase a recent review that examined the role of NSOM in cell biology, the most important technical challenge is the ability of an NSOM instrument to perform under physiologically relevant conditions.36 By topographically imaging lithographically proteinpatterned substrates in liquid, we determined the physical features that a cell encounters in vitro. With the imaging information, specific cell responses to surfaces can be predicted and then the surfaces engineered to optimize cell-surface interactions. This paper describes the novel use of NSOM for noncontact topographical imaging of proteins patterned onto glass substrates, where the imaging was performed in solution. By observing the surfaces after the individual processing steps involved in depositing protein on micropatterned surfaces, we identified the key steps that create the resulting topographical features. This paper discusses the optical and topographical analysis of stepwise construction of patterned glass substrates and the resultant protein pattern that serves to regulate cellular attachment and migration. (24) Dunn, R. C. Chem. Rev. 1999, 99, 2891-2927. (25) Paesler, M.; Moyer, P. Near-Field Optics: Theory, Instrumentation, and Applications, 1st ed; John Wiley & Sons: New York, 1996. (26) Garfias, L.; Siconolfi, J. J. Electrochem. Soc. 2000, 147, 25252531. (27) James, P.; Garfias-Mesias, L.; Moyer, P.; Smyrl, W. J. Electrochem. Soc. 1998, 145, L64-L66. (28) Garfias-Mesias, F.; Smyrl, W. J. Electrochem. Soc. 1999, 146, 2495-2501. (29) Yang, Z.; Calloway, J.; Yu, H. Langmuir 1999, 15, 8405-8411. (30) McMaster, T.; Miles, M.; Shewry, P.; Tatham, A. Langmuir 2000, 16, 1463-1468. (31) Cappella, B.; Baschieri, P.; Ruffa, M.; Ascoli, C.; Relini, A.; Rolandi, R. Langmuir 1999, 15, 2152-2157. (32) Cacciafesta, P.; Humphris, A.; Jandt, K.; Miles, M. Langmuir 2000, 16, 8167-8175. (33) Mueller, H.; Butt, H.-J.; Bamberg, E. Langmuir 2000, 16, 95689570. (34) Camesano, T.; Natan, M.; Logan, B. Langmuir 2000, 16, 45634572. (35) Lister, T.; Pinhero, P. Langmuir 2001, 17, 2624-2628. (36) de Lange, F.; Figdor, C. J. Cell Sci. 2001, 114, 4153-4160.
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Materials and Methods Sample Preparation. Microlithography. No. 1 glass coverslips (12-545-101, Fisher Scientific, Springfield, NJ) were cleaned with Alconox and rinsed three times with 18 MΩ water. Cleaned coverslips were acid-etched in 9:1 v/v sulfuric acid and 30% hydrogen peroxide for 20 min. Next, the samples were rinsed twice with 18 MΩ water, followed by a third rinsing in ethanol. Acid-etched samples were dried under a filtered stream of air and stored in Parafilm-sealed Petri dishes prior to use. Lithography. In a clean room at the Rutgers University Microelectronics Research Laboratory, acid-etched coverslips were spin-coated with 180 µL of hexamethyldisilazane (HMDS; 34020-B, PCR Inc., Gainesville, FL) for 30 s at 3000 rpm using a Laurell Technologies WS 400 process controller spin-coater. Next, 180 µL of photoresist (PR; Shipley Microposit S1318, Microlithography Chemical Corp., Newton, MA) was spin-coated onto the surface for 30 s at 3000 rpm. Coated samples were baked at 110 °C for 10 min to remove residual solvents. The PR-coated samples were placed into a Karl Suss MJB 3 Vt contact-exposure tool and irradiated with light at 254 nm for 9 s through a 10 µm (transparent)/20 µm (opaque) striped photomask (Infinite Graphics, Salem, NH). Photoexposed regions of PR were removed by gently swirling the sample for 30 s in a 150 mL beaker of aqueous base developer (Microposit M319, Microlithography Chemical Corp.). Patterned samples containing alternating regions of glass and PR were dried with filtered N2, placed in Petri dishes, sealed with Parafilm, and covered in aluminum foil for protection from further exposure to light. Sample Mounting. Individual PR-patterned coverslips were secured to the bottom of a glass dish with epoxy (Extra Fast Setting epoxy, Harcros Chemicals Inc., Belleville, NJ). After the epoxy was mixed, it was sparingly applied to the bottom of the micropatterned glass coverslip and then placed in a glass dish. The mounted sample was placed under vacuum (∼30 mmHg) for 30 min, which allowed the sample to cure and prevented any residual solvent in the epoxy from depositing on the micropatterned sample. Following mounting, the sample was stored in an aluminum covered, Parafilm-sealed Petri dish overnight. Near-Field Scanning Optical Microscopy. Tip Preparation. All NSOM probe tips were prepared from type F-AS optical fiber (SiO2, 3.7 µm in core diameter with 125 ( 2 µm of cladding and 245 ( 15 µm of polymer coating) obtained from Newport Corp. The optical fibers were pulled with a commercial pulling machine (Sutter Instruments) and then observed under a microscope using a 63× objective to monitor the probe shape before mounting on the tuning fork. The apex of the probes was between 50 and 100 nm. More details about the preparation of the nanoprobes used to perform these tests will be reported in the future.37 Imaging. The topographical and optical images were performed on a near-field scanning optical microscope with a unique, custommade head incorporating a tuning fork transducer. This modification was described in earlier publications and allows for imaging in liquids with high resolution.26-28 The experimental setup is shown in Figure 1. An optical fiber nanoprobe is used for obtaining both the surface topography and the optical images while focusing a 480 nm laser beam onto the sample surface. The optical image is obtained by using a three-way connector that allows the laser light go through the nanoprobe to illuminate the sample; the reflected light from the sample travels into the tip and back into the same fiber and is finally focused into the photodetector. By collecting the light in this way, one can avoid the “shadowing” effect encountered while collecting in the reflection mode in the far-field38 if the sample is dry. Collecting in the far-field while the sample is immersed in liquids yields poor imaging because of the reduced light collected caused by scattering and by artifacts created by any liquid movement. The nanoprobe was glued onto one arm of the tuning fork in a manner similar to the feedback procedure developed by Karrai and Grober39,40 and van Hulst.41,42 The fork was rigidly attached (37) Garfias-Mesias, L.; Weaver, J.; Buechler, M.; Smyrl, W. J. Electrochem. Soc., in preparation. (38) Weston, K.; Buratto, S. J. Phys. Chem. B 1997, 101, 5684-5691. (39) Karrai, K.; Grober, R. Appl. Phys. Lett. 1995, 66, 1842-1844. (40) Karrai, K.; Grober, R. Ultramicroscopy 1995, 61, 197-205.
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Figure 1. Schematic, simplified drawing of the near-field scanning optical microscope used for optical and topographical measurements. to a piezoelectric vibrator (the driving piezo), which normally vibrates at a frequency of 33 kHz with an amplitude of nearly 0.01 nm. The driving piezo vibrates the tuning fork and the optical fiber nanoprobe together, with the latter positioned perpendicular to the sample plane. The shear-force feedback method28,38 was used to maintain a constant distance between the nanoprobe and the sample, avoiding any contact.23,43 The tip-to-sample distance was regulated by monitoring the damping of the nanoprobe oscillations through a feedback control loop. To achieve feedback, the driving piezo signal and the generated electric signal in the fork were synchronized with the lock-in amplifier. The frequency at which this electrical signal is maximum is normally referred to as the “resonance frequency”. When a dry sample was imaged, the resonance frequency for this tip-fork configuration was normally 22 kHz. In liquids, the tip encountered greater viscous damping in the near-field and as a consequence, the resonance frequency was 100-200 Hz lower than those frequencies measured in air. This method is noncontact with a typical interaction distance between 10 and 30 nm in air.25 The tip-to-sample interaction distance in liquids was still maintained at 10-20 nm, approximately the same order of magnitude as imaging in air. In the case of the in situ experiments, the NSOM tip was “incubated” (i.e., wetted) in 2 mL of 18 MΩ water overnight to ensure that the tip was fully wetted prior to imaging. In preparation for imaging, the mounted and wetted sample was fixed to the NSOM scanner, and then the tip was brought into feedback. Once the tip was within stable feedback range (∼9 µm), the tip was retracted and more liquid was added to prevent evaporation during scanning, as significant volume changes affect the feedback after several hours.28 With this method, feedback control in liquids is robust and remains stable for hours.27 A 60 × 60 µm image was then obtained at each step with 200 pixel resolution. Typically, scanning rates were 6 µm/s when feature heights were in the nanometer range as noted for samples containing undeveloped PR. Scanning rates were 3 µm/s when feature heights were in the micrometer range, typical for samples with PR. The driving piezo was set between 0.5 and 1.0 mV, and the resonance frequency was 20 kHz.28,40 All measurements were performed at room temperature. Atomic Force Microscopy. Atomic force microscopy was performed with a commercial system (Explorer from Thermomicroscopes-Veeco). To directly compare the data obtained with AFM and NSOM, the same scanning system described in the (41) Ruiter, A.; Veerman, J.; van der Werf, K.; van Hulst, N. Appl. Phys. Lett. 1997, 71, 28. (42) Rensen, W.; van Hulst, N.; Ruiter, A.; West, P. Appl. Phys. Lett. 1999, 75, 1640-1642. (43) Toledo-Crow, R.; Yang, P.; Chen, Y.; Vaez-Iravani, M. Appl. Phys. Lett. 1992, 60, 2957-2959.
Figure 2. Schematic of the in situ process for creating defined protein patterns. Step 1: The micropatterned glass substrate (A) with alternating regions of glass and photoresist is incubated in AMPS solution. Step 2: The AMPS-rich sample (B) is incubated in a glutaraldehyde solution to cross-link the AMPS. Step 3: The substrate (C) is incubated in a laminin solution. Step 4: The remaining photoresist on D is removed with acetone. Step 5: BSA is adsorbed onto the surface of E. Following processing, the surface of F contains alternating 10 µm lamininrich and 20 µm BSA-rich stripes. previous paragraphs was used to obtain the AFM images. By use of the identical scanning system, changes in topography will be due only to the tip-surface interaction or the environment (dry vs wet) and not to potential differences in calibration in the X-Y or Z-direction as both techniques used the same scanning system. For the AFM studies, the conventional feedback method26,37 was used in contact mode using a cantilever made of silicon nitride (Si3N4). A sensor response curve is obtained immediately after engaging into feedback to generate a straight line with a slope of 0.28 nN/nm. Substrate Imaging and Preparation. A schematic of processing steps 1-5 described in detail below, is depicted in Figure 2. Step 1: (Aminopropyl)triethoxysilane Treatment. The mounted sample was rinsed with 18 MΩ water and imaged at 3 µm/s prior to any processing step to obtain a baseline measurement of surface features (Figure 3). After imaging, the water was removed and the sample was shaken (600 rpm) in approximately 2 mL of 1% (aminopropyl)triethoxysilane (AMPS; A3648, Sigma Chemical, St. Louis, MO) aqueous solution for 2 min. The AMPS solution was removed, and the sample was rinsed with 18 MΩ water and imaged in water at 3 µm/s resulting in sample B (Figure 2).
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Figure 3. The image is a micropatterned glass substrate (A) with alternating 20 µm wide PR stripes and 10 µm wide regions of exposed glass. The NSOM instrument was operated in a noncontact mode in an aqueous environment to image a 60 µm square area. (A) Topographical image. (B) Optical image confirming the topographical features. (C) Three-dimensional topographical image where L is the width of laminin-rich stripes, B is the width of PR or BSA-rich stripes, H is the differential height between laminin and PR or between laminin and BSA, and Z is the height differential between laminin-rich and alternate regions. Step 2: Incubation in Glutaraldehyde. The AMPS-coated sample was incubated in approximately 2 mL of a 2.5% glutaraldehyde (G-6257, Sigma Chemical) in phosphate buffered saline (PBS; 17-517Q, BioWhitaker, Walkersville, MD) solution for 1 h at room temperature. Following incubation, the sample was rinsed with 18 MΩ water three times and then imaged in water at 3 µm/s (sample C, Figure 2). Step 3: Laminin Deposition. Laminin (CB-40232, Collaborative, Bedford, MA) was thawed on ice and diluted in Hank’s balanced salt solution (HBSS; 14185-052, Life Technologies, Grand Island, NY) to a concentration of 50 µg/mL. The mounted sample was incubated in 2 mL of the diluted laminin solution at room temperature for 1 h. The NSOM tip was placed in 18 MΩ water during this incubation to prevent the tip from drying out and protein from depositing on the tip. Next, the sample was rinsed once with PBS and twice with 18 MΩ water and then imaged in water at 3 µm/s (sample D, Figure 2). In one experiment, the sample remained at room temperature overnight and was reimaged in water at a scan rate of 3 µm/s. Step 4: Removal of Photoresist. To remove the remaining PR, the sample was sonicated in acetone. The orientation of the mounted sample was determined by etching the glass holder with a scribe. The mounted sample was removed from the NSOM instrument, placed in a beaker with 150 mL of acetone, and sonicated for 8 min. During the sonication, the NSOM tip was again stored in 18 MΩ water to keep the tip well hydrated. Following sonication, the sample was rinsed with 18 MΩ water four times to remove residual acetone. The sample was placed on the NSOM instrument in the correct orientation and imaged at a scan rate of 6 µm/s (sample E, Figure 2). Step 5: Bovine Serum Albumin Deposition. A 0.5% solution of bovine serum albumin (A464-02, J.T. Baker, Phil-
lipsburg, NJ) was prepared in PBS. The samples were incubated in approximately 2 mL of 0.5% BSA for 1 h at room temperature, rinsed with 18 MΩ water, and then imaged in water at 6 µm/s. The tip was incubated in 18 MΩ water to maintain hydration and prevent protein deposition (sample F, Figure 2). Immunofluorescent Staining. Laminin was visualized by immunofluorescent staining. Samples were rinsed three times in PBS and incubated in primary polyclonal anti-laminin (L9393, Sigma Chemical) diluted 1:100 for 1 h. Following incubation the samples were again rinsed three times with PBS and then incubated with a secondary antibody, fluoroisothiocyanateconjugated IgG (F-9887, Sigma Chemical) diluted 1:100, for 1 h. The samples were analyzed with a Zeiss LSM 410 (Germany, Thornwood, NY) confocal laser scanning microscope with a computer-controlled laser scanning assembly attached to a microscope. An Omnichrome 3 line argon/krypton laser operating at 488, 568, and 647 nm was used as the excitation source. The images were processed with Zeiss LSM control software. Image Analysis. The images were individually analyzed. The four main features measured were width of laminin-rich stripes (L), width of PR or BSA-rich stripes (B), differential height between laminin and PR or between laminin and BSA (H), and height differential between laminin-rich and alternate regions (Z) (Figure 4). In each 60 µm × 60 µm image, 12 regions were selected and measured in triplicate. Six areas spanned the laminin-rich stripes, and six areas spanned the alternate stripes. The in situ experiment was repeated to ensure reproducibility. Data Analysis. Measurements from the image analysis were entered into Microsoft Excel and then exported for statistical analysis into Statistical Analysis Software (SAS) version 6.12 for the Macintosh. A SAS program was written to determine the average feature measurement and standard deviation for each
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Figure 4. This cartoon diagram illustrates the cross section of a micropatterned surface with feature measurements as noted: width of the laminin-rich stripe (L), width of the PR or BSA-rich stripe (B), ridge height (H), and height differential between laminin-rich and alternate stripes (Z). PR is denoted by horizontal stripes, BSA by black dots (white background), and laminin by white dots (gray background). (I) In substrates A-D, PR was present on the coverslip in the regions eventually enriched in BSA. Thus, L is the distance between PR stripes, B is the width of the PR stripes, H is the height of the PR at the interface, and Z is the height measured from the midpoint of the PR stripe. (II) In substrates E and F, ridges appeared at the laminin edges following sonication in acetone to remove the PR. In this case, L and B are measured as the distance between the highest point of adjacent ridges, H is the ridge height relative to the BSA-rich stripes, and Z is the height difference between the laminin-rich stripe and the alternate stripe. processing step. Student’s t-test (R ) 0.05) was used to determine statistically significant changes for each step.
Results and Discussion Following the procedure outlined in Figure 2, the final substrates (F) presented alternating regions of 10 µm wide laminin-rich stripes and 20 µm wide BSA-rich stripes (Figure 5A) separated by phase-dark boundaries. To confirm that the laminin was confined to the 10 µm regions, the substrates were incubated with a fluorescently labeled antibody to laminin. The laminin antibody confirms that adsorbed laminin was confined to 10 µm stripes (green), whereas the 20 µm wide BSA-rich stripes (black) were devoid of laminin as observed with immunofluorescence imaging (Figure 5B). Clearly, the lithographic procedure creates a distinct pattern of biologically active proteins. The lithographically prepared samples are then observed under several conditions (Figure 6). Specifically, the topographies in air and in water of the samples with both protein stripes (i.e., substrate F) are compared. The first image (Figure 6A) was acquired with a very thin layer of water remaining on the sample in an attempt to image the proteins in their natural hydrated form using AFM in contact mode to attain feedback. In this experiment, the topography delineates the narrow stripes of laminin and wider stripes of BSA. The white lines on top of the laminin stripes (corresponding to higher topographical regions) may correlate to residual photoresist. In contrast, the BSA stripes (wider stripes) show no evidence of photoresist. The height differential between the laminin and the BSA was as high as 80 nm in some samples. The image in Figure 6B was acquired by using NSOM in air under the same sampling conditions as when imaged by AFM, that is, with a thin layer of water on the sample surface. In this experiment, the same features observed with AFM were observed in the topographic image using NSOM (optical fiber). However, the height difference between the laminin and BSA regions was determined as
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approximately 150 nm even though the scanning systems were identical in Figure 6A,B. Therefore, the height differences cannot be due to differences in the Z calibration but instead are due to height differentials between the laminin and BSA regions analyzed on the “dry” surface. The NSOM topographic image for the sample immersed in liquid (Figure 6C) shows a distinct set of features when compared with the AFM and the NSOM images for the “dry” samples (i.e., thin layer of water for Figure 6A,B). In solution, the difference in height between the laminin and BSA stripes is around 240 nm. This value indicates that either laminin significantly expanded while immersed in water or, more likely, both proteins expanded upon immersion in water. An interesting phenomenon was the appearance of circular features that appeared on immersed samples. These features were as high as 360 nm with respect to the BSA regions. These features are not yet understood but may be a conglomerate of proteins that inflate in liquid. The optical images acquired in the near-field using the reflection mode are shown in Figure 6D,E. The optical image in air (Figure 6D) shows streaks that are most likely due to the interference from the light scattering while the sample was dried by the incident light (480 nm laser with ∼1 mW of power). This effect was not observed in the NSOM optical image while the sample was immersed in water. Using the same experimental conditions as in the “dry” experiments, the optical image (Figure 6E) showed a more homogeneous surface without artifacts created during the imaging process. The optical image in water (Figure 6E) showed a clear differentiation of the two protein regions in contrast to the optical images obtained “dry”, that is, with a thin film of water (Figure 6D). Imaging in liquids has three fundamental advantages. First, a more stable feedback signal is obtained (i.e., more homogeneous damping of the nanoprobe oscillations avoids external interference like air current and noise). Second, drying of the sample by incident light is prevented. And last, the solution reduces scattered light in the NSOM chamber, thereby decreasing the probability for artifacts in the optical image, clearly demonstrated by comparing the optical NSOM images of Figure 6B,C. Once the features observed by confocal laser scanning microscopy (Figure 5) were confirmed by NSOM (Figure 6), the goal was to determine how and when the topographical features were created. Taking advantage of NSOM’s superior capabilities in liquid, images were taken for all six substrates (A-F) depicted in Figure 2. For each image, four separate features were analyzed as represented by the cartoon in Figure 4. Regions designated B correspond to the PR stripes in substrates A-D (e.g., I in Figure 4) and to the BSA-rich stripes that predominantly appear in substrates E and F (e.g., II in Figure 4). The regions designated L for laminin correspond to the laminin-rich stripes between PR in substrates A-D and between BSA in substrates E and F (Figure 4). The height differences between the PR and glass regions of substrates A-D and between the PR and laminin-rich regions of substrates E and F were denoted H. The height differences between the laminin-rich and PR regions in substrates A-D and between the laminin-rich and BSA-rich regions in substrates E and F were denoted Z. Prior to the processing steps described in Figure 2, the micropatterned glass substrates (A) were imaged. Figure 3 illustrates representative topographical (Figure 3A), optical (Figure 3B), and three-dimensional (Figure 3C) images. This figure shows side-by-side topographic (Figure 3A) and optical (Figure 3B) images obtained concurrently from the same area of sample immersed in water. From
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Figure 5. Confocal laser scanning micrograph of micropatterned substrates with alternating 10 µm wide laminin-rich and 20 µm wide BSA-rich regions observed by phase contrast microscopy (A) and fluorescence imaging (B).
Figure 6. Representative images of micropatterned substrates, E and F. The AFM image (A) was obtained in contact mode on a sample with a thin film of water over a 60 µm square area. The NSOM instrument was operated in the noncontact mode on a “dry” sample (i.e., thin film of water) resulting in topographical (B) and optical (D) images as well as in an aqueous environment with both topographical (C) and optical (E) images confirming the topographical features. The NSOM instrument also scanned a 60 µm square area.
the topographic image (Figure 3C), patterns of light (which correspond to higher) regions and dark (lower) regions that are 10 and 20 microns wide, respectively, are observed. Although the patterns were also observed in the optical image (Figure 3B), the patterned stripes were not as clearly defined as in the topographical images. The three-dimensional image (Figure 3C) shows a reason for this lack of definition: the edges of the photoresist stripes slope, a feature that is not obvious from the flat topographical image (Figure 3B). From the pattern dimensions, the light and dark regions were assigned to PR and glass, respectively. Widths of the exposed and unexposed regions (i.e., glass and PR) were 8.5 and 21.2 µm, roughly corresponding to the expected pattern dimensions of 10 µm glass (L) and 20 µm
PR (B). The PR and exposed glass regions had an average height differential of 2.1 µm (H and Z, Figure 3C) as shown in Tables 1 and 2 for substrate A. Silylation of Glass Surface (Step 1). In the first processing step, the mounted lithographically patterned coverslip was sonicated in AMPS solution (step 1) to silylate the exposed glass substrate (B). This step effectively silylates the hydroxyl groups, generating free amino groups for further reactions. Resolution of this sample was limited to approximately 300 nm in the x- and y-directions and 11 nm in the z-direction due to topographic imaging parameters such as image resolution (200 pixels per line), scanning rate (3 µm/s), scanning area (60 µm), and probe size (e100 nm). The height difference on substrate B slightly decreased
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Table 1. Change in Height (H) as a Function of Processing, Standard Deviation, Percent Change from the Previous Processing Step, and Significance of the Height Change height, H (µm) substrate
height
SD
% change
siga
A B C D E F
2.138 2.011 2.005 1.731 0.104 0.149
0.049 0.028 0.145 0.062 0.010 0.37
-6.23 -0.30 -13.7 -94.0 +43.3
yes no yes yes yes
a Significance based on 95% confidence level using Student’s t-test.
Table 2. Change in Bulk Height Differential (Z) as a Function of Processing, Standard Deviation, Percent Change from the Previous Processing Step, and Significance of the Height Change bulk height difference, Z (µm) substrate
height
SD
% change
siga
A B C D E F
2.115 1.918 1.918 1.804 0.022 0.017
0.057 0.053 0.188 0.043 0.005 0.005
-9.31 0.00 -11.4 -98.8 -22.7
yes no yes yes yes
a Significance based on 95% confidence level using Student’s t-test.
after incubation in AMPS from 2.1 µm (Tables 1 and 2), which was statistically significant based on Student’s t-test (R ) 0.05). The decreases in the height differential, H and Z, are attributed to AMPS accumulating in greater quantities on the glass surface. For substrate B, the measured widths of L and B were close to the original values and the differences calculated were within the detection limits of the NSOM instrument. Cross-Linking with Glutaraldehyde (Step 2). In the second step, the AMPS-treated sample (C) was incubated in glutaraldehyde solution. Glutaraldehyde cross-links the free amino groups of the AMPS, enhancing protein adsorption in subsequent steps. Statistical analysis of the topographical features of substrate C indicated an insignificant change in feature height differences (Tables 1 and 2), essentially remaining near 2.0 µm. Similarly, the pattern widths were relatively unchanged. Because glutaraldehyde only cross-links the AMPS, measurable feature changes at this stage were not anticipated. Laminin Deposition (Step 3). In step 3, laminin was absorbed to the micropatterned surface to yield substrate D. Both height parameters, H and Z, significantly decreased from the original height of 2.1 µm after incubation with laminin (Tables 1 and 2, D). For example, the bulk height differential (Z) slightly decreased from 1.9 µm (C) to 1.8 µm (D). Laminin adsorbed onto the entire surface, as demonstrated by fluorescence microscopy (unpublished data). Yet, the statistically significant decrease in the height differential indicates that laminin either (i) accumulated in greater quantities on the modified glass surface or (ii) adopted a different conformation on the glass surface. The patterned stripe widths, L and B, increased, and the measurements were still within instrumental detection limits. To evaluate the stability of the laminin-coated surface (D), samples were left overnight on the NSOM stage at room temperature and then reimaged. Feature widths and height differences were unaffected by prolonged incubation.
Removal of Photoresist (Step 4). In step 4, removal of the remaining PR by sonication in acetone drastically changed the sample profile on substrate E. The purpose of this processing step was to generate a surface that consisted of alternating regions of exposed glass (after PR removal) and laminin. After sonication in acetone, ridges appeared at the laminin edge (illustrated in Figure 4, profile II). The height differential (H) significantly decreased to 0.10 µm (Table 1, E) and varied depending on the sample and region. These edges or ridges may be remnants of PR not removed by sonication in acetone or may be excess laminin accumulating at the interface. The measurable height difference (Z) between the laminin and the newly exposed glass (Table 2, E) indicates that the NSOM instrument readily detects submicron topographical features. However, this difference (Z) was very close to the detection limit in the z-direction for the NSOM under the operating conditions selected. At this processing stage, the increased statistical variability in the L and B widths indicates that the removal of the PR generates a high degree of variability. Bovine Serum Albumin Deposition (Step 5). In the final step, BSA was added to create regions that discouraged cell attachment on substrate F. Specifically, regions of glass on substrate E exposed by removal of PR were covered by BSA to create alternating regions that are less permissive (BSA) and more permissive (laminin) to cells. Following incubation with fluorescently conjugated antilaminin, the antibody specifically bound to the laminin such that fluorescence was confined to the anticipated laminin-rich regions of the micropatterned substrate (see Figure 5B). At the laminin/BSA interface in substrate F, the ridge that first appeared following removal of the PR in substrate E remained and the relative height difference (H) increased following the BSA deposition. Figure 6 shows representative topographical (Figure 6C) and optical (Figure 6E) images of the same area of a water-immersed substrate with laminin-rich and BSA-rich patterns. From the pattern spacing, the light regions in Figure 5 were assigned as edges bordering the laminin (∼10 µm wide) and BSA (∼20 µm wide) regions. The consistently slight increase in widths of the laminin-rich (L) and alternate stripes (B) were statistically significant, yet still within the detection limits of the instrument. In contrast, the marked increase in the relative height differential was extremely variable (Tables 1 and 2, F) and statistically significant. The height increase may be attributable to preferential adsorption of BSA to the interface edges or to PR that was not removed by sonication in acetone. Conclusions The processing steps outlined in the schematic of Figure 2 are successfully utilized by many research groups to evaluate cell attachment, growth, and function on micropatterned glass. Previous research predominantly focused on the chemical cues provided by the protein micropatterns, while the impact of surface topography that may result from microlithographic patterning has largely been ignored. Our NSOM analysis of lithographically prepared samples clearly illustrates the topographical component to micropatterned glass, notably, the appearance of ridges at the laminin/BSA interface after the apparent “removal” of PR. While changing the surface energy, adhesivity, or cell receptors (i.e., chemical cues) of the substrate undoubtedly plays the major role in directing cellular behavior, the physical cues likely affect
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motile behaviors such as cell spreading, navigation, and alignment. Ignoring the cell interactions will limit one’s ability to optimize the protein-patterned surfaces. Our findings do not invalidate work cited herein but offer an alternative perspective on the cellular response to the chemical and topographical cues provided by the micropatterned surfaces. NSOM is a powerful tool for analyzing the physical or topographical features of the protein patterns. With this tool, high-resolution optical images are concurrently obtained with topography while both substrate and probe are immersed in solution. Thus, protein micropatterns are observed in aqueous solution with accurate and noninvasive imaging over relatively large scanning areas as compared to AFM. The micron-sized features described herein are approaching the detection limit of the NSOM technique, because scanning large areas
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lowers the resolution. Nonetheless, we are able to image micron-sized features under physiologically relevant conditions. Acknowledgment. The authors thank the following funding sources: Johnson and Johnson Fellowship (D.M.T.), American Association for University Women Selected Professions Dissertation Fellowship (D.M.T.), Rutgers University/UMDNJ NIH Biotechnology Training Program (K.E.S., D.M.T.), New Jersey Center for Biomaterials Summer Fellowship (K.E.S.), Charles and Johanna Busch Memorial Fund (H.M.B.), and DuPont (K.E.U.). L.G. acknowledges useful discussions with R. B. Comizzoli and J. D. Sinclair. LA0204038
