© Copyright 1999 American Chemical Society
AUGUST 17, 1999 VOLUME 15, NUMBER 17
Letters Shear Force and Phase Imaging of Protein Boundaries Giles H. W. Sanders, Martyn C. Davies, Clive J. Roberts,* Saul J. B. Tendler, and Philip M. Williams Laboratory of Biophysics and Surface Analysis, School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, U.K. Received November 18, 1998. In Final Form: May 12, 1999 A scanning probe microscope employing a shear force feedback mechanism as used in most near-field scanning optical microscopes has been utilized to provide phase images of a patterned protein boundary. The microscope employed a dithering fiber optic tip and tuning fork method of tip-sample distance control. The effect of tip vibration amplitude upon the phase contrast is investigated, and possible contrast mechanisms are discussed.
1. Introduction In recent years the family of scanning probe microscopes has expanded considerably. Two of the most important of these additions have been phase contrast1,2 and shear force microscopy,3,4 the latter through its use in near field scanning optical microscopy (NSOM).5 In this Letter we introduce a combination of the two techniques displaying phase contrast between uncoated polymer and proteincoated polymer. This has been achieved through the measurement of the phase shift of a NSOM probe using shear force feedback. NSOM has found applications in a wide variety of systems ranging from the biological6-9 to polymers10,11 to (1) Tamayo, J.; Garcı´a, R. Langmuir 1996, 12, 4430-4435. (2) Magonov, S. N.; Elings, V.; Whangbo, M.-H. Surf. Sci. Lett. 1997, 374, L385-L391. (3) Betzig, E.; Finn, P. L.; Weiner, J. S. Appl. Phys. Lett. 1992, 60, 2484-2486. (4) Kirsh, A. K.; Meyer, C. K.; Jovin, T. M. J. Microsc. (Oxford) 1997, 185 (3), 396-401. (5) Pohl, D. W.; Denk, W.; Lanz, M. Appl. Phys. Lett. 1984, 44, 651653. (6) Haydon, P. G.; Marchese Ragona, S. P.; Basarsky, T. A.; Szulczewski, M.; McCloskey, M. J. Microsc. (Oxford) 1996, 182 (3), 208216. (7) Enderle, T.; Ha, T.; Ogletree, D. F.; Chemla, D. S.; Magowan, C.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94 (2), 520-525. (8) Gheber, L. A.; Hwang, J.; Edidin, M. Appl. Opt. 1998, 37 (16), 3574-3581.
semiconductors.12,13 In NSOM a subwavelength probe is rastered over the sample of interest within the optical near field (experimentally between 5 and 10 nm). The probe either provides illumination that is collected in the far field by a microscope or collects light that has interacted with the sample. In one particular configuration the probe both provides the illumination and collects the reflected light.14 In the majority of cases at present the NSOM probe is a tapered fiber optic which can be coupled to a laser to provide illumination. With such a configuration it is necessary to maintain the probe within the optical near field of the sample but without making contact. Several different feedback mechanisms have been used, including tunneling15,16 and optical,17,18 but the most successful has proved to be shear force.3 With shear force feedback the (9) Garcia Parajo, M. F.; Veerman, J. A.; Ruiter, A. G. T.; van Hulst, N. F. Ultramicroscopy 1998, 71 (1-4), 311-319. (10) Higgins, D. A.; Kerimo, J.; Vandenbout, D. A.; Barbara, P. F. J. Am. Chem. Soc. 1996, 118 (17), 4049-4058. (11) Rucker, M.; DeSchryver, F. C.; Vanoppen, P.; Jeuris, K.; DeFeyter, S.; Hotta, J.; Masuhara, H. Nucl. Instrum. Methods Phys. Res. Sect. B 1997, 131 (1-4), 30-37. (12) Hallen, H. D.; Larosa, A. H.; Jahncke, C. L. Phys. Status Solidi A 1995, 152 (1), 257-268. (13) Xu, Q.; Gray, M. H.; Hsu, J. W. P. J. Appl. Phys. 1997, 82 (2), 748-755. (14) Jalocha, A.; Moers, M. H. P.; Ruiter, A. G. T.; van Hulst, N. F. Ultramicroscopy 1995, 61, 221-226. (15) Lieberman, K.; Lewis, A. Appl. Phys. Lett. 1993, 62, 1335-1337.
10.1021/la981621x CCC: $18.00 © 1999 American Chemical Society Published on Web 07/23/1999
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fiber optic probe is oscillated parallel to the sample surface (in direct contrast to most other resonant scanning probe modes where a tip is oscillated normal to the sample). The oscillation amplitude of the probe is decreased by damping arising from long-range interactions when it moves close (ca. 10 nm) to the surface. In ambient conditions these interactions will probably be dominated by capillary forces arising from hydration layers between the probe and the sample. It is this damping that provides the feedback signal, and in an ideal situation, shear force provides true noncontact imaging that is not typically achieved with other SPM imaging modes in ambient conditions. Phase imaging is a recent development of intermittent contact mode AFM. In this mode the tip is tapped into the surface and the amplitude signal is employed to maintain feedback. However a signal relating to the phase shift of the tip oscillation in comparison to the driving signal is also obtained. This provides the phase image from which further information about the sample properties can be obtained19 since the phase shift is related to energy dissipation of the tip due to interactions with the sample.20 Recent work by Noy et al.21 has advanced phase imaging to chemical sensitivity by employing functionalized probes. In this Letter we combine both the above techniques to obtain phase contrast of biological molecules on a polymeric sample employing a shear force feedback mechanism. Although the measurement of a phase signal can be seen as analogous to tapping mode phase imaging, it is important to note that the physical origin of the phase contrast is not totally anologous for shear force. In tapping mode the probe oscillates normal to the sample with intermittent contact between the probe and the sample. In shear force imaging the probe is oscillated parallel to the sample surface away from the surface. Therefore in tapping mode both long-range and short-range interactions between the tip and the sample will affect the energetics of the probe and thus the phase contrast, but in shear force only a limited region of tip-sample interaction will affect the probe oscillation and the phase contrast seen. Several groups have noted that shear force microscopy can be used to obtain material contrast between metals22 or between metals and insulators.23 This paper introduces the first report of shear force noncontact chemical contrast on biological systems. 2. Materials and Methods 2.1 Scanning Probe Microscopy. Shear force microscopy was undertaken employing a Topometrix (Saffron Walden, U.K.) Lumina NSOM. This instrument is a second generation commercially available transmission mode NSOM. The sample is scanned under the probe, and a shear force feedback mechanism employing a tuning fork24 is used to maintain tip-sample separation. The probes (16) Volgunov, D. G.; Gaponov, S. V.; Dryakhlushin, V. F.; Klimov, A. Y.; Lukyanov, A. Y.; Mironov, V. L.; Panfilov, A. I.; Petrukhin, A. A.; Revin, D. G.; Rogov, V. V. Instruments and Experimental Techniques 1998, 41, 269-274. (17) Kramer, A.; Hartmann, T.; Stadler, S. M.; Guckenberger, R. Ultramicroscopy 1995, 61, 191-195. (18) Cline, J. A.; Barshatzky, H.; Isaacson, M. Ultramicroscopy 1991, 38, 299-304. (19) Bar, G.; Thomann, Y.; Brandsch, R.; Cantow, H. J.; Whangbo, M. H. Langmuir 1997, 13, 3807-3812. (20) Cleveland, K. B.; Anczykowski, B.; Schmid, A. E.; Elings, V. B. Appl. Phys. Lett. 1998, 72, 2613-2615. (21) Noy, A.; Sanders, C. H.; Vezenov, D. V.; Wong, S. S.; Lieber, C. M. Langmuir 1998, 14, 1508-1510. (22) Durkan, C.; Shvets, I. V. J. Appl. Phys. 1996, 80, 5659-5664. (23) Bereton, L. Personal communication. (24) Karrai, K.; Grober, R. D. Appl. Phys. Lett. 1995, 66, 1842-1844.
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are bought commerciallly from Topometrix preglued to the tuning fork. They have a nominal Q factor of between 250 and 500. An attempt to measure the amplitude to the tip vibration was made through coupling the probe to a argon ion laser and monitoring change in the shape of the transmitted light profile. Large oscillation amplitudes would give rise to a line of length equivalent to the oscillation amplitude. In the case of tuning fork oscillation, it was not possible to measure the vibration amplitude of the tip even with maximum instrumental driving voltage of 20 V as no measurable change in the light profile was seen (using 40 × 0.85NA lens). In these studies the amplitude signals were fed into the feedback circuit to regulate the tip-sample separation. The phase signal was measured as an output from the microscope control unit and fed back to obtain the phase contrast images. The signal obtained is a voltage which is related to the phase shift, the oscillation amplitude, and the shape of the phase spectrum. It is possible to convert the signal into a true phase angle by calibrating each tip (and each oscillation amplitude) through measuring the voltage signal at a number of phase angles (set in software). However, this has not been undertaken here, and only the input voltages are quoted. Prior to entering feedback the phase angle was set in the instrument software (Topometrix SPM lab version 4.01) such that the phase voltage signal was zerosthis corresponds to the linear zone of the phase spectrum such that the voltage measured is approximately linearly proportional to the phase shift. All images were taken using a 50 µm scanner unit attached to the base plate of a Nikon Diaphot 300 inverted microscope and in constant distance mode with a scan rate between 0.5 and 1.5 Hz. The probes employed were commercially available (Topometrix) pulled and aluminumcoated fiber optics mounted on the tuning fork. Atomic force microscopy was undertaken using a Digital Instruments (Santa Barbara, CA) Nanoscope IIIa MultiMode atomic force microscope in tapping mode and employing a E type scanner throughout. 2.2. Sample Preparation. Protein boundaries were formed upon polycarbonate (HybriSlip) cover slips (Sigma). Masks were formed by placing polydimethylsiloxane, PDMS (Dow Corning), blocks onto the Hybrislips. The PDMS block formed a impermeable interface with the polycarbonate. Avidin-fluorescein isothiocyanate (FITC) (Sigma) solution (1 mg/mL) was then dropped around the block and left for 1 h to incubate forming a protein boundary between the unmasked and masked area. During this time the avidin solution physisorbs to the polycarbonate. After the sample was washed in deionized water, the PDMS block was peeled off the Hybrislip. The resulting protein distribution was confirmed using fluorescent optical microscopy which showed FITC labeled protein had adsorbed onto the Hybrislip, but not in areas which had been masked by PDMS. Lines of avidin (12 µm wide) were formed by a microfluidic flow method25,26 in which avidin solution flowed up microcapillaries formed from a PDMS mold placed on the Hybrislip. This method is described in more detail elsewhere.27 3. Results and Discussion Figure 1a displays an AFM image of the protein coated/ (25) Delamarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science 1997, 276 (5313), 779-781. (26) Delamarche, E.; Bernard, A.; Schmid, H.; Bietsch, A.; Michel, B.; Biebuyck, H. J. Am. Chem. Soc. 1998, 120 (3), 500-508. (27) Patel, N.; Padera, R.; Sanders, G. H. W.; Canizarro, S.; Davies, M. C.; Langer, R.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Shakesheff, K. M. FASEB J. 1998, 12 (14), 1447-1454.
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Figure 1. Tapping mode AFM image of a boundary between avidin-coated and uncoated regions on a Hybrislip: (a) topographical image with the inset displaying invidual protein molecules; (b) concurrently obtained phase image of the same region. The insert in (a) displays a 1 µm × 1 µm area of the boundary.
uncoated boundary on the Hybrislip obtained in air. Hard tapping (high driving amplitude and low setpoint) was employed, and Figure 1b displays the concurrently obtained phase image. The contrast seen arises from differences in the viscoelastic properties of the protein layer compared to the polycarbonate of the Hybrislip. The insert in Figure 1a displays the boundary at higher resolution (1 µm × 1 µm). Single molecules and arregates of protein can be seen. The images also show that the protein generally forms a monolayer on the polycarbonate. Figure 2a displays 50 µm square topographical image of the same system obtained employing shear force feedback as the imaging mechanism. The large lump visible in the images can be attributed to a fault in the Hybrislip and not an aggregate of protein. This was confirmed by concurrent fluorescence microscopy, which showed that this point did not fluoresce and thus was not protein. This image was obtained using the amplitude of the probe oscillation as the feedback signal. The feedback point was set at 95% of the free oscillation value. It should however be noted that the feedback point here is not necessarily the actual tip oscillation amplitude with respect to the free amplitudesthere is a contribution from the tuning fork itself. This was confirmed by amplitude-distance measurements which displayed that the oscillation amplitude is not zero even when the tip is in actual contact with the surface but found to be roughly 75% of the free amplitude. The feedback point chosen, however, ensured that, in the majority of cases, the tip did not appear to touch the surface during scanning, and thus the images were true noncontact and not a result of any tapping on the surface. Using lower feedback points the tip was found to touch the surface during scanning. This has been confirmed experimentally by fluorescence microscopy, which showed that the protein had been swept away (data not shown). In parts b-e of Figure 2 phase signal images acquired simultaneously with amplitude feedback shear force topography of the same area are shown. In all the images the phase contrast is noted in volts and darker points correspond to higher phase shift. Figure 2b is the phase image acquired with an voltage of 1.6 V oscillating the tuning fork. This is equivalent to an oscillation amplitude in the namometer range. The image shows very little phase contrast with only the edge of the protein boundary being visibly different to the rest of the image. The contrast seen here is likely to be a result of contact between the probe and the edge of the proteinsthis is a result of decreased feedback efficiency through using amplitude rather than phase measurement as the shear force
Figure 2. Shear force images of the protein boundary: (a) topographical image obtained using shear force feedback; (be) phase images of the same area taken with dithering voltages of 1.6, 0.16, 0.04, and 0.02 V, respectively. The contrasts (black to white) seen in images (b), (c), (d), and (e) correspond to 0.216, 0.932, 1.439, and 1.384 V, respectively. The amplitude of this voltage is directly related to the phase shift.
feedback monitor.28,29 This figure does illustrate the extent of the crosstalk between the topographic and phase images. It is unlikely that the other images will show significantly more crosstalk than that observed in Figure 2b as similar imaging conditions were employed. Parts c, d, and e of Figure 2 display the same boundary imaged with a drive amplitude of 0.16, 0.04, and 0.02 V, respectively. In these images the edge of the protein again shows some crosstalk between the topography and phase images, but a phase shift is also visible beyond the edge which can be attributed to true phase contrast. It is immediately obvious that decreasing the drive amplitude increases the phase contrast with a definite contrast between the proteincoated and uncoated area and not just at the boundary. At the lowest drive amplitude it can be seen that the signalto-noise is reaching a practical limit. The oscillation amplitude used in these cases is much less than those typically employed by systems employing optically monitored shear force. (28) Ruiter, A. G. T.; vanderWerf, K. O.; Veerman, J. A.; Garcia Parajo, M. F.; Rensen, W. H. J.; vanHulst, N. F. Ultramicroscopy 1998, 71, 149-157. (29) Ruiter, A. G. T.; Veerman, J. A.; vanderWerf, K. O.; vanHulst, N. F. Appl. Phys. Lett. 1997, 71, 28-30.
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Figure 3. Topography (a) and shear force phase images (b) of a single protein line formed by microfluidic patterning.
Figure 3 illustrates an image of a 12 µm line of protein formed through microfluidic flow of avidin solution over a Hybrislip with an oscillation drive amplitude of 0.04 V. Clear phase contrast between the uncoated polycarbonate and the avidin is again visible. There is also some crosstalk visble in the images. It has recently been reported23 that phase contrast is only possible with shear force microscopy when there is a change of both the probe Q factor and also the position of the resonant frequency peak. Such a situation requires both change in dissipation of energy and in the restoring elastic force acting on the probe.20 Changes in damping can occur through solvation, viscosity, or adhesional changes, and variations in the restoring forces can arise from these and also van der Waals and electrostatic interactions. It is not clear at present which of these are contributing
Letters
to the phase contrast observed in the images. It is feasible that some contribution to the contrast might arise from the interaction of the hydration layers of the sample and the tip, giving rise to a frictional force. A further possibility is that the tip had picked up some protein (for example during scanning over the boundary edge) and that the phase contrast is a result of long-range hydrophobic protein-protein interactions perturbing the tip vibration. In both cases a decrease in drive amplitude (and thus lower probe velocity) would give rise to longer interaction times, as the probe would be oscillating less so spending more time within an interaction zone with the sample, and thus the greater phase shifts seen. In the future both of these mechanisms will be tested through monitoring the phase shift as a function of humidity and through the use of functionalized probes. The results outlined in this Letter correspond to the first report of shear force phase contrast observed on biological samples. Since shear force feedback provides no or very little contact between the probe and the sample, this technique should allow for chemical discrimination of different surface entities with much less perturbation of the sample compared to other scanning probe microscopy imaging modes. Acknowledgment. The authors thank the BBSRC for Postdoctoral funding for G.H.W.S. We also thank Topometrix Corporation and the BBSRC for joint funding of the NSOM instrument. LA981621X
