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Noncontact Near-Field Scanning Optical Microscopy Imaging Using an Interferometric Optical Feedback Mechanism Hitoshi Shiku, Jeffrey R. Krogmeier, and Robert C. Dunn* Department of Chemistry, University of Kansas, Lawrence, Kansas 66045 Received September 8, 1998. In Final Form: January 7, 1999 An interferometric optical feedback mechanism is explored for tip-sample distance control in near-field scanning optical microscopy. An optical signal, based on the interference between the light exiting the tip aperture and that reflecting off the sample surface, is used to implement a feedback scheme to regulate tip-sample distance. The noncontact nature of this feedback mechanism may provide greater flexibility in imaging soft or fragile samples. To characterize the performance of the optical feedback mechanism, images are analyzed of a calibration standard, fluorescently doped lipid monolayers, latex spheres, and fixed cells. Images taken of these samples using optical feedback and the standard tapping-mode feedback are comparable in quality. These samples also demonstrate the ability of optical feedback to follow both large and small height changes and accurately reflect the sample topography. In a nonscanning mode, the interferometric signal can be used to noninvasively probe small dynamic height changes of a sample with nanometric spatial resolution. Using a piezo ceramic bimorph to simulate sample movement, we show that nanometric height changes can be detected with millisecond time resolution. This may provide a unique way to probe protein conformational changes free of tip-sample interactions.
Introduction Near-field scanning optical microscopy (NSOM) is a powerful scanning probe technique which offers high spatial resolution by positioning a nanometric light source close to a sample surface.1-3 Solid-state devices, thin films, biological samples, and single molecules have all been studied at the submicrometer level using NSOM.1 As the technique evolves, new feedback mechanisms that allow constant tip-sample separation during scanning continue to appear. Methods relying on tunneling current,4,5 impedance,6 shear force,7,8 and tapping mode9-11 have been used to control the tip-sample distance in NSOM applications. The latter methods, based on force feedback mechanisms, do not depend on the conductive properties of the sample, which makes them particularly attractive for biological samples. However, force feedback mechanisms retain problems associated with tip-induced sample degradation if the forces involved are not sufficiently small. For experiments on fragile samples such as biological specimens or films suspended at the air/water interface, (1) Paesler, M. A.; Moyer, P. J. Near-Field Optics: Theory, Instrumentation, and Applications; John Wiley and Sons: New York, 1996. (2) Pohl, D. W. Scanning Near-Field Optical Microscopy (SNOM), In Advances in Optical and Electron Microscopy; Mulvey, T., Sheppard, C. J. R., Eds.; Academic Press: London, 1991; Vol. 12, pp 243-312. (3) Betzig, E.; Trautman, J. K.; Harris, T. D.; Weiner, J. S.; Kostelak, R. L. Science 1991, 251, 1468-1470. (4) Durig, U.; Pohl, D. W.; Rohner, F. J. Appl. Phys. 1986, 59, 33183327. (5) Harootunian, A.; Betzig, E.; Isaacson, M.; Lewis, A. Appl. Phys. Lett. 1986, 49, 674-676. (6) Hsu, J. W. P.; Lee, M.; Deaver, B. S. Rev. Sci. Instrum. 1995, 66, 3177-3181. (7) Betzig, E.; Finn, P. L.; Weiner, J. S. Appl. Phys. Lett. 1992, 60, 2484-2486. (8) Toledo-Crow, R.; Yang, P. C.; Chen, Y.; Vaez-Iravani, M. Appl. Phys. Lett. 1992, 60, 2957-2959. (9) Talley, C. E.; Cooksey, G.; Dunn, R. C. Appl. Phys. Lett. 1996, 69, 3809-3811. (10) Lieberman, K.; Lewis, A.; Fish, G.; Shalom, S.; Jovin, T. M.; Schaper, A.; Cohen, S. R. Appl. Phys. Lett. 1994, 65, 648-650. (11) Muramatsu, H.; Chiba, N.; Homma, K.; Nakajima, K.; Ataka,T.; Ohta, S.; Kusumi, A.; Fujihira, M. Appl. Phys. Lett. 1995, 66, 32453247.
the forces involved in these methods can become extremely problematic. Therefore, a continued development in the techniques available to reduce tip-sample interactions is essential for these important, but demanding, applications. Among the many scanning probe microscopies, NSOM is unique in that light can be delivered and collected with the tip. This capability adds additional avenues through which feedback schemes can be implemented for controlling the tip-sample distance. For instance, photon tunneling between an evanescent field in a sample and the tip has been used extensively in NSOM to position the tip close to the sample.12,13 The nonlinear dependence in photon tunneling with tip-sample distance makes this a very sensitive method of feedback, analogous to electron tunneling in scanning tunneling microscopy. However, waveguides are necessary to setup the evanescent field in the sample, which is sometimes inconvenient, and coupling between the force and optical images can sometimes complicate these measurements. Other feedback schemes have successfully exploited the interferences experienced by the light exiting the NSOM tip aperture and that reflected from the sample surface to control the tip-sample distance using straight NSOM probes.14-16 Here we extend these previous results to show that both small and large topography changes can be accurately imaged using a cantilevered NSOM probe employing an interferometric optical feedback method. The detection scheme incorporates a long working distance microscope to monitor the interferences between light exiting the NSOM tip aperture and that reflecting off the (12) Courjon, D.; Vigoureux, J. M.; Spajer, M.; Sarayeddine, K.; Leblanc, S. Appl. Opt. 1990, 29, 3734-3740. (13) Reddick, R. C.; Warmack, R. J.; Ferrell, T. L. Phys. Rev. B 1989, 39, 767-790. (14) Kramer, A.; Hartmann, T.; Eschrich, R.; Guckenberger, R. Ultramicroscopy 1998, 71, 123-132. (15) Kramer, A.; Hartmann, T.; Stadler, S. M.; Guckenberger, R. Ultramicroscopy 1995, 62, 191-195. (16) Guttroff, G.; Keto, J. M.; Shih, C. K.; Anselm, A.; Streetman, B. G. Appl. Phys. Lett. 1996, 68, 3620-3622.
10.1021/la981198s CCC: $18.00 © 1999 American Chemical Society Published on Web 02/17/1999
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Figure 1. Schematic of the near-field scanning optical microscope capable of both tapping-mode and optical feedback imaging. For both feedback methods, the cantilevered NSOM tip is oscillated at its resonant frequency with a bimorph. In tapping-mode feedback, the amplitude of the oscillation is monitored by reflecting a laser off the bend of the NSOM tip and into a split photodiode (not shown). Under optical feedback, the light exiting the NSOM tip and reflecting off the surface is collected from the side with a long working distance microscope and detected on a PMT. The signal from the PMT is sent to a lock-in amplifier referenced to the bimorph frequency and then to the AFM controller.
sample surface. The method can sense the position of the tip relative to the sample when the tip is several micrometers above the surface. The technique is easy to incorporate into NSOM, does not require an additional light source, and can be implemented in a noncontact mode which is completely noninvasive toward the sample. The latter is important for planned experiments on films at the air/water interface and may be useful for studies on living cells. We find that the technique accurately tracks surface topography on a variety of samples and can sense sample height changes less than 5 Å. In a nonscanning mode, the interferometric signal can be used to detect small dynamic height changes in a sample. With the tip positioned above a piezoelectric bimorph, we show that nanometric motions can be followed on the millisecond time scale. These results offer a promising new method for noninvasively monitoring small protein conformational changes in real time, without the need for external tags or markers. Materials and Methods The experiments reported here were carried out with a customdesigned near-field scanning optical microscope, the details of which have been described previously.9 Briefly, the NSOM is built around an inverted fluorescence microscope (Zeiss Axiovert 135TV) equipped with a fluar 40x, 1.3 NA oil immersion objective lens. For NSOM measurements, a cantilevered NSOM probe is held in a Dimension AFM head (Digital Instruments), which is mounted on the fluorescence microscope. The sample is raster scanned under the NSOM tip using a separate x-y closed-loop piezo scanner (Physik Instrumente) which keeps both the feedback and fluorescence signals aligned on their respective detectors. The 514 nm line from an argon ion laser (Liconix 5000) is passed through a λ/4 and a λ/2 waveplate (Newport) to control
the polarization before being coupled into the NSOM probe. Sample fluorescence is detected with an avalanche photodiode detector (EG&G, SPCM-200). The output of the detector is sent to a Nanoscope IIIa controller (Digital Instruments), which controls all aspects of the NSOM operation. In contrast to the more commonly employed shear-force method of feedback, tapping-mode feedback uses a cantilevered NSOM probe vibrated normal to the sample surface at its resonant frequency, typically between 20 and 60 kHz. The amplitude of the tip motion is monitored using the conventional technique in which light is reflected off the tip bend and sent into a split photodiode. For the optical feedback experiments, the light emerging from the NSOM probe is collected from the side at an angle of approximately 30° from the surface with a long working distance microscope (Infinity Photo-Optical Co., model K2) and detected with a photomultiplier tube (PMT) (Hamamatsu, R1527). The output of the PMT is sent to a lock-in amplifier (Stanford Research Systems, model SR830) referenced to the bimorph modulation signal used to oscillate the NSOM probe. The feedback signal from the lock-in amplifier was maximized by controlling the polarization of the light exiting the NSOM probe with the waveplates. Langmuir-Blodgett films were formed from L-R-dipalmitoylphosphatidylcholine (DPPC) (Sigma) and 1,1′-dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine perchlorate (diIC18) (Molecular Probes) which were used without further purification. DPPC was dissolved in spectral grade chloroform (1 mg/mL) into which small volumes of a concentrated solution of diIC18 in methanol were added to a final diIC18 concentration of 0.25 mol %. Approximately 50 µL of the DPPC/0.25 mol % diIC18 solution was dispersed onto a 10 mM aqueous MgCl2 subphase and compressed at a rate of 100 cm2/min using a computer-controlled Langmuir-Blodgett trough (Nima Technology, model 611) equipped with a Wilhelmy pressure sensor. The films were transferred onto freshly cleaved mica surface at a dipping velocity of 25 mm/min while maintaining a constant surface pressure. For the microsphere experiments, 260 nm carboxylate-modified
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latex microspheres (Molecular Probes) were spin-coated onto freshly cleaved mica. For experiments on cells, normal diencephalon astrocytes taken from 1 day old Sprague-Dawley rats (American Type Culture Collection, CRL-2005) were cultured on glass coverslips.17 The cells were cultured in Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose, supplemented with 10% fetal bovine serum, to a coverage of approximately 50%. The astrocytes were fixed in 5% paraformaldehyde for 10 min and then incubated in 2 mL of Hanks HEPES buffer solution for 30 min. The coverslips were rinsed with cold Nanopure water and allowed to dry overnight.
Results and Discussion Figure 1 shows a schematic of the near-field microscope configured for both tapping mode and optical feedback. In the typical tapping-mode arrangement, a laser source is reflected off the bend of a cantilevered NSOM probe and detected on a split photodiode (not shown). The tip is oscillated at its resonance frequency using a bimorph, and the drop in amplitude of the tip oscillation is monitored to sense the sample surface. A typical approach curve for tapping-mode NSOM is shown in Figure 2A. As the tip approaches the sample, the signal remains constant until interactions with the sample surface cause the signal to drop to the baseline. The distance over which the signal drops is tip dependent but typically reaches the baseline within 50 nm. In the optical feedback arrangement, the light exiting the NSOM probe is detected from the side with a PMT coupled to a long working distance microscope (Figure 1). The PMT detects both the light directly exiting the NSOM probe and that which reflects off the sample surface. As the NSOM tip approaches the surface, the change in path length for the two beams causes constructive and destructive interferences in the intensity observed by the PMT. Figure 2B shows the optical approach curve measured simultaneously with the normal force curve seen in Figure 2A. Unlike the force approach curve which only senses the sample when the tip is nanometers from the surface, the optical approach curve begins sensing the sample while the tip is several micrometers away.12,14-16,18-21 This feature provides a mechanism for tip feedback that inherently has more flexibility in the choice of tip-sample separation. The peak separation in the optical approach curve is approximately 500 nm, corresponding to λ/(2 sin θ), where λ is the wavelength of the light source and θ is the angle of the detection system from the sample surface.21 Near the surface, the magnitude of the oscillations increases and finally drops to zero when the tip reaches the sample. As has been previously reported, the optical signal is tip dependent and sensitive to tip shape, aperture size, and coating quality.19-21 It is very important that the NSOM tips have well-defined apertures and be free of pinholes to obtain good optical signals. Similar tapping and optical approach curves are obtained for a variety of supported samples. However, for samples at the air/water boundary, the tapping signal is unable to detect the interface. This prevents the implementation of a stable feedback mechanism for controlling the tip-sample separation. The optical signal, on the other (17) Radany, E. H.; Brenner, M.; Besnard, F.; Bigornia, V.; Bishop, J. M.; Deschepper, J. M. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 64676471. (18) Fischer, U. Ch.; Durig, U. T.; Pohl, D. W. Appl. Phys. Lett. 1988, 52, 249-251. (19) Bozhevolnyi, S. I.; Keller, O.; Xiao, M. Appl. Opt. 1993, 32, 48644868. (20) Weston, K. D.; Buratto, S. K. J. Phys. Chem. B 1997, 101, 56845691. (21) Durkan, C.; Shvets, I. V. J. Appl. Phys. 1997, 83, 1171-1176.
Figure 2. Comparison of approach curves taken under (A) tapping-mode and (B) optical feedback. In contrast to the tapping-mode approach curve in (A) which remains unchanged until the tip is near the sample, the optical approach curve in (B) begins to oscillate with the tip micrometers above the surface. In (C), a topography image of a calibration standard is shown. The image was collected using optical feedback with the NSOM tip held at position a in (B). The 5 µm × 5 µm × 200 nm deep pits in the calibration standard are accurately followed by the NSOM tip.
hand, has the same qualitative characteristics as the approach curve shown in Figure 2B. This provides a mechanism for tip feedback at the air/water interface as demonstrated previously.14 Moreover, we find equally stable optical signals for samples under liquid solution, which may provide a means of tip feedback for fragile samples such as living cells. To demonstrate that the optical feedback arrangement can be used to track sample features, a topographical image of a calibration standard is shown in Figure 2C. The standard consists of 5 µm × 5 µm pits that are 200 nm deep. The tip-sample separation was set at position a in Figure 2B. The NSOM topographical image shown in Figure 2C accurately tracks the surface contours, and the height contrast measured is the same as that obtained using the normal tapping-mode method. However, there is a false height change reported by the optical signal on the right-hand side of the pits as the edge is reached. This arises from complications encountered from the sample geometry, which will be explored in more detail later in the paper. To further compare optical feedback with normal tapping feedback methods, Figure 3 shows NSOM fluo-
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Figure 3. Tapping-mode fluorescence (A) and topography (B) images of 15 µm × 15µm region of a DPPC lipid monolayer doped with 0.25 mol % fluorescent diIC18. The Langmuir-Blodget film was transferred onto mica substrate in a region of the pressure isotherm at which both liquid condensed (LC) and liquid expanded (LE) lipid phases coexist in the film. The fluorescence in (A) marks areas of LE phase in the lipid monolayer which can also be seen as regions of lower height in the NSOM topography image (B). Images C and D show the NSOM fluorescence and topography images, respectively, of the same area of the film collected using optical feedback.
rescence and topographical images of a DPPC lipid monolayer doped with 0.25 mol % diIC18 fluorescent dye. Images A and B of Figure 3 show the NSOM fluorescence and height images, respectively, taken using tapping-mode feedback, and images C and D of Figure 3 show the same area imaged using optical feedback. The LangmuirBlodgett monolayer was transferred onto a freshly cleaved mica surface in a region of the pressure isotherm at which both liquid condensed (LC) and liquid expanded (LE) lipid phases coexist in the film. As shown previously, both the NSOM fluorescence and topographical images can be used to map the distribution of the lipid phases.22-24 The bright regions in the NSOM fluorescence images shown in images A and C of Figure 3 map the locations of the LE lipid phase into which the fluorescent diIC18 dye partitions. These regions can be compared with the NSOM height images shown in images B and D of Figure 3, which are sensitive to the small 5-8 Å height differences between the two lipid phases. The LE phase is lower in height than the surrounding LC phase. The direct correlation between (22) Hollars, C. W.; Dunn, R. C. Biophys. J. 1998, 75, 342-353. (23) Hollars, C. W.; Dunn, R. C. J. Phys. Chem. 1997, 101, 63136317. (24) Shiku, H.; Dunn, R. C. J. Phys. Chem. B 1998, 102, 3791-3797.
both the NSOM fluorescence and topographical images can, therefore, be used to assign the phase structure present in the film. When the images shown in Figure 3 are compared, the small lipid domains seen in the fluorescence and force images under normal tapping-mode feedback (A and B) are also detectable when imaging using the optical feedback mechanism (C and D). The NSOM fluorescence image taken with optical feedback (Figure 3C) reveals the same small LE domains as seen in the tapping-mode fluorescence image (Figure 3A), although the contrast is somewhat diminished. This can be attributed to partial photobleaching of the sample from previous scans and from the increased tip-sample separation used in the optical feedback method. The small 5-8 Å height differences between the LE and LC phases remain visible in the optical feedback height image shown in Figure 3D, which illustrates its sensitivity to small height changes in the sample. These images demonstrate the capabilities of optical feedback for use in high-resolution NSOM fluorescence and topographical measurements. One potential drawback of this feedback mode arises from its sensitivity to refractive index changes in the sample.20,25 Refractive index changes can potentially
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Figure 4. (A) 3 µm × 3 µm tapping-mode NSOM topography image of 260 nm latex microspheres spin-coated onto a mica surface. (B) Optical reflection signal recorded simultaneously with the force image shown in (A). The contrast seen in (B) reveals a coupling between the reflection signal and the topography of the sample. (C) Height image taken using optical feedback of the same sample region imaged in (A). Because of the coupling between the surface features and the reflection signal, the height image in (C) does not reflect the same surface contours as measured in the tapping-mode image.
couple into the feedback signal and report false topography in the optical feedback image. For the lipid film images shown in Figure 3, this can be checked by subtracting the height images collected under both feedback mechanisms. Any changes in height between the two images will show up as contrast in the subtracted image. Subtracting image D from B in Figure 3 results in a homogeneous image that only contains contrast arising from the different noise levels of the two images. The lack of features shows that our experimental system is not sensitive to the small refractive index differences between the two lipid phases and that an accurate mapping of the surface topography is obtained under optical feedback. Fluorescence and height images were also collected in optical feedback with the tip held further from the sample. With the tip held in position b (Figure 2B), resolution and contrast in the fluorescence image diminished but still clearly showed the larger domains. In the height image, no contrast was observed but the system did remain in feedback with the tip actively held at a constant distance from the sample. This demonstrates the noncontact imaging capabilities of the optical feedback mechanism. In addition to refractive index changes in the sample, differences in the collection efficiency arising from sample topography20,21,26,27 can also complicate the height images measured under optical feedback. This is not a problem for samples such as the lipid film shown in Figure 3 that contain nearly constant refractive index and low topography. However, for samples that exhibit sharp topographical features on the order of the probe tip diameter, false height changes can arise in the optical feedback topographic image. To investigate this effect, 260 nm diameter latex microspheres spin-coated onto a mica substrate were imaged under tapping and optical feedback. Figure 4A shows a NSOM tapping-mode image of the latex spheres and their close packed arrangement on the mica surface. The height changes observed are approximately 30 nm, which reflects a convolution between the true sample topography and the shape of the particular NSOM tip. Figure 4B shows the reflection signal monitored while the tapping-mode image in Figure 4A was collected. If the optical signal is unaffected by geometric considerations in the sample, then this image should not exhibit any (25) Cline, J. A.; Isaacson, M. Appl. Opt. 1995, 34, 4869-4876. (26) Durkan, C.; Shvets, I. V. J. Appl. Phys. 1996, 79, 1219-1223. (27) Weston, K. D.; DeAro, J. A.; Buratto, S. K. Rev. Sci. Instrum. 1996, 67, 2924-2929.
contrast. Instead, there is clear evidence that the optical signal is responding differently across the spheres. Figure 4C shows the same area of the sample imaged under optical feedback. While the latex spheres are still observable in this image, the contours do not follow the sample topography measured in tapping mode. Moreover, in the upper part of the image, a sudden change in the optical signal causes the tip position to jump from the first node of the optical approach curve to the second node, further complicating the image and degrading the spatial resolution. These results point to some of the problems that can arise through the optical feedback mechanism and what types of sample features may be particularly problematic. The motivation for exploring this new feedback mode arises from its potential in reducing the tip-sample interactions in experiments on soft biological samples. The previous examples demonstrate that samples exhibiting small height changes and nearly constant refractive index can easily and accurately be imaged under optical feedback. However, for biological specimens that often exhibit complicated topography changes, the results shown in Figure 4 suggests that these samples may be problematic. As an initial test to see if refractive index changes or the structure of cells complicates the optical feedback mechanism, astrocyte cells that have been cultured on glass coverslips, fixed, and dried were imaged using both tapping and optical feedback. Figure 5 compares topographical images of a fixed astrocyte imaged using tapping-mode feedback (A) and the optical feedback mechanism (B and C). Both feedback modes clearly track the contours of the cell. Comparison of the heights measured by both techniques reveals similar dimensions for the cell indicating that refractive index or structural features of the cell are not significantly contributing to the optical feedback signal. Of course, fixed and dried cells are much different than living cells and these initial results are far from conclusive, but they do show the ability of the optical feedback method to accurately track complicated surfaces such as cells. Perhaps some of the more interesting applications of this technique will not arise from incorporating it into a tip feedback scheme at all but, instead, use the optical signal as a tool for measuring small dynamic height changes in microscopic sample areas. With the NSOM tip positioned close to a sample surface and held stationary, the optical signal can report height changes occurring directly below the aperture. This nonscanning mode of
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Figure 6. In a nonscanning mode, the optical signal can be used to measure small dynamic height changes in a sample. To characterize this, the NSOM tip was held stationary above a piezo ceramic bimorph to which a sine wave was applied. In both (A) and (B), the upper trace shows the sine wave applied to the bimorph and the bottom trace plots the optical signal. In (A), the bimorph was oscillated with an amplitude of 60 mV (6 nm) at a frequency of 100 Hz. To demonstrate the ability to track motions on the millisecond time scale, the bimorph frequency was increased in (B) to 1 kHz. The amplitude of the sine wave was also increased to 100 mV, which corresponds to a bimorph movement of less than 12 nm.
Figure 5. (A) 20 µm × 20 µm tapping mode topography image of an astrocyte cell that has been cultured on a glass cover slip, fixed, and dried. (B) Image of the same area of the cell taken using the optical feedback mechanism. The contours of the cell observed by both tapping and optical feedback are the same, indicating the applicability of optical feedback for these types of applications. (C) 40 µm × 40 µm optical feedback topography image of the same cell.
operation may be useful in studying processes such as protein conformational dynamics without the need for external tags or contact with the sample. The optical signal, therefore, provides a completely noninvasive method for monitoring small nanometer height changes on a fast time scale. To characterize the amplitude and time scales accessible with this technique, the NSOM tip was positioned above a piezo ceramic bimorph to which a sine wave of variable amplitude and frequency was applied. The tip was held without feedback at a position approximately corresponding to b in Figure 2B. Clearly, there are no tip-sample force interactions at position b in the optical approach
curve which can be seen through comparison with the corresponding position in the tapping approach curve. Figure 6 shows the results from two trials. In Figure 6, the upper trace plots the sine wave applied to the bimorph and the bottom trace shows the optical signal probing the motion of the bimorph. In Figure 6A, the bimorph was oscillated at 100 Hz with an applied voltage of 60 mV. Calibration of the bimorph displacement using atomic force microscopy indicates that this voltage corresponds to a maximum displacement of 6 nm. The optical trace shows that the interferometric signal correctly tracks the sine wave motion of the bimorph. In Figure 6B, the bimorph was oscillated faster with a frequency of 1 kHz and an amplitude of 100 mV, which corresponds to a displacement of 12 nm. These results show that the optical signal can accurately track nanometric movements on the millisecond time scale, which offers interesting implications for probing biological motions in real time.28,29 The amplitudes and time scales accessible with this interferometric technique are sufficient to probe many significant biological motions, free from any tip-sample force interactions that can influence the observed movements. (28) Rousso, I.; Khachatryan, E.; Gat, Y.; Brodsky, I.; Ottolenghi, M.; Sheves, M.; Lewis, A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 79377941. (29) Radmacher, M.; Fritz, M.; Hansma, H. G.; Hansma, P. K. Science 1994, 265, 1577-1579.
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Conclusion An optical interferometric technique for sensing the tipsample distance in NSOM is reported. Topographical images collected with the optical feedback method were compared with similar images collected using the standard tapping-mode method. The optical method is shown to accurately track surface topography for most of the samples studied. Images of lipid monolayers collected under optical feedback have comparable high resolution fluorescence and topography characteristics as that obtained under tapping-mode operation. However, for samples exhibiting topography changes on the order of the NSOM tip, such as the latex spheres, geometric considerations can lead to false height contrast in the optical feedback topography image. As shown for fixed cells though, both the tapping-mode and optical feedback images report similar height information. These results illustrate the feasibility of using this noncontact technique
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for imaging fragile samples such as living cells or for carrying out experiments at the air/water interface. In a nonscanning mode of operation, we have demonstrated the ability of the interferometric signal to track small nanometer motions on the millisecond time scale. This noninvasive method of probing changes in height does not require any force interactions between the tip and the sample or the need for external tags. This capability should prove useful in monitoring dynamic height changes at specific locations of a sample, which may be particularly informative for many biological processes. Acknowledgment. Support for these projects was provided by NSF (CHE-9612730), NSF-CAREER (CHE9703009), and the Searle Scholars Program/The Chicago Community Trust. LA981198S
