Near-field scanning optical microscopy (NSOM) is

Extending Near-Field Scanning Optical Microscopy for Biological Studies Olivia L. Mooren, Elizabeth S. Erickson, Nicholas E. Dickenson, and Robert C. Dunn* University of Kansas, Lawrence, KS Keywords: near-field scanning optical microscopy (NSOM), single molecule, heating, live cell, atomic force microscopy (AFM) Near-field scanning optical microscopy (NSOM) is a scanning probe technique that enables optical measurements to be conducted with nanometric spatial resolution. In addition to high-resolution optical information, NSOM yields a topographic mapping of sample features that enables a direct comparison to be made between surface topography and the optical signal. For the biological sciences, where sophisticated fluorescence labeling protocols have been developed, the simultaneously collected fluorescence and topography information measured with NSOM offers a potentially powerful tool. The progress in implementing NSOM on unfixed, viable samples, however, has been slowed over concerns regarding sample heating and problems associated with damaging forces generated during imaging. Here, we discuss recent measurements that show sample heating to be modest and not limiting for NSOM applications on viable tissues. In addition, we highlight recent work on developing new NSOM probes that have been demonstrated to be amenable with imaging unfixed samples under buffered conditions. These developments now enable the high resolution of NSOM to be applied to areas in the biological sciences that were previously inaccessible. ( JALA 2006;11:268 72) *Correspondence: Robert C. Dunn, Ph.D., University of Kansas, Department of Chemistry, Multidisciplinary Research Building, 2030 Becker Dr., Lawrence, KS 66047; Phone: þ1.785.864.4313; Fax: þ1.785.864.5396; E-mail: rdunn@ku.edu 1535-5535/$32.00 Copyright c 2006 by The Association for Laboratory Automation doi:10.1016/j.jala.2006.05.012 INTRODUCTION The desire to probe biological samples on the length scale over which important structures and substructures exist has led to the development of a number of high-resolution techniques. Historically, optical microscopy has proven to be the most useful given its noninvasiveness, specificity, speed, low cost, and ease of use. The optical diffraction limit, however, generally limits the spatial resolution to approximately 250e300 nm, which prevents the direct observation of many important biological structures. This limitation has led to the development of other techniques such as electron microscopy (EM) and scanning probe microscopy (SPM), which have atomic resolution and are able to directly visualize proteins. Although having higher spatial resolution, these methods unfortunately lose many of the inherent advantages of optical approaches. For example, EM usually requires long and relatively harsh sample preparation procedures and operates under vacuum, which precludes measurements on viable samples. SPM, on the other hand, provides high-resolution topographical information and is compatible with viable tissues, but largely lacks the specificity of optical techniques, such as immunofluorescence. These limitations led to the development of near-field scanning optical microscopy (NSOM), 1,2 which simultaneously measures sample topography and fluorescence with nanometric spatial resolution. Here, we briefly discuss recent developments in NSOM that have enabled the imaging of viable biological samples with high resolution. 268 JALA August 2006

NSOM PRINCIPLES AND IMPLEMENTATION The basic principle that led to the development of NSOM was first proposed by E.H. Synge in 1928. 3 Synge proposed an approach to circumvent the fundamental resolution limit in light microscopy by eliminating the lens normally used to focus light. Instead of using a lens to focus light, Synge proposed passing light through a subwavelength aperture in an opaque screen held very close to the sample surface. In this arrangement, light passing through the aperture in the screen is forced to interact with the sample before diffracting away from the aperture. By scanning the aperture across the surface, an optical image of the sample can be measured with a spatial resolution only limited by the size of the aperture. Today, aperture-based NSOM is a scanning probe technique that uses a tapered fiber optic probe to form the nanometer-sized aperture through which light is delivered to the sample. 4,5 Apertureless NSOM probes have also been developed, 6,7 however, our discussion will focus on aperture-based NSOM. Figure 1A shows a typical fiber optic NSOM probe with light exiting the aperture. Several methods have been developed for fabricating NSOM probes. The most common involves heating and mechanical pulling of the optical fiber to form the taper. The sides of the taper are coated with 50e100 nm of aluminum to confine the light such that it only exits the aperture at the distal end of the probe. A magnified image of a fiber optic NSOM probe is shown in Figure 1B. Visible in Figure 1B are the grains in the aluminum coating and the aperture at the end, where light exits the probe. With the nanometric light source formed, the NSOM probe must be scanned within nanometers of the sample surface to obtain high spatial resolution. Several force feedback methods similar to those developed for other SPM methods have been successfully adopted in NSOM to control the sample-tip gap. 5,8 To illustrate the high spatial resolution and favorable detection limits of NSOM, Figure 2 shows an NSOM fluorescence image of single molecules. Each peak in Figure 2 represents the fluorescence from a single fluorescent diic 18 molecule in a lipid monolayer, and the feature size indicates a spatial resolution of w25 nm. This illustrates that the spatial resolution of NSOM can be an order of magnitude better than that possible using conventional optics. In addition to the high resolution and low detection limits, NSOM measures sample fluorescence and topography simultaneously. Because a feedback method is implemented to keep the NSOM probe close to the sample surface, a force mapping of the sample surface is measured along with the optical signal. 5 Figure 3 shows the simultaneously measured NSOM fluorescence (Fig. 3A) and force (Fig. 3B) measurements of a fixed cell cultured onto a coverslip. This one-toone mapping of sample fluorescence and topography provides another useful aspect of NSOM for probing biological samples. However, the progress in applying NSOM in the field of biological sciences has been slow. One concern revolves around the heating that is known to occur at the NSOM aperture as light is lost into the metal coating of the probe. This occurs in the taper region of the probe where the fiber no long acts as a waveguide. Heating Figure 1. (A) Magnified view of a typical metal-coated fiber optic NSOM probe with light emerging from the aperture at the end. (B) Electron microscopy image of an NSOM probe. The grains arise from the aluminum coating used to confine the light. Visible at the tip is the subwavelength aperture through which light passes. The end of this probe was milled using a Focused Ion Beam (FIB) instrument. JALA August 2006 269

It is important to stress that this reflects the maximal heating at high output powers, and generally lower powers are used in NSOM experiments. As NSOM output powers are lowered, the temperature decreases and may be further reduced under aqueous conditions where heat can be transferred into the solution. Therefore, for biological applications, heating by the NSOM probe should not be a limiting factor. Another serious concern involves the forces generated during imaging that can damage fragile, viable samples. In the past, this has prevented NSOM measurements on viable samples with the NSOM probe in active feedback. MODIFICATIONS TO NSOM PROBES Figure 2. A 1.7 mm 1.7 mm NSOM fluorescence image of a field of single molecules. Each peak in the image represents the fluorescence from one molecule of diic 18 in a lipid monolayer, and the peak width, w25 nm, illustrates the high spatial resolution. effects at the aperture are difficult to characterize due to its small size, but significant heating could lead to sample damage in biological applications. It is well known that NSOM probes eventually fail as more light is coupled through the probe due to heating, and previous studies suggested that heating could reach hundreds of degree Celsius. 9,10 However, these measurements were taken along the taper of the probe, 9 micrometers from the aperture, so it remained unclear what heating the sample experienced. We recently reported measurements using a thermochromic sample to precisely quantify the amount of heat transferred to the sample. 11 This study found that sample heating is considerably less than previously reported and a maximum of 55e65 C is reached. As illustrated in Figure 3, NSOM measurements on fixed biological samples are very informative and have been used to study questions on protein colocalization, 12 the spatial distribution of cellular components, 13,14 and the orientation of molecules within models of the biological membrane. 15 NSOM measurements have also been made in aqueous environments, and several techniques for implementing these measurements have been developed. 16e18 Progress in implementing NSOM measurements on unfixed, viable biological samples under buffered conditions, however, has been a formidable challenge. Typical fiber optic NSOM probes have large spring constants (w200 N/m) that lead to damaging forces while imaging soft biological samples in active force feedback. 19 A tremendous effort has been made, therefore, to develop new approaches for reducing the spring constant of the NSOM probe. 17,20,21 This has recently been demonstrated and shown to enable live cell NSOM measurements. 20 Because of the small spring constant of conventional atomic force microscopy (AFM) probes (w0.06e0.6 N/m), AFM measurements on biological samples are easily Figure 3. Simultaneously collected 40 mm 40 mm NSOM (A) fluorescence and (B) force images of a cell that has been cultured onto a glass coverslip and fixed. A cell surface receptor has been immunofluorescently labeled. 270 JALA August 2006

Figure 4. Two NSOM/AFM hybrid tips fabricated in our laboratory. (A) A small hole is milled into a conventional triangular AFM cantilever and a fiber optic NSOM probe is inserted and glued into place. Further milling with the FIB removes the excess fiber and produces a flush face that enables efficient coupling of light into the probe. (B) NSOM/AFM probe fabricated without the use of FIB milling. Fiber optic NSOM probes are fabricated using the chemical etching technique that produces tapers with reduced aspect ratios while maintaining the flat surface necessary for coupling light into the probe. The NSOM probe is glued onto the end of a conventional rectangular AFM cantilever. implemented, and these studies are well established. 22,23 It has long been recognized that incorporating an NSOM light source into an AFM cantilever is a possible avenue for imaging living tissues. 21,24,25 One method developed for fabricating hybrid NSOM/ AFM probes involves coupling conventional fiber optic NSOM probes with AFM cantilevers. 20 A7e20 mm hole is milled into a triangular AFM cantilever using a focused ion beam (FIB) instrument. A conventional fiber optic NSOM probe is carefully inserted into the hole using micromanipulators and secured into place using an ultraviolet curable adhesive. The hybrid tip is then returned to the FIB where the excess fiber is removed, such that the fiber is flush with the surface of the cantilever. A magnified view of a finished fiber optic NSOM/AFM probe is shown in Figure 4A. Using these hybrid probes, NSOM images of living cells have recently been demonstrated. 20 Human arterial smooth muscle cells, in which the adrenergic receptors in the cellular membrane were fluorescently labeled, were imaged under buffered conditions. Having demonstrated the utility of these hybrid NSOM tips for live cell imaging, recent work has centered on developing protocols for tip fabrication that do not rely on FIB milling. FIB instruments are expensive and the milling is time consuming. Developing protocols that avoid the use of the FIB are therefore desirable for this approach to be of wide utility. Developing a hybrid NSOM/AFM probe that does not rely on FIB milling presents several challenges. The biggest obstacle involves reducing the size of the fiber optic NSOM probe to be attached to the AFM cantilever. The total probe size must be minimized to keep the hybrid tip mechanically stable. Moreover, the fiber optic probe not only has to have a small aperture at one end, but the other must be cleaved flat for efficient coupling of light into the probe. These metrics are hard to satisfy by the traditional approach for taper formation using fiber optic heating and mechanical pulling. Therefore, tapers were formed using the chemical etching approach. 26 This method produces tapers with reduced aspect ratios, which increases throughput efficiency 27 and allows very short fiber probes to be fabricated while maintaining a cleaved end for efficient coupling. Figure 4B shows an NSOM/AFM hybrid tip fabricated in our laboratory using this approach. The total length of the fiber optic probe attached to the AFM cantilever is w300 mm, which we are currently working to reduce further. Apparent in Figure 4B is the sharp taper that forms the NSOM aperture, and the flat cleave on the opposite end where light is coupled into the probe. It is important to stress that this probe was produced without the use of FIB milling, which greatly reduces the cost and time associated with fabrication. These new hybrid NSOM/AFM probes have higher light throughput than those produced by the heating and pulling method, and yield good topographical resolution in initial imaging tests. These tips are currently being further characterized for applications in the biological sciences, but offer a promising new approach for tip fabrication in NSOM. CONCLUSIONS NSOM offers a potentially powerful new tool for application in the biological sciences. Its nanometric spatial resolution and simultaneous detection of both optical and topographical signal seems ideally suited for biological applications. NSOM has already made important contributions in solidstate applications, probing polymer systems, and model membranes. 5 Progress on biological systems has been slowed by concerns over sample heating and due to problems implementing the technique on unfixed tissues. Recent experiments JALA August 2006 271

have shown, however, that sample heating is modest and not likely to cause problems. 11 Reducing the forces involved in imaging, such that fragile samples are not damaged, has proven to be a much greater challenge. However, the recent development of hybrid NSOM/AFM tips has enabled NSOM measurements on living cells under buffered conditions. 20 Further developments in tip fabrication that increase the efficiency, and reduce the time and cost involved are currently underway and should significantly enhance the impact that NSOM will have in the field of biological sciences. ACKNOWLEDGMENTS The authors gratefully acknowledge funding from NIH (GM55290) and the Madison and Lila Self Foundation. REFERENCES 1. Pohl, D. W.; Denk, W.; Lanz, M. Optical stethoscopy: image recording with resolution l/20. Appl. Phys. Lett. 1984, 44, 651e653. 2. Lewis, A.; Isaacson, M.; Harootunian, A.; Muray, A. Development of a 500 A spatial resolution light microscope. Ultramicroscopy 1984, 13, 227e232. 3. Synge, E. H. A suggested method for extending microscopic resolution into the ultra-microscopic region. Philos. Mag. 1928, 6, 356e362. 4. Betzig, E.; Trautman, J. K.; Harris, T. D.; Weiner, J. S.; Kostelak, R. L. Breaking the diffraction barrier: optical microscopy on a nanometric scale. Science 1991, 251, 1468e1470. 5. Dunn, R. C. Near-field scanning optical microscopy. Chem. Rev. 1999, 99, 2891e2927. 6. Novotny, L.; Stranick, S. J. Near-field optical microscopy and spectroscopy with pointed probes. Annu. Rev. Phys. Chem. 2006, 57, 303e331. 7. Krug, J. T. II; Sánchez, E. J.; Xie, X. S. Design of near-field optical probes with optimal field enhancement by finite difference time domain electromagnetic simulation. J. Chem. Phys. 2002, 116, 10895e10901. 8. Edidin, M. Near-field scanning optical microscopy, a siren call to biology. Traffic 2001, 2, 797e803. 9. Staehelin, M.; Bopp, N. A.; Tarrach, G.; Meixner, A. J.; Zschokke- Graenacher, I. Temperature profile of fiber tips used in scanning nearfield optical microscopy. Appl. Phys. Lett. 1996, 68, 2603e2605. 10. La Rosa, A. H.; Yakobson, B. I.; Hallen, H. D. Origins and effects of thermal processes on near-field optical probes. Appl. Phys. Lett. 1995, 67, 2597e2599. 11. Erickson, E. S.; Dunn, R. C. Sample heating in near-field scanning optical microscopy. Appl. Phys. Lett. 2005, 87, 201102. 12. Enderle, T.; Ha, T.; Ogletree, D. F.; Chemla, D. S.; Magowan, C.; Weiss, S. Membrane specific mapping and colocalization of malarial and host skeletal proteins in the Plasmodium falciparum infected erythrocyte by dual-color near-field scanning optical microscopy. Proc. Natl. Acad. Sci. USA 1997, 94, 520e525. 13. Burgos, P.; Yuan, C.; Viriot, M. L.; Johnston, L. J. Two-color near-field fluorescence microscopy studies of microdomains ( rafts ) in model membranes. Langmuir 2003, 19, 8002e8009. 14. Hwang, J.; Gheber, L. A.; Margolis, L.; Edidin, M. Domains in cell plasma membranes investigated by near-field scanning optical microscopy. Biophys. J. 1998, 74, 2184e2190. 15. Hollars, C. W.; Dunn, R. C. Probing single molecule orientations in model lipid membranes with near-field scanning optical microscopy. J. Chem. Phys. 2000, 112, 7822e7830. 16. Gheber, L. A.; Hwang, J.; Edidin, M. Design and optimization of a near-field scanning optical microscope for imaging biological samples in liquid. Appl. Opt. 1998, 37, 3574e3581. 17. Koopman, M.; de Bakker, B. I.; Garcia-Parajo, M. F.; van Hulst, N. F. Shear force imaging of soft samples in liquid using a diving bell concept. Appl. Phys. Lett. 2003, 83, 5083e5085. 18. Talley, C. E.; Lee, M. A.; Dunn, R. C. Single molecule detection and underwater fluorescence imaging with cantilevered near-field fiber optic probes. Appl. Phys. Lett. 1998, 72, 2954e2956. 19. Talley, C. E.; Cooksey, G. A.; Dunn, R. C. High resolution fluorescence imaging with cantilevered near-field fiber optic probes. Appl. Phys. Lett. 1996, 69, 3809e3811. 20. Kapkiai, L. K.; Moore-Nichols, D.; Carnell, J.; Krogmeier, J. R.; Dunn, R. C. Hybrid near-field scanning optical microscopy tips for live cell measurements. Appl. Phys. Lett. 2004, 84, 3750e3752. 21. Krogmeier, J. R.; Dunn, R. C. Focused ion beam modification of atomic force microscopy tips for near-field scanning optical microscopy. Appl. Phys. Lett. 2001, 79, 4494e4496. 22. Dvorak, J. A. The application of atomic force microscopy to the study of living vertebrate cells in culture. Methods 2003, 29, 86e96. 23. Horber, J. K. H.; Miles, M. J. Scanning probe evolution in biology. Science 2003, 302, 1002e1005. 24. Oesterschulze, E.; Rudow, O.; Mihalcea, C.; Scholz, W.; Werner, S. Cantilever probes for SNOM applications with single and double aperture tips. Ultramicroscopy 1998, 71, 85e92. 25. Eckert, R.; Freyland, J. M.; Gersen, H.; Heinzelmann, H.; Schürmann, G.; Noell, W.; Staufer, U.; de Rooij, N. F. Near-field fluorescence imaging with 32 nm resolution based on microfabricated cantilevered probes. Appl. Phys. Lett. 2000, 77, 3695e3697. 26. Hoffmann, P.; Dutoit, B.; Salathe, R. P. Comparison of mechanically drawn and protection layer chemically etched optical fiber tips. Ultramicroscopy 1995, 61, 165e170. 27. Burgos, P.; Lu, Z.; Ianoul, A.; Hnatovsky, C.; Viriot, M. L.; Johnston, L. J.; Taylor, R. S. Near-field scanning optical microscopy probes: a comparison of pulled and double-etched bent NSOM probes for fluorescence imaging of biological samples. J. Microsc. 2003, 211, 37e47. 272 JALA August 2006