279 High resolution imaging of native biological sample surfaces using scanning probe microscopy Andreas Engel∗†, Cora-Ann Schoenenberger∗ and Daniel J Muller ¨ ∗‡ The possibility of acquiring high resolution topographs using scanning probe microscopes under physiological conditions allows the observation of biomolecules at work. Progress has recently been made in imaging protein–DNA complexes, individual oligomers and protein arrays. Scanning probe microscopes are now tools that complement X-ray crystallography and electron microscopy. Addresses ∗ME Muller ¨ Institute for Structural Biology, Biozentrum, Klingelbergstr 70, CH-4056 Basel, Switzerland †e-mail: aengel@ubaclu.unibas.ch ‡Forschungszentrum Julich, ¨ IBI-2: Structural Biology, D-52425 Julich, ¨ Germany Current Opinion in Structural Biology 1997, 7:279–284 Electronic identifier: 0959-440X-007-00279 Scanning near field optical microscopes (SNOMs) measure the local optical properties of a sample using an aperture as a probe. Significant progress in the underlying technology has lead to the detection of single fluorophores [3], but the resolution achieved is at least an order of magnitude less than that of the STM. For the latter, however, the sample must be electrically conducting, which precludes biological structures as they are notorious insulators. High resolution scanning probe microscopy of biological samples is primarily achieved using the AFM. This instrument also allows samples to be scanned in solution, opening up the fascinating possibility of observing biomolecules at work. Excellent reviews are now available that illustrate various applications of the AFM to biological research [4–7]. This review concentrates, with a single exception, on high resolution images acquired with the AFM.  Current Biology Ltd ISSN 0959-440X Abbreviations 2D two-dimensional 3D three-dimensional AFM atomic force microscope SNOM scanning near field optical microscope STM scanning tunneling microscope Introduction Invented more than a decade ago [1,2], scanning probe microscopes are powerful tools to study the surfaces of solids in vacuum, gas or liquid at atomic-scale resolution. A small probe (with an atomic size of up to a few tens of nanometers) is scanned over the sample surface and the signal produced by the sample–probe interaction is acquired. The height (z) of the probe relative to the sample can either be kept constant during the raster-scan in the (x,y)-plane, or it can be controlled by a servo system to keep the interaction signal constant. In the latter mode, the surface corrugations are contoured. To achieve high resolution, the decay length of the sample–probe interaction must be small. The tunneling current between the probe tip and the conducting sample typically changes by a factor of ten when the tip–sample distance changes by 1 A˚. Whereas van der Waals and electrostatic static interactions between the tip and the sample extend over several nanometers, shell–shell repulsion forces rise very sharply (by a factor >10 A˚−1) when the tip is in contact with the sample surface. Atomic resolution, therefore, has been obtained using scanning tunneling microscopes (STMs) and using atomic force microscopes (AFMs). Instrumentation The heart of the AFM is a pyramid-shaped stylus (with an apex radius of 2–10 nm) mounted on a flexible cantilever (spring constant k = 0.1–1 Nm−1). A laser beam reflected from the back of this lever onto a multifaceted photo diode allows deflections of < 1 nm to be detected (Fig. 1). Hence, an AFM may be operated in contact mode with a force applied to the stylus of about 10−10 N. The resonance frequency of the cantilever sets a limit to the speed for contouring a surface; this speed is slower in liquids than in vacuum as result of damping [8]. A piezo scanner is actuated to translate the sample relative to the tip in x, y and z directions. The corresponding signal is provided by the computer that drives the microscope and acquires the images. Multiple images may be collected in parallel by taking advantage of different imaging modes. A useful image, akin to differential interference contrast in light microscopy, is generated by the error or deflection signal [9]. When flat samples are scanned slowly, the piezo scanner is able to follow the surface corrugation precisely, thus counteracting the cantilever deflection. On corrugated samples, however, the servo speed is too slow and a deflection is inevitable. This deflection signal is valuable for low magnification work, in which the AFM is operated with a high scan speed. To monitor the absolute height information, the z-signal (or height signal) driving the piezo scanner is recorded simultaneously. In addition, it is possible to reconstruct the topography from the deflection signal, approximating the latter as the first derivative of the height signal along the fast scan direction [10••]. 280 Macromolecular assemblages Figure 1 Physical constraints of scanning probe microscopy. (a) Simple geometric model to estimate the resolution. A spherical particle of diameter D (shown on the left) exhibits a full width at half maximum (FWHM =2√ (RD + D2/4) when imaged with a spherical tip of radius R. The true height of the particle is measured. An array of spherical particles scanned with the same tip (shown on the right) yields a corrugation amplitude, h ≈ (D/8R)1/f, using the spatial frequency of the array, f = 1/D. Thus, a scanning probe microscope has approximately an 1/f transfer function. This is a qualitative description, however, as the imaging process is strictly nonlinear, as illustrated by the small gray sphere on the left that does not produce a signal when close to the larger particle but that would be seen when isolated. Such hard sphere models do not take sample flexibility and long range tip–sample interaction into account. (b) Interactions between substrate and sample in electrolyte solutions. The interactions are symmetric to those between the sample and the tip, unless the sample is covalently bound to the support. Immobilization may therefore require different buffer conditions than imaging. For adsorption, the Debye layer thickness λD needs to be minimized (λD =0.304/√ [ec] nm for monovalent electrolyte concentrations, ec; [34•]) to allow sample adsorption by van der Waals attraction [14]. For imaging, however, the electrolyte should be selected to equalize electrostatic repulsion and van der Waals attraction. For large sheets, it is possible to change the buffer without detachment of the sample. Substrate–sample and sample–stylus interactions Operating the AFM in contact mode induces friction; in order to withstand friction forces, samples need to be immobilized. Large structures adhere well to substrates or can simply be pushed into small holes. Small structures (e.g. single protein complexes, thin filaments), however, exhibit only a small contact area for interaction with the substrate. Protocols have been developed to covalently bind biomolecules to the support [11,12]. Alternatively, friction forces can be minimized by oscillating the cantilever vertically; using this ‘tapping mode’ [13] weakly adsorbed samples can be observed, albeit at somewhat reduced resolution. Samples may firmly bind to freshly cleaved mica that exposes a weakly (negatively) charged, chemically inert surface. In this case, the sum of electrostatic repulsion and van der Waals attraction determines whether samples adsorb. Electrostatic forces depend on the surface-charge density of sample and substrate, and on the electrolytes in the buffer solution. Sample adsorption is thus controlled by the nature and concentration of electrolytes, as recently demonstrated using a variety of samples [14]. Interestingly, the interactions between sample and stylus involve to a large extent the same forces that occur between sample and support. Only the very tip that is in contact with the surface atoms will sense the shell–shell repulsion. It is this short range interaction that confers high resolution structural information. The symmetry in the interactions between the substrate, sample and stylus implies that not necessarily the same buffers should be used for adsorption as for imaging. In addition, height measurements are profoundly affected by the electrolytes, as the surface-charge density of the sample and the support are, in general, different (DJ Muller, ¨ A Engel, unpublished data). It is important to note that orders of magnitudes smaller that occur when samples are hydrophilic surface is covered molecules [15,16]. these forces are several than the capillary forces observed in air, as every by several layers of water High resolution imaging Filamentous structures DNA was an attractive sample with which to test the capability of the STM in the early days of scanning probe microscopy. The first results fostered hopes that it would become possible to sequence DNA using an STM. This goal has not been achieved, and many results have subsequently been identified as artefacts. Nevertheless, double-stranded DNA molecules adsorbed to mica can be imaged in a humid atmosphere at high resolution, using an STM capable of detecting tunneling currents of 10−12 Amp (Fig. 2a) [16]. Clear images of DNA can be obtained at ambient pressure using the AFM when it is flooded with dry nitrogen to minimize capillary forces, or when the tapping mode is used [7]. For the imaging of DNA and native protein–DNA complexes in buffer solution, much effort has been invested in the immobilization of DNA on the High resolution imaging of native biological sample surfaces Engel, Schoenenberger and Muller ¨ 281 Figure 2 Scanning probe microscopy of DNA. (a) Plasmid DNA (pUC 18) on mica imaged by an STM at high resolution (inset) in a humidity chamber (at 66% relative humidity) [16]. The thin water layer coating on the sample has a thickness of ≈ 0.4 nm and exhibits a high conductivity of up to five magnitudes greater than bulk water. The water layer, therefore, enables the imaging process with an STM. (b) AFM image of double stranded l HindIII DNA attached onto mica [17]. To reduce the capillary forces that occur in air, the image is recorded in propanol. (c) The same molecule recorded after one minute demonstrates the reproducibility of the imaging process. Gray shades from dark to bright correspond to a vertical distance of 2.5 nm in (a), 1.8 nm in the inset of (a), and 0.5 nm in (b) and (c). STM images courtesy of R Guckenberger, Max-Planck Institute, Martinsried, Germany; AFM images courtesy of H Hansma, University of California, Santa Barbara, USA. Figure 3 Atomic force microscopy of single protein complexes. (a) CryoAFM image of human IgG monitored at 85K in a vacuum [18•]. The characteristic Y shape of IgG is clearly visible. After adsorption onto mica, the sample is dried in a nitrogen stream and transported into the AFM chamber. (b) Surface plot of an AFM image of GroES oligomers imaged in solution [19••]. After adsorption onto mica, the molecules are fixed with 2% glutaraldehyde. The fine features at the top of the dome reveal seven subunits, which correlates well with the atomic structure of GroES [35]. Gray shades from dark to bright correspond to a vertical distance of 6.5 nm in (a), and 3.5 nm in (b). Images courtesy of J Mou and Z Shao, University of Virginia, Charlottesville, USA. substrate, but improved images have been obtained by employing the tapping mode (Fig. 2b) [17]. Oligomeric complexes Antibodies are among the most interesting biomolecules that are studied by scanning probe microscopy, because imaging these biomolecules at high resolution under physiological conditions opens an important field of applications. The results have been rather disappointing. As antibodies are relatively bulky and flexible structures, they are seen mainly as blobs without substructure. The immobilization of IgGs using cooling techniques has led to images that reveal the Y-shaped molecules (Fig. 3a) [18•]. Higher resolution can be obtained on less corrugated 282 Macromolecular assemblages Figure 4 Regular protein arrays The tightest molecular packing is achieved with regular protein arrays and the highest resolution images have been acquired on such 2D crystals [21]. The contours determined with the AFM have been compared with 3D structural information from electron microscopy [10••,22] and X-ray crystallography [23••]. These experiments have confirmed that the data acquired using the AFM under ¨ native conditions agree to within a few Angstroms with data obtained by other methods. A thoroughly studied example is the Escherichia coli porin OmpF (Fig. 4). AFM image of OmpF. The periplasmic surface of OmpF trimers reconstituted into a rectangular 2D crystal is a suitable sample with which to study the reliability of the AFM. The crystalline sheets adsorb readily to freshly cleaved mica in 300 mM KCl, 10 mM Tris-HCl, pH 7.4. The topograph shown has been recorded on a Nanoscope III, using a Olympus cantilever (k = 0.1 N m−1), applying a force of 100 pN to the stylus. The trimers exhibit a tripartite protrusion consisting of a triangular mass with an indentation about the threefold axis and three arms. This structure protrudes by 0.5 nm from the bilayer surface and separates the three elliptical channels. The striking feature of this topograph is its signal-to-noise ratio: the substructure of each trimer is clearly resolved. In some instances, damaged trimers are distinct (indicated by arrows). The tip used to acquire this image has an elongated apex, which leads to a minor astigmatism. A rectangular unit cell (a = 13.8 nm, b = 7.9 nm) is marked; gray shades from dark to bright correspond to a vertical distance of 1.5 nm. Surface topographies of purple membranes have revealed discrepancies with respect to the initial atomic model of bacteriorhodopsin in that the cytoplasmic loop connecting helices C and D exhibited the same height as loop AB, and that loop EF was not visible (Fig. 5a) [24]. It has subsequently been observed that loop EF becomes visible when the force applied to the stylus is < 200 pN (Fig. 5b) [25••]. In addition, the new atomic model of bacteriorhodopsin reveals that the AB and DC loops are at comparable heights [26]. Purple membranes are, therefore, a suitable sample to test the quality of an AFM, the preparation protocol, and the operator’s skill: if loop EF can be seen, the overall system is tuned to detect rather delicate protein structures. The highest resolution achieved is with samples that have a corrugation amplitude < 0.5 nm, such as the extracellular surface of purple membranes (Fig. 5c) [21]. High resolution imaging is not restricted to 2D crystals. Recently, promising data have been recorded on 3D protein crystals allowing the study of crystal packing and its defects [27•]. Conclusions biomolecules because they are less flexible and can be contoured precisely by the probe tip. When the stability is further improved by glutaraldehyde fixation, single oligomers can be imaged at 1 nm resolution (Fig. 3b) [19••]. Although these images exhibit superb resolution, the preparation steps required for a sample impeach observation of biological activities. Densely packed proteins The geometry of the probe tip restricts access to the topmost fraction of a spherical or cylindrical sample. Tightly packed oligomers appear to be an ideal sample: proteins laterally support each other, hiding surfaces that the tip cannot reach in any case. Membrane proteins can often be reconstituted in lipid bilayers at high density or can be inserted in supported monolayers. Such examples include various toxins, whose surface features have been resolved with the AFM [20]. Scanning probe microscopes are tools that entail interesting applications for structural biologists; in particular, the AFM has demonstrated its capability of acquiring surface topographies with a lateral resolution of 0.5–1 nm and a vertical resolution of 0.1–0.2 nm when the biological sample is in a buffer solution. Conformational changes can now be detected at subnanometer resolution, which opens a new avenue to monitor the relationship between structure and function of biomolecules [28,29•]. Such exciting observations suggest that structure–function determinations may become a major application of AFM. The high sensitivity of force measurements has initiated an ever growing variety of experiments that tackle the question of interaction forces between macromolecules [30–32,33••]. The knowledge gained from this work will help to increase the sensitivity of the instruments to allow samples to be scanned at even lower forces. Combined with the progress in understanding the tip–sample interactions [15,34•], these developments are expected to increase not only the resolution, but also the reproducibility and ease of scanning probe microscope operation. High resolution imaging of native biological sample surfaces Engel, Schoenenberger and Muller ¨ Figure 5 283 References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. Binnig G, Rohrer H: Scanning tunneling microscope. Helv Phys Acta 1982, 55:726–735. 2. Binnig G, Quate CF, Gerber C: Atomic force microscope. Phys Rev Lett 1986, 56:930–933. 3. Betzig E, Chichester RJ: Single molecules observed by near-field scanning optical microscopy. Science 1993, 262:1422–1425. 4. 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