Biological applications of scanning probe microscopes.

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1991.20:79-/08

Annu. Rev. Biophys. Biophys.

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Annu. Rev. Biophys. Biophys. Chem. 1991.20:79-108. Downloaded from www.annualreviews.org by McMaster University on 05/01/13. For personal use only.

BIOLOGICAL APPLICATIONS OF SCANNING PROBE MICROSCOPES Andreas Engel

M. E. Muller-Institute for High-Resolution Electron M icroscopy at the Biocenter, University of Basel, CH-4056 Basel, Switzerland KEY WORDS:

scanning probe microscopy, atomic force microscopy, scanning tunneling microscopy, biological samples, specimen preparation

CONTENTS PERSPECTIVES AND OVERVIEW......

....... ...................................... .....................................

INSTRUMENTATION AND THEORETICAL BACKGROUND......................................................

Resolution .. ................ . ............ ... .......... . . . . . . . . . . . . . . . .............. ........ ...... .......... . . . . . . . . . . Servo System, Scan Devices, and Scan Modes ..... ........ .................. .................. Probes Scanning Tunneling Microscopy . . .......... ... . . ............ .... Scanning Force Microscopy.......... . . . . .............................................. . . . . ...................... SPM for Biological Applications . . . . . . ............................ .......... ....... .. . .. ............ . .. . . . .

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....... . . . .............................. ........................................... Protein Structure ....... . ....... ... .............. .................. ................ ........... . . . . . . . . . ................ Preparation Art ifacts During Immobilization and Dehydration . ... ..... Specimen Supports .................................................. .......... . . ....................................

SAMPLE PREPARATION FOR SPM

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79 81 81 83 84 86 87 87 88 88

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88 89

. ............................ . ............................... . . .. ........................................... Scanning Tunneling Microscopy ................ ........ . .................................................... Scanning Force Microscopy ......................................................................................

90 90 100

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APPLICATIONS

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CONCLUSIONS AND PROSPECTS

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PERSPECTIVES AND OVERVIEW The invention of the scanning tunneling microscope (STM) in the early 1 980s by Nobel Prize winners G. Binning and H. Rohrer (2 1 -26, 52, 64, 96) has initiated an exciting series of novel local probe microscopes that imagc the surfaces of conducting as well as insulating solids with atomic 79 0883-9 1 82 /9 1 /06 1 0-0079$02.00

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resolution. In studies of the silicon surface properties, for example, the capabilities of the STM have been used to depict the Si(1 l l )-(7 x 7) recon struction for the first time (26), to visualize the surface density-of-states of Si ( l l l ) and Si(IOO) for different energies ( 60, 6 1 ), to investigate the initial stage of Ag condensation on Si (1 l l ) surfaces (see 1 1 7) (Figure I), and to study surface disorder at atomic scale ( 1 27). Atomic scale surface structure analysis of insulators, however, only became possible after the develop ment of the scanning force microscope (SFM) (4, 5,1 9 , 20, 54, 63, 65, 66, 85, 86, 1 23). These novel local-probe microscopies provide complementary structural and chemical information not accessible by other methods such as conventional microscopies or X-ray diffraction. One generates scanning probe micrographs by moving a nanometer-sized sensor (the probe) along an x, y-raster over a solid and writing the sensor signal to a storage device. Depending on the sensor and the mode of operation, micrographs provide information on the topography, the electronic structure and the mech anical or thermal properties of solid surfaces (1 20, 1 25, 1 26) . The resolution is determined by the sharpness of the sensing tip, which can ultimately consist of a single atom, and by the precision of the scanning device. Scanners are manufactured from piezo ceramic materials and permit sub Angstrom control of the probe position. Most sensors allow images to be recorded in different environments such as vacuum, ambient pressure,

Figure 1 Initial stage of Ag-condensation. The Ag-islands cover approximately one third of the Si ( l l l) (7 x 7) surface. Single Si atoms spaced by approximately 0.8 nm are imaged by the STM with unprecedented clarity. Shape and dimensions of the triangular Ag-islands are determined by the underlying Si surface structure. The topograph was recorded at U 2 V, IT 3 nA; the scale bar represents 2 nm. Reprinted from Reference 1 1 7 with permission. =

=


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physiological buffers, and other liquids (e.g. oil, liquid nitrogen, liquid helium). Clearly, these possibilities make scanning probe microscopies particularly attractive [or biological research because they may ultimately permit investigations of biological surfaces at molecular resolution in their native environment. While the application of the STM in material sciences has progressed substantially, breakthroughs have been sparse in biological sciences, although initial results were promising ( 1 2) . These breakthroughs are the intensely debated high-resolution (better than 1 nm in a few cases) DNA images ( 10, 14, 3 1, 37, 38, 76, 79) and several low-resolution (3 nm at best) topographs from air-dried uncoated protein assemblies (7, 32, 42, 59, 71, 1 12, 124). On the other hand, a series of promising results have most recently been obtained using the SFM (36, 4 1, 12 1, 122, 13 1), indicating that molecular resolution in physiological buffers is feasible. Although somewhat premature, a review of this young and very rapidly moving field of structural research seems timely in view of rather divergent opinions among the expcrts in scanning probe methodology and instrumentation and the structural biologists more faimilar with data gathered with con vention a l microscopes. I believe that thc potentials of scanning probc microscopes (SPM) will eventually become routinely exploitable and that SPMs will play a decisive role in the direct assessment of structure-function relati onships of bio logical systems. Two key aspects beyond the refinement of the instruments need to be considered: First, biological macromolecules and their supra molecular assemblies are very fragile structures requiring special sample preparation procedures. Second, direct comparison with results from con ventional imaging methods such as electron microscopy appears to be more important to enhance the understanding of scanning probe micro graphs than commonly anticipated. Therefore, I present in this review comparative examples from electron microscopy whenever possible. The physics of SPM are discussed, and specimen preparation is given special emphasis. I then concentrate on practical examples in an attempt to unveil trends and to provide as complete an insight into current developments as possible. INSTRUMENTATION AND THEORETICAL BACKGROUND Resolution

Tersoff & Hamann ( 1 1 6) supplied the theoretical foundation of STM . Recently, Rohrer (97) provided suitable approximations t o estimate the resolution of different SPMs. The heart of an SPM is the probe s tip '

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(Figure 2). Its geometry determines the width L of the interaction filament that contains a fraction (1 -1 /11) of the total interaction power according to Equation 1 (97): L

�

2 j2.P ('1) J( s+R) s

L

�

2 j2.Jln ( '1) J( s+R) /K

for fed) � d-n, for fed)

�

n

�

2

exp ( -Kd) ,

1.

whcre R is the tip radius, s the gap between tip and sample, P (I1) is of order one and varies slowly with 11, fed) describes the distance dependence of the interaction, and l/K represents its decay length (Figures 3b and c) . The practical resolution, however, is also determined by the sensitivity of the microscope, i.e. the noise level, as discussed for the STM (75, 1 1 5, 1 1 6) and for the SFM (1 34). As a rule of thumb, the vertical position of a tip scanned over a row of tightly packed spheres of diameter D is modulated by h (110): h

�

D2j (SR)

=

Dj (SRv)

for D

=

(lj v) «R

and

R»

s.

2.

Provided that the mechanical and electronic stability of the microscope allows detection of e.g. O.O l -nm height differences, we estimate from Equa tion 2 that the tip radius R needs to be smaller than 3 nm to resolve spheres of O.5-nm diameter. Although imaging with local probes is not a linear, spatially invariant process, linear system theory has been applied for weak corrugations ( 1 1 5, 1 1 6) . Equation 2 illustrates qualitatively that periodic

b

a

L

Figure 2 Principles of sensor microscopy. (a) Tip radius R, gap size s, and the profile f(d) describing the interaction between local probe and sample determine the lateral resolution L. (b) A scanning probe microscope (SPM) consists of a sensor (5), a scanner (x,y, z), a servo system, and a storage device. (c Several sensors can be used: (1) a mechanically sharpened or etched metal tip with or without insulation for tunneling; (2) a cantilever with or without a pyramid-shaped tip for force measurements; (3) a nm-sized thermojunction; and (4) a small pipette for measuring ion currents.

)

83

SPM IN BIOLOGY Tunnel

gap


a

II mr R ITLL

Tunnel current

pA

IT

40

Force

nN

F

20

C

�_attractive 30

0

20

-20

10

-40

-

repulsive

U

0.2

0.3

nm

2

3

nm

Figure 3 Interaction profiles for tunneling and atomic forces. (a) Electrons may tunnel from occupied states below the Fermi level EF I of metal M 1 into empty states above the Fermi level EF2 of metal M2 if the corresponding wavefunctions overlap. The bias voltage U determines which occupied states in Ml participate in the tunneling, hence explaining discontinuities in the IT-U plot. (b) The exponential dependence of tunneling current IT versus distance reflects the spatial extension of the wa vefunctions. The current and distance values given in the IT-s plot represent typical operation conditions for STM. (c) Approaching a clean surface with a sensitive cantilever reveals attraction due to dispersion forces at distances of several tens of nm, while closed-shell interactions produce a strong repulsion at distances below 0.2 nm. The values given in the F-s plot are typical for SFM .

corrugations are attenuated with l /v, where v represents the spatial fre quency (1 1 0). This transfer characteristic is similar to that of the dark field mode in scanning transmission electron microscopy (STEM) but different from the coherent axial bright-field mode in conventional trans mission electron microscopy (TEM), in which high frequencies are enhanced (48). Servo System, Scan Devices, and Scan Modes

While the probe scans a sample surface in x and y, the servo system moves it in the z direction to keep the sensor signal constant (constant signal mode). In this mode, the servo signal is written to the storage device (Figure 2b). Formerly used tripod scanners (2 1 , 25) are now replaced by piezo-tubes with an outer electrode that is segmented into four quadrants to allow for bending (27). The servo signal for contracting or expanding the tube along its axis is either applied to the inner electrode or added to the outer electrode signals keeping the inner electrode grounded. Static calibration of scanners in all directions with subnanometer precision is possible with heterodyne interferometry (1 1 0, 1 1 1 ), while a dynamic cali bration is done by homodyne interferometry (1 1 0). Tube scanners have characteristic deflection as well as contraction resonance frequencies in the


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range of 1 to 1 0 kHz. The contraction resonance frequency limits the scan velocity on strongly corrugated surfaces because a direct mechanical contact between sample and tip must be avoided by the servo system, ensuring stable operation within a frequency range below contraction resonances only. Higher scan speeds are possible on flat surfaces. In this case, the servo system is used to approach the tip, but is disabled for scanning so that the tip moves at constant height over the sample while the sensor signal is written to the storage device [constant height mode (24)]. M icrofabricated scanners have higher resonance frequencies (3) but, as yet,have not found widespread application . Probes

Different probes have been described (for reviews see 1 25, 1 26) (Figures 2c and 4). Cutting a thin Pt-Ir wire at an oblique angle suffices for tunneling on atomically flat surfaces at ambient pressure. Mechanically sharpened tips frequen Lly exhibit multiple protrusions (1 1 4) (Figure 4a),leading to a characteristic duplication of sample features (47, 57) (Figures 4e and f). For bulky structures protruding some tens of nanometers from the supporting surface, tips of well-defined geometry are required such as those commonly obtained by electrochemical etching (57, 1 1 2) (Figure 4b). Controlled submicron protrusions can be deposited on electropolished tips by exposure to the electron beam of a scanning electron microscope (SEM) (2). Frequently, tips are processed for optimum performa n ce by pulses of typically 5-Y height and 1 JiS width during tunneling or even by collision of the tip with the substrate. While the tip structure is difficult to control under ultra-high vacuum (UHY) conditions in which field ion microscopy allows the direct assessment of the structure (for a discussion see 33), the situation is worse for STM of organic materials: continuous tip alterations during scans of biological samples at ambient pressure are likely to be the rule rather than the exception. Tips suitable for STM in conducting solutions are glass- or epoxy-coated Pt-Ir wires (Figure 2c; available from Longreach Scientific Resources, Orr's Island, Maine, USA) or are made from etched metal wires by coating with Apiezon wax (90) (available from Angstrom Technology, Mesa, AZ, USA). Technically more involved is the manufacturing of force-sensing cantilevers that should have a spring constant C::::; 1 Njm for high sensitivity, as well as a small mass rno for a high resonance frequency w ::::; JC/rno. In addition,cantilevers (Figure 2c) should carry a fine tip at their end that ultimately touches the sample surface or measures magnetic or electrostatic forces. M icrofabricated can tilevers (5, 1 9) as displayed in Figure 4d are commercially available (Park Scientific Instruments, Mountain View, CA, USA). M inute deflections of the cantilever are measured by the tunneling effect (4, 1 9, 20 65, 85,86), ,


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Figure 4 Sensors for STM and SFM. (a) A mechanically sharpened tungsten wir� r�veals several sharp protrusions after tunneling. The protrusion marked by an arrow is displayed at higher magnification in the insets and has a radius of 35 nm. (b) Rotating a gold wire during electrochemical etching in HCI yields a single tip with a radius below 50 nm. (c) Very sharp but frequently jagged tips are obtained by electrochemical etching and mechanical disruption. (d) Microfabricated cantilevers carry a pyramid-shaped tip at their end.

(e,J)

Multiple tips yield images with a characteristic duplication of the sample structure at a distance that corresponds to the separation of the tips; Pt-C layer on mica (e), and TMV rods (f). Scale bars represent 3 11m in a; 500 nm and 100 nm, respectively, in the insets of a; 4 11m in d; 40 nm in e; and 1 00 nm in! Reprinted from References 114 (a), 1 1 2 (b), 57 (c, e), 92 (d), and 47 (f), with permission.


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a capacitive displacement detector (51), resonance heterodyne inter ferometry (83, 125, 126), the optical fiber interferometer technique (98), or the optical levei technique (6, 123). Patch-clamp micropipettes can be pulled to exhibit cylindrical openings of some 50 nm (Figure 2c), but microfabricated nanopipettes with a diameter of less than 10 nm appear feasible too, promising scanning ion-conductance microscopy (62) capable of resolving single ion channels. Incidently, such pipettes have also been applied in scanning near field optical microscopy (77, 95). Last but not least, microfabrication was required to build a 50-nm thermocouple junc tion (125,126,129) (Figure 2c) that was used to record maps of the thermal properties of solid surfaces at a resolution of approximately 10 nm. Scanning Tunneling Microscopy

For tunneling on conductors or semiconductors under ideal conditions, the tunneling current has the form (see 11) (Figure 3b): IJ...s)

=

Io (U)exp ( -A j¢s),

3.

where Io(U) is proportional to the density of states contributing to the tunneling current filament at bias voltage U (116), A is 10.25 nm I eV- 1/2, and ¢ is the local barrier height. Therefore, STM allows three parameters to be assessed. First, the tunnel effect is used to guide the probe at a fixed gap distance s over the sample surface, thereby recording its topography. For a barrier height of typically 4 eV, IJ...s ) changes by one order of magnitude for ,1s 0.1 nm (Figure 3b), a sensitivity permitting one to resolve sub- Angstrom height differences. However, a true topography is only obtained if neither Io(U) nor ¢ changes as the tip is scanned. Second, ¢(x,y) can be assessed by oscillating the tip in z direction with a frequency w beyond the cutoff of the servo system, thus modulating s with ,1z cos (wt) (see 97). In this mode, the tip still tracks the surface contour, but in addition, a modulation flh is then detected using a lock-in amplifier. This provides an estimate of ¢(x,y), as derived from Equation 3 under the assumption that ,1s ,1z: -

=

=

4. A quantitative interpretation of barrier height profiles is difficult because the tip geometry (i.e. changes of the tunneling filament shape resulting from the tip's displacement along z) and forces between tip and sample that induce deformations (i.e. ,1s =1= ,1z) cannot easily be assessed. In fact, repulsive interaction forces tend to reduce the actual gap modulation (29, 30), yielding an apparently low barrier height, whereas attractive forces increase ,1s, thereby leading to a larger ,1IT. The latter explains a pheno-

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menon observed on graphite termed giant corrugations ( 1 07). Therefore, I refer to the dIT/dz-profile as z-modulation response in the following. Third, the tip can be fixed at height z and position x, y and the voltage ramped to determine Io(U), thus yielding information on the density of states versus their energy (60,6 1 ) (Figure 3a).


Scanning Force Microscopy

Forces can be attractive or repulsive between 2 nm and several tens of nm and are strongly repulsive for distances below some 0.2 nm because of the closed-shell repulsion (Figure 3c) . This is approximated by the Lennard Jones potential, which holds for point-like objects (67):

V(d)

=

C1 - C2

dl2

5.

d6'

Model calculations confirmed the experimental finding that atomic resolu tion is possible with SFM (4, 1 9, 85, 86) only in a small force window around 10- 8 N and depends critically on the presence of a single atom at the tip of the probe (1 34). Stronger forces [ 5 x 10- 8 N ( 1)] would destroy both the sample and the sensor, whereas single atoms do not produce detectable deflections of the cantilever if the forces are too weak ( 1 34). Dispersion forces acting over tens of nm represent in most cases the dominant term of van der Waals interaction and exhibit a potential pro portional to r-6 (see Equation 5). Attractive forces of approximately 5 x 1 0- 9 N were observed when advancing a cantilever to a mica surface submerged in water ( 1 23). In contrast, the adhesion between two metals separated by a vacuum gap extends over a much shorter distance (�0.4 nm) but is of similar magnitude (39). However, thin water films building a liquid bridge between sample and tip induce attractive capillary forces (69, 70) of 4 x 1 0-7 N on mica in air ( 1 23). Other researchers (83, 86) have reported similar attractive forces, indicating a potential problem of SPM at ambient pressure. �

SPM for Biological Applications

Whereas in material sciences SPM experiments can be done in DHV, yielding reproducible, quantitative results that are interpretable within the current theoretical framework, experiments on biological structures lack this advantage. In many cases, the experimenter is even not sure that what he observes results from what he thinks is under the tip. Therefore,SPMs must meet several prerequisites to be suitable for structural biology. First, they should be rugged and easy to operate. Tip changes must be straight forward because tip crashes are frequent. Second, large-area scans should


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be available because well-preserved areas tend to be sparse on biological samples. Third, hybrid instruments such as SPM-light microscopes (57, III) or SPM -SEM systems (50, 113) appear to be of particular interest, as samples are searched much more efficiently with conventional microscopes, allowing parallel imaging or TV scan rates and because the tip can be positioned with submicron precision on sample areas known to be of good quality. Several technical approaches have been presented in the past to meet such requirements (18, 43, 71, 87). Last but not least, special attention must be given to the calibration of the SPM concerning x, y and z deflection and should be repeated frequently to prevent misinterpretation of images (110). SAMPLE PREPARATION FOR SPM Protein Structure

The ultimate goal of biological SPM is the direct visualization of structural changes related to changes in the biological activity. Therefore, pre paration procedures should induce minimal alterations of the biological structure while allowing for reproducible SPM. One should keep in mind that the three-dimensional (3D) structure of proteins representing proto type biomacromolecules depends on the aqueous environment for which they have been optimized by evolution (see 34). The folding of a protein is determined by the sequence and nature of amino acid residues and their environment. Polar or charged residues tend to shield hydrophobic residues from the polar aqueous medium. Disulfide and hydrogen bonds stabilize the fold, the latter involving water molecules in many cases. In addition, watcr molecules are adsorbed at protein surfaces, clustering over charged residues, but also forming crystalline cages over surface areas of reduced hydrophilicity. This hydration layer contributes towards the specific configuration and stability of supramolecular assemblies. Preparation Artifacts During Immobilization and Dehydration

As outlined in a classical review by Kellenberger & Kistler (72), several phenomena need to be considered: (a) specimen-support interactions, (b) surface-tension collapse during drying in air, and (c) thermal collapse of the destabilized supramolecular structure after freeze-drying. Because the immobilization of the sample on a suitable substrate is an indispensable prerequisite for SPM, the first phenomenon is relevant for scanning samples in an aqueous environment. Reproducible and controlled im mobilization is feasible on chemically activated surfaces that allow covalent crosslinking of soluble proteins, supramolecular assemblies, or mem-


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brancs. Such currently devcloped techniques promise efficient immo bilization without significant loss of enzymatic activity. They involve, for example, modification of glass surfaces with carbene-generating hetero bifunctional reagents that are photoactivated by light in the 320-nm to SSO-nm range after adsorption of the specimen (3S, 1 00). Alternatively, monolayer techniques are used to prepare surfaces with known properties that appear to be suitable for the controlled adsorption of biological structures (41, 1 2 1 , 1 22). For ambient pressure SPM, the surface tension collapse is likely to induce the most severe of all possible artifacts. In fact, surface tensions at air-water interfaces are sufficiently large to break covalent bonds, as demonstrated by mass measurements of unfixed and glutaraldehyde-fixed protein oligomers (44). In general, air-dried supramolecular structures are spread-flattened, distorted, and often disrupted. Only few particularly robust or thin structures such as DNA or the bacterial hexagonally packed intermediate (HPI) layer ( 1 3, 1 28) preserve their native 3D structure remarkably well during dehydration in air. Electron microscopists found that embedding biomacromolecules in a heavy-metal salt solution pro duces a solid cast that surrounds the proteinous s tructure, thereby pre venting the surface tension collapse. In addition, this heavy-metal cast also provides the contrast required to outline the envelope of proteins. Unfortunately, commonly used negative stains are bad conductors, other wise they could well serve for sample preparation for STM at ambient pressure. The surface tension can be reduced by small amounts of sur factants; however, such treatments may impair the integrity of mem braneous structures or denature proteins. Surface-tension artifacts can be circumvented by freeze-drying in vacuum, a technique often applied in electron microscopy. Freeze-dried samples are unstable, as demonstrated by their sensitivity to the loss of the hydration layer (55) as well as their sensitivity towards electron bombardment (46). In addition, freeze-dried samples cannot be transferred to ambient pressure, as rehydration immediately leads to structural rearrangements. Thus, although freeze-drying is clearly the best procedure for dehydrating biomacromolecules, freeze-dricd samples have to be sta bilized for observation at ambient pressure. Optimum stability and good conductance have been obtained by coating samples with a I-nm Pt-Ir-C layer (9, 1 1 2), whereas carbon layers alone do not exhibit sufficient stability and conductance unless they have a thickness of 3 nm ( 1 1 2). Specimen Supports

Highly oriented pyrolytic graphite (HOPG) has been the support of choice for many STM experiments, as it provides atomically flat areas, good


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Figure 5 Highly oriented pyrolytic graphite (HOPO). STM topographs of graphite exhibit a trigonal lattice with unit cell dimensions a b 0.246 nm. (a) Lattice irregularities sometimes interrupt atomically flat areas. (b) At the edges of flat plateaus, rich periodic structures are often found. Topograph a was recorded at U = -0. 1 5 V, IT 8 nA, and b at -0.1 V, IT 3 nA. Scale bars represent 2 nm in a and 10 nm in b. The scale from black U to white is 2.5 nm in b. The maxima along the line marked in b are spaced hy 1.6 nm. Reprinted from References 57 (a) and 99 (b), with permission. =

=

�

=

=

mechanical stability and conductance, and a well-characterized top ography with a hexagonal modulation ofO.246-nm periodicity (1 07) (Fig ure 5a). However, graphite often exhibits structures at step edges that are unrelated to the 0.246-nm repeat (99) (Figure 5b) and may lead to misinterpretation of the topographs. In addition, HOPG possesses rather hydrophobic surfaces to which biological samples tend to adsorb poorly. Although less flat than HOPG, Pt-C-coated mica or glass surfaces (9, 57, 58, 1 1 1 , 1 1 2), as well as thin carbon films mounted on Au-coated fen estrated plastic films (1 12) were found to be suitable supports for STM. In addition, the latter allows identical structures to be imaged using STM and transmission electron microscopy. Freshly cleaved mica, either un coated or sometimes coated by Langmuir-Blodgett films with specific properties, is frequently used for SFM (36, 4 1 , 54, 1 2 1 , 1 22) (see below). Finally, Au surfaces have been used for imaging DNA in the STM (78, 79). APPLICATIONS Scanning Tunneling Microscopy

As discussed above, the tunnel effect transports electrons from the metal tip to the sample or vice versa. This process requires that the sample is


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conducting. Conductivity of proteins has been sparsely studied, although electron transport is of central importance in energetics of biological systems (see 94). The conductivity of various amphiphilic model com pounds has been measured in monolayer experiments and indicates that tunneling is the primary charge transport mechanism along linear alkanes, while unsaturated bonds constitute molecular wires (see 74). Recent model calculations for proteins involved in electron transport whose structures are known at atomic resolution consider tunnel probabilities along the peptide backbone, through disulfide and hydrogen bridges, and through van der Waals contacts (16, 17, 68). Electron paths have thus been identi fied that indicate that hydrogen bonds contribute significantly towards protein conductivity, providing a short-cut of the protein fold, thus increas ing conductivity by orders of magnitude (17). These model calculations suggest that the conductivity of proteins largely depends on their fold and the integrity of hydrogen bridges. In addition, proton transport within the hydration layer may contribute to protein conductivity as well. These suggestions may explain the observation that protein hydration is a pre requisite for conductance (7, 59, 112). STM has been possible on conducting organic crystals (101) and on very thin organic layers as illustrated with topographs of phtalocyanin (80, 89) (Figure 6), liquid crystals (103, 104) (Figure 7), and poly(y-benzyl-L glutamate) (PBLG) (84) (Figure 8). Several alkylcyanobiphenyls (mCB)

a

Figure 6 Phtalocyanin. (a) The fiat, hydrophobic ring-shaped structure exhibiting four fold symmetry makes it an ideal test sample for STM. (b) The topograph of a single Cu phtalocyanin molecule adsorbed to a GaAs ( 1 00) surface displays a strong depression at the position of the Cu atom and a pronounced four-fold symmetry. Recording conditions: U 2.5 V. TT 0.3 nk (c) A high-resolution electron micrograph of a chlorinated Cu phtalocyanin crystal reveals rich structural details. The frame in b has a size of 3 nm by 4.5 nm, the contour lines arc separated by 0.025 nm, and the scale bar in c represents I nm. Reprinted from References 89 (b) and 1 1 9 (c), with permission. =

=


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Figure 7 4' -n-decyl-4-cyanobiphenyl ( IOCB). Atomic resolution is documented by this STM topograph of the smectic liquid crystal IOCB deposited on HOPG. Every second C atom of the alkane chain is distinct (arrows). Recording conditions: U 0.8 V, I, 0. 1 nA. The frame has a size of 1 1 .2 nm by 1 1 .2 nm and the scale from black to white is 0.2 nm. Reprinted from Reference 1 03, with permission. =

-

=

were imaged at atomic resolution and the STM contrast was found to match the densities of the highest occupied and lowest unoccupied molec ular orbitals, allowing the differentiation of alkyl and cyanobiphenyl moieties (103). This quantitative interpretation differs from the hypothesis that the topography of mCB contoured by STM results from changes of the substrate work function ( 1 09). STM topographs of Langmuir-Blodgett Cd-arachidate bilayers have been recorded in constant height mode and document the possibility of STM on insulating layers (49, 1 02), but molec ular resolution has been obtained exclusively by using HOPG as substrate. Topographs of thick paraffin crystals (88) illustrate the surprising feasi bility of STM on organic materials that are thought to be perfect insulators and document the fact that dynamic processes such as melting can be followed by STM. Although various electron transfer mechanisms were considered in this work, none could account for the currents through multilayered alkane films required to record topographs. The first biomolecule studied by STM was double-stranded DNA. A range of topographs have been presented ( l 0, 1 4, 1 5, 22, 3 1 , 37, 38, 76, 78, 79, 1 1 8), but the contrasts of these topographs and z-modulation responses (if available) are contradictory (see 1 1 2). Several DNA images presented in Figure 9 allow for a comparison between electron micrographs


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Figure 8 Poly(y-benzyl-L-glutamate) (PBLG). This compound represents a model for poly peptides. PBLG apparently assumes different conformations when adsorbed to HOPG from different solvents. (a) Helical bundles reveal a repeat of 1.6 nm when PBLG is deposited from chloroform, while (b) a side-by-side association of helices is observed when PBLG is deposited from dimethylformamide. The arrow in a marks a discontinuity of the helix (see 84). Recording conditions: (a) U 50 m Y, IT 0.1 nA; (b) U 0.5 Y, IT 0.1 nA. The noise in b has been eliminated by Fourier filtering. Frame sizes are (a) 30 nm by 24 nm, (b) 31 nm by 25 nm. Reprinted from Reference 84, with permission. =

=

=

=

and STM topographs of various DNA preparations. Linear arrays of bumps regularly spaced by 2-5 nm have frequently been observed as illustrated in Figures 9c,e,j. The structure in Figure 9dhas been interpreted as a hairpin fold of a double-stranded DNA molecule (14). Unstained double-stranded DNA recorded at low dose in the STEM exhibits intensity maxima spaced by approximately 3 nm as well, but electron statistics and irregularities of the carbon support film reduce the visibility of these maxima (Figure 9a). The object, however, is identified through the direct measurement of mass-per-length from the number of scattered electrons (45), as well as by the restriction enzyme discernible at the correct distance from the end of the DNA molecule. Attempts have been made to sequence DNA by heavy-metal labeling (28). Distinct single Os atoms spaced by I nm indicatcd that on averagc cvery third base of the poly(U) polymer was labeled and allowed atom movement to be quantitatively assessed (Figure 9b). The STM, on the other hand, has provided a spectacular view of the Z-DNA helix ( 1 0) or that of poly(dA) (38) (Figure 9f). But when we compare the graphite in Figure 5b with the PBLG in Figure 8a or some of the published DNA images, we may appreciate the criticism that HOPG artifacts appear to create structures that are hard to discriminate from those of the biomacromolecule (99). Therefore, it would be desirable to have topographs of DNA adsorbed to different supports to see whether the spectacular information content of published DNA topographs can be

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reproduced. Alternatively, atomic-scale resolution that allows unam biguous identification of the DNA appears feasible, as illustrated by a single STM topograph of a short mouse B-cell DNA fragment recorded recently under URV-conditions (37). As outlined above, thicker samples are likely to suffer from surface tension artifacts, hence freeze-drying followed by metal-carbon coating has provided the best STM topographs (8, 9, 57, 58, 112). The Pt-Ir-


SPM IN BIOLOGY

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C coat recently used (9, 1 1 2) stabilizes the 3D structure of freeze-dried specimens and warrants good conductivity, two prerequisites for repro ducible results. As shown in Figure 1 0, individual recA molecules [Mr 37 kilodaltons (kd)] are sometimes visible at the surface of the helical recA DNA complex. Interestingly, a characteristic loop of uncovered double stranded DNA is clearly visible at the end of the fiber. Single recA mol ecules cannot be discerned on the negatively stained recA-DNA filament recorded in the electron microscope (Figure lO, inset), but a full 3D reconstruction of the filament was accomplished from such projections (40), which then facilitated interpretation of the STM topograph (see 1 1 8). Stacked double-disks of the RNA-depleted tobacco mosaic virus (TMV) exhibit a deep groove every 4.6 nm (see 9 1 ) (Figure I1 h) that is distinct on the STM topograph ofPt-C--coated freeze-dried TMV (1 1 2) (Figure I l a). Close inspection reveals the fine groove separating the two disks, indicating a resolution of 2.3 nm. The development of supports suitable for both STM and transmission electron microscopy (TEM) has enabled imaging of identical areas in both microscopies (1 1 2). Although this imaging will likely be feasible on metal-coated specimens only, it nevertheless allows the performance ofSTM to be evaluated quantitatively (1 1 0), as illustrated by images of phage T4 polyheads recorded by STM (Figure 1 2a) and TEM (Figure 1 2c) . Polyheads are aberrant tubular structures assembled from the major capsid protein gp23 of phage T4 mutants unable to produce mature heads (73). These tubular arrays collapse during adsorption to a hydrophilic support film, thus exposing a planar hexagonal lattice with unit cell dimensions a = b = 13 nm. The analysis in Figure 12 shows that the STM topograph exhibits disorder of the polyhead lattice (Figure 1 2d), although the electron micrograph recorded afterwards demonstrates that

Figure 9 DNA. (a) An unstained phage ).-DNA-restriction enzyme complex adsorbed to a thin carbon film and recorded in the STEM at 800 e- Inm2 exhibits a typical curved con formation. Periodic intensity maxima are marked by arrows. The restriction en zyme is discernible near the top of the frame. Bar is 10 nm. (b) A STEM high-dose dark-field image shows single-stranded poly(U) molecules specifically labeled with Os-bipyridine as an irregular, curved filament (left side) revealing single Os atoms spaced by I nm distinctly after high pass filtering (right side). Bar is 2 nm. (c-f) Variations of double-stranded DNA and single stranded poly(dA) conformations, all adsorbed to HOPG for observation by STM. (c) pBR322 DNA recorded at U - 0.18 V, II 0.5 nA. Bar is 20 nm (in x and y direction) and I nm in z direction. (d) A hairpin loop of a calf thymus DNA fragmen t recorded at U - 9 7 mY, IT � 3.3 nA. Bar is to nm, and grey-tone range represents a z-range of 13.2 nm. (e) Calf thymus DNA recorded at U 81 mV shows regular bumps at a distance of 2.9 nm (arrows) . Bar is 5 nm. U) Poly(dA) preparation recorded at U 0.18 V, IT = �

�

�

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=

0.16 nA. Bar is 2 nm, and grey-tone range represents a z-range of 5.4 nm. Reprinted from References 45 (a), 28

(b), 15 (c), 14 (d),

76 (e) , and 38 (f), with permission.


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F(qure 10 recA-DNA complex. The STM (opograph of freeze-dried Pt-Ir-C-coated recA DNA complexes reveals the helical surface and in some instances individual recA monomers. A DNA loop that has not been covered by recA molecules starts at the end of the helical complex (arrow). A negatively stained recA-DNA helix recorded by a STEM is displayed in the inset. Bars represent 20 nm. The topograph is reprinted from Reference 9, with permission.

Figure II Tobacco mosaic virus (TMV). (a) A Pt-C-coated freeze-dried RNA-depleted TMV rod reveals pronounced grooves separated by 4.6 nm at its cylindrical surface, which separates the paired disks. (b) The same sample recorded in the STEM after negative staining shows deep as well as fine grooves, the latter outlining individual disks within the pairs. Fine grooves are also visible on the STM topograph, indicating a resolution of 2.3 nm. The surface relief in a has a size of 96 urn by 150 nm. Reprinted from Reference 112 (a), with permission.


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i5 Figure 12

STM and TEM on identical areas. The STM topograph (a) and the TEM micrograph (c) ora Pt-Ir-C-coated phage T4 polyhead reveal

a surprising similarity that is quantitated by their cross-correlation function (b). Panel

a

is a subframe of the polyhead in

e

that exhibits some

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Biological applications of electron probe microanalysis in France.

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