Visualizing molecular charging High-resolution atomic force microscopy (AFM) has been used to control and image the charge state of organic molecules adsorbed on multilayer sodium chloride films. Fatayer et al. biased an AFM probe tip with a voltage to charge and discharge molecules such as azobenzene and porphine from cations to anions. Subsequent imaging with carbon monoxide–functionalized tips revealed changes in the conformation, bond order, and aromaticity of the organic molecules resulting from charge-state changes. Science, this issue p. 142

Abstract The charge state of a molecule governs its physicochemical properties, such as conformation, reactivity, and aromaticity, with implications for on-surface synthesis, catalysis, photoconversion, and applications in molecular electronics. On insulating, multilayer sodium chloride (NaCl) films, we controlled the charge state of organic molecules and resolved their structures in neutral, cationic, anionic, and dianionic states by atomic force microscopy, obtaining atomic resolution and bond-order discrimination using carbon monoxide (CO)–functionalized tips. We detected changes in conformation, adsorption geometry, and bond-order relations for azobenzene, tetracyanoquinodimethane, and pentacene in multiple charge states. Moreover, for porphine, we investigate the charge state–dependent change of aromaticity and conjugation pathway in the macrocycle. This work opens the way to studying chemical-structural changes of individual molecules for a wide range of charge states.

The charge-induced changes of a molecule have fundamental implications for chemical reactions, catalysis, electrochemistry, photoconversion, and charge transport. Charged molecules can be studied in various environments (1), such as gas phase, solution, and solid. Experimentally, structural information is mostly obtained from x-ray diffraction for molecular solids (2) and from vibrational and optical spectroscopy for molecules in solution and gas phase (3). Typically, counterions are used to stabilize charged molecules and avoid fragmentation or charge leakage in solids and solutions (4). Hence, the crystallization and the counterions may affect the geometry of the molecules.

The aforementioned experimental techniques predominantly probe a large number of molecules. The ability to detect single-molecule changes upon charging with atomic resolution would allow previously unknown phenomena to be observed (5) and the influence of the environment to be quantified. Moreover, it would also affect applications such as devices based on single-electron transfer as well as thin-film devices and provide fundamental insights into redox reactions and charge-carrier injection.

Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) imaging of adsorbed molecules in different charge states have been shown in specific cases on ultrathin insulating films, such as bilayer NaCl, in which tunneling to the substrate and STM operation is possible. For some molecules, two charge states can be stabilized under special conditions of energy-level alignment and relaxation energy, that is, when reorganization in the film/molecule leads to level shifting across the Fermi level. In such special cases, molecular conformational switching (6), tunnel barrier changes (7), and formation of a metal-molecule complex (8) have been demonstrated.

On thicker insulating films, electrons cannot be exchanged with the substrate, and thus multiple charge states can be stabilized in general (9) and reorganization energies determined (10). In this study, we complement single-electron sensitivity (11–13) and charge-state control (9) with atomic resolution of AFM by using CO tips (14). We resolve molecules in multiple charge states (oxidation states) with atomic resolution, including bond-order analysis. The molecular charge states are controlled by deliberately exchanging electrons between tip and molecule on an insulating substrate (9), accessing multiple charge states, including doubly charged ions. The changes in the molecular geometry of charged species can often be rationalized by using classical organic chemistry approaches such as the evaluation of Lewis resonance structures or Clar’s sextet rule.

In this study, we investigated four model compounds with different properties and applications related to their charge-state transitions. We resolved minute changes in adsorption geometry, bond-order relations, and aromaticity. All of the experiments were performed on an NaCl film thicker than 20 monolayers (MLs) on Cu(111) by using a qPlus-based (15) AFM under ultrahigh vacuum at 5 K (materials and methods). The CO-functionalized tip approached the multilayer NaCl area under frequency-shift (Δf) feedback, in which constant Δf AFM imaging was used to locate molecules. To identify and manipulate the charge state of molecules, we used Kelvin probe force spectroscopy, that is, Δf as a function of sample voltage V. Transitions in the molecular charge state were observed as steps between different Δf(V) parabolas (11–13), in which each step upon ramping V in the positive (negative) direction corresponded to a decrease (increase) in charge state by attaching one electron (hole).

We assign the charge states by comparison to the ion resonances and pristine charge states on NaCl(2ML)/Cu(111) by using the scattering of surface-state electrons and/or interface-state localization as indications (16). Moreover, the separation (gap) between transition voltages on the insulating film provided another indicator (fig. S8). To avoid exchange of electrons with the tip during imaging, we obtained AFM images at voltages between different charge transitions, in different charge states. The AFM images were taken in constant-height mode, at a tip height z near the minimum of Δf(z) above the molecule, which roughly corresponded to a distance of 3.9 Å between the molecule imaged and the oxygen atom of the tip (17). The atomic contrast of the AFM images confirmed that the tip remained CO functionalized, even when biases of several volts were applied.

Azobenzene (A) (Fig. 1A) is an archetypical molecular electronics building block (18) and photomechanical actuator (19) that can be switched between cis and trans isomers by light (20, 21), tunneling electrons (22), and electric fields (23). The AFM image of neutral A0 (Fig. 1B) showed that it was adsorbed with both phenyl rings slightly tilted out of plane with respect to the NaCl surface. The phenyl rings were approximately parallel (figs. S2 to S4), indicated by the same sides of the phenyl rings appearing bright and with comparable Δf contrast in the AFM image.

Fig. 1 Measurements and calculations on azobenzene. (A) ∆f(V) spectrum recorded on top of an azobenzene molecule. V was ramped from 1 to 3 V. The inset shows the chemical structure of azobenzene. (B) Constant-height AFM image of A0 at V = 0.5 V. (C) Constant-height AFM image of A−1 at V = 2.5 V, tip-sample distance reduced by 0.3 Å with respect to (B). (D and E) Simulated AFM images of on-surface A0 and A−1, respectively. All scale bars correspond to 5 Å. (F and H) Top view of the atomic models of A0 and A−1, respectively. (G and I) Chemical structures of A0 and A−1, respectively, with wedged bonds representing out-of-plane conformations.

An electron was attached to A0 when V was ramped above 2 V (Fig. 1A), creating the anion A−1. In A−1 (Fig. 1C), the phenyl rings were also tilted out of plane but in opposite directions, indicated by opposite sides of the two phenyl rings appearing bright in the AFM image (fig. S5). Hence, the conformation changed when A0 was reduced to A−1. A sequence of AFM images of A while alternating its charge state demonstrated the reversibility and reproducibility of this charge-conformation switching (fig. S6).

The AFM measurements were in excellent agreement with AFM simulations (24) of the adsorbed molecule (Fig. 1, D and E) performed in the respective geometries obtained by density functional theory (DFT) calculations (see supplemental material for details and tables S1 and S2) of A0 (Fig. 1F) and A−1 (Fig. 1H). For both oxidation states, A was in the trans conformation, but there were small differences in the molecular geometry. A0 was planar, and the molecular plane was tilted by 17° with respect to the surface plane. Conversely, A−1 was nonplanar with the phenyl rings tilted in opposite directions by ~4° with respect to the surface plane. The switch from a planar to a nonplanar conformation can be rationalized by the reduction of the azo group (N=N) when switching from A0 to A−1, which alters the π conjugated system and induces the distortion from planarity. The example of azobenzene showed that charge-induced changes in the adsorption geometry and molecular conformation—that is, tilts of a few degrees of molecular moieties—could be detected.

Bond-order analysis can be a powerful tool for resolving structural and electronic properties of molecules in different charge states. In general, bond-order differences can be qualitatively resolved by AFM as different brightness (∆f values) and as different apparent bond length (17, 25). Brighter ∆f contrast is caused by increased Pauli repulsion caused by increased electron density and thus indicates smaller bond length. At small tip-sample distances, the tilting of the CO at the tip strongly affects the images and leads to a magnifying effect, increasing bond-length differences by about one order of magnitude (17). Moreover, background forces related to the local potential landscape contribute to the CO tilting and thus also affect the apparent bond length (17, 24). Thus, bonds should be compared only if they are in similar local environments.

Before applying this method to resolve bond-order relations of molecules with oxidation state–dependent functions, we characterized the well-studied model system of pentacene (P). The ∆f(V) spectrum of P (fig. S8) revealed four different oxidation states, cation (P+1), neutral (P0), anion (P−1), and dianion (P−2). The differences observed in the AFM images (Fig. 2, A to D, and fig. S9) revealed an apparent contraction (elongation) of the molecule along its short (long) axis with increased negative charge, which agrees with the calculations (fig. S10). However, when comparing apparent bond lengths obtained at different voltages, the electric field changes with different V and affects the spring constant and tilt of the CO molecule at the tip apex (24). In fact, we observed an overall compression of the molecular image of P0 with increased V (fig. S11 and supplementary text SM1). However, differences between individual bonds in one image, measured at identical voltages, could be compared without such systematic error. For bond-order analysis, we compared only the contrast within individual AFM images and bonds with a similar local environment.

Fig. 2 Measurements and calculations on pentacene. Constant-height AFM images of the (A) cationic (P+1), (B) neutral (P0), (C) anionic (P−1), and (D) dianionic (P−2) molecule. Scale bars represent 5 Å. (B) and (C) are imaged at a tip-sample distance that was 0.3 Å smaller than in (A) and (D). V is (A) −3.3 V, (B) 0.5 V, (C) 2.5 V, and (D) 3.6 V. (E) DFT-calculated average C-C bond length of each hydrocarbon ring for different charge states. (F) Possible resonance structures of P0 and P−2.

In the images of P−1 and P−2, we observed a ∆f modulation with the centers of the second and fourth ring appearing darker than the centers of the other rings, indicating that these rings increased in diameter and/or exhibited a reduction in electron density. DFT calculations of the average C-C bond length in individual rings (Fig. 2E) showed that P−1 and P−2 featured second and fourth rings with an increased average bond length compared with the other rings. For the dianion P−2 , the effect could be rationalized by considering the reduction of pentacene to form radical anions in the second and fourth rings (Fig. 2F), leading to a structure with three Clar aromatic sextets distributed in the first, third, and fifth rings. The measurements of pentacene show that small charge-induced effects in the bond-length relations can be resolved by AFM. Importantly for redox reactions, we obtain the locations of sites with increased radical anion character upon charging.

We applied our method to the electron acceptor tetracyanoquinodimethane (T) (Fig. 3A), relevant for doping in organic electronics (26). The ∆f(V) spectrum of T (fig. S14) revealed three different oxidation states, neutral (T0), anion (T−1), and dianion (T−2). Consistent with its large electron affinity, we observed the anion T−1 even at 0 V. The AFM image of T0 (Fig. 3B) showed four attractive lobes, two large ones and two small ones (fig. S15), which we assigned to an upstanding adsorption conformation (27) for T0 (fig. S16 and supplementary text SM2). AFM measurements of T0 at smaller tip-molecule distances than in Fig. 3B resulted in the molecule being picked up by the tip.

Fig. 3 Tetracyanoquinodimethane model and measurements. (A) Chemical structure of T. Constant-height AFM images of the (B) neutral (T0), (C) anionic (T−1), and (D) dianionic (T−2) molecule. Panels (C) and (D) are imaged with a tip-sample distance that is 1.9 Å smaller than in (B). Scale bars represent 5 Å. V is indicated in each image. (E) Major resonance structure proposed for T−2.

In contrast, the AFM image of T−1 (Fig. 3C) resolved the central carbon ring adsorbed parallel to the surface. The adsorption orientation switch between T0 and T−1 was reversible and did not result in lateral movement of the molecule (fig. S17). The contrast on the central ring of T−1 indicates bond-length alternation (BLA). Increased ∆f, indicative of shorter bond length, was observed above the double bonds of the ring as depicted in its neutral chemical structure (Fig. 3A). The BLA in the ring suggests that T−1 does not have a perfect benzenoid character (28). However, in T−2, the ∆f contrast of the central ring became homogeneous (Fig. 3D), indicating no BLA and thus a change to a benzenoid character (Fig. 3E), in agreement with previous calculations (29). In addition, comparing T−2 with T−1, the regions of the carbons connecting the cyano groups showed increased ∆f for the dianion. Possible explanations include higher electron density at these carbons or adsorption-height changes in the termination of the molecule upon charge-state change. DFT calculations (tables S9 and S10) indicated that the cyano groups were more bent for T−1 than for T−2. In the observed adsorption orientation changes upon reduction of T0, the prevalent phenyl character changed from quinoid in T−1 to benzenoid in T−2 and was accompanied by small geometry changes in the dicyano moieties.

We also applied our method to porphyrins, which in multiple oxidation states fulfill essential functions in medicine, biology, chemistry, and physics (30). The aromaticity and conjugation pathway of porphyrins in different oxidation states are debated (31). The parent compound of porphyrins is porphine (F), a fully conjugated substituent-free planar macrocycle (Fig. 4A). According to the [18]annulene model, the neutral molecule has an aromatic conjugation pathway involving 18π electrons (4n + 2), indicated by the red-colored bonds of the resonance structures shown in Fig. 4A, bypassing the NH in the pyrroles and the outer CH=CH groups of the azafulvene rings. Upon double reduction, the macrocyclic conjugation pathway changes to a formally antiaromatic 20π-electron (4n) system, encompassing the whole periphery of porphine (Fig. 4B). The change in the macrocyclic π conjugation can influence the global aromaticity of the molecule, although local heterocyclic π circuits (pyrrole subunits, 6π-electron, 4n + 2) would contribute as well (31).

Fig. 4 Analysis of porphine and its conjugation pathway. Chemical structure of (A) neutral (F0) and (B) dianionic (F−2) porphine. The red path shows the expected annulene-type conjugation pathway for each charge state. Constant-height and corresponding Laplace-filtered AFM images of (C and D) F0, (E and F) F−1, and (G and H) F−2. The constant-height AFM images in (E) and (G) are taken at tip-sample distances larger by 0.5 Å and 0.4 Å, respectively, than the AFM image in (C). All scale bars correspond to 5 Å. V is indicated in each image. (I) Highlighted bonds in F. Measured apparent bond lengths of (J) a and c and (K) l 1 and l 2 of F as a function of charge state.

The ∆f(V) spectrum of F (fig. S21) revealed three different oxidation states, neutral (F0), anionic (F−1), and dianionic (F−2). The AFM images and the respective Laplace-filtered images are shown in Fig. 4, C to H, and in figs. S22 and S23. The location of the hydrogens inside the cavity can be inferred from AFM images at increased tip-sample distance (fig. S24) and is indicated in Fig. 4C. We observed tautomerization switching (32) of F only at V > 3.7 V (fig. S25). In the individual AFM images, we observed apparent bond-length differences in the pyrrole and azafulvene rings as well as in the methine bridges, which connect the five-membered rings. The outer C-C bonds of the pyrrole and the azafulvene ring, labeled a and c (Fig. 4I), respectively, can be compared within an image (SM1) because of the similar environments (Fig. 4J).

For F0, a appeared longer than c in conformity with the resonance structures shown in Fig. 4A, in which c is a double bond and not included in the conjugation pathway. For F−2, a and c appeared with the same apparent length within the measurement accuracy, in agreement with the conjugation pathway highlighted in Fig. 4B including a and c. For F−1, bonds a and c had qualitatively the same relation as in F0—that is, a larger than c—indicating that the conjugation pathway of the neutral molecule was maintained in the anion, although a less stable 19π-electron system was formed.

Another possible comparison is in each bond of the methine bridge, labeled l 1 and l 2 (Fig. 4K). These bonds change appreciably for each charge state, but a simple picture of resonance Kekulé structures cannot rationalize their behavior. For both F0 and F−2, l 1 was shorter than l 2 , with the difference between l 1 and l 2 being larger in F−2 than in F0. The latter indicates increased BLA for F−2 and thus likely a reduced aromaticity with respect to F0. For the anion F−1, l 1 was longer than l 2 ; the asymmetry at the meso-carbon position of the methine bridge was inverted with respect to both F0 and F−2. Additional insight was gained by DFT calculations of F adsorbed on NaCl in different charge states (tables S11 to S13), in qualitative agreement with the experiment (fig. S26). In porphine, we observed substantial changes of the bond-order relations upon charging and used them to track the evolution of the macrocycle’s structure, aromaticity, and conjugation pathway as a function of its oxidation state. With the demonstrated bond-order resolution and charge-state control, our method effectively complements redox potential measurements (10) and mapping of orbital densities (33) for the investigation of charged molecules on surfaces.

Supplementary Materials science.sciencemag.org/content/365/6449/142/suppl/DC1 Materials and Methods Supplementary Text Figs. S1 to S29 Tables S1 to S14 References (34–41)

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Acknowledgments: We acknowledge G. Meyer, R. Allenspach, J. Repp, and S. J. Garden for discussions. Funding: The project was supported by the European Research Council Consolidator grant AMSEL (contract no. 682144). D.P. thanks Agencia Estatal de Investigación (MAT2016-78293-C6-3-R), Xunta de Galicia (Centro singular de investigación de Galicia, accreditation 2016–2019, ED431G/09), and the European Regional Development Fund for financial support. Author contributions: S.F. and L.G. designed the experiments. S.F. carried out the experiments with support from F.A. S.F. and N.M. carried out the DFT calculations. All authors analyzed the data. S.F. and L.G. drafted the manuscript and finalized it with the input from F.A., Y.Z., D.U., D.P., and N.M. Competing interests: The authors declare no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials.