Significance We report a method to image and reveal structural details of proteins on a truly single-molecule level. Low-energy electron holography is used to image individual proteins electrospray deposited on freestanding graphene. In contrast to the current state of the art in structural biology, we do away with the need for averaging over many molecules. This is crucial because proteins are flexible objects that can assume distinct conformations often associated with different functions. Proteins are also the targets of almost all the currently known and available drugs. The design of new and more effective drugs relies on the knowledge of the targeted proteins structure in all its biologically significant conformations at the best possible resolution.

Abstract Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.

Most of the currently available information on structures of macromolecules and proteins has been obtained from either X-ray crystallography experiments or cryo-electron microscopy investigations by means of averaging over many molecules assembled into a crystal or over a large ensemble selected from low signal-to-noise ratio electron micrographs, respectively (1). Despite the impressive coverage of the proteome by the available data, a strong desire for acquiring structural information from just one individual molecule is emerging. The biological relevance of a protein lies in its structural dynamics, which are accompanied by distinct conformations. For a protein to fulfill its vital functions in a living organism, it cannot exist in just one single and fixed structure, but needs to be able to assume different conformations to carry out specific functions. Conceptually, at least two different conformations, just like in a simple switch, are needed. In view of oxygen transport to cells for example, binding oxygen in one specific conformation and releasing it again in a different conformation are needed. To address the “physics of proteins” as described by Hans Frauenfelder in his pioneering review (2), one needs to realize that proteins are complex systems assuming different conformations and exhibiting a rich free-energy landscape. The associated structural details, however, remain undiscovered when averaging is involved. Moreover, a large subset of the entirety of proteins, in particular from the important category of membrane proteins, is extremely difficult, if not impossible, to obtain in a crystalline form. If just one individual protein or protein complex can be analyzed in sufficient detail, those objects will finally become accessible.

For a meaningful contribution to structural biology, a tool for single-molecule imaging must allow for observing an individual protein long enough to acquire a sufficient amount of data to reveal its structure without altering it. The strong inelastic scattering cross-section of high-energy electrons as used in the state-of-the-art aberration-corrected transmission electron microscopes (TEMs) inhibits accumulation of sufficient elastic scattering events to allow high-resolution reconstruction of just one molecule before it is irremediably destroyed (3). The recent invention of direct detection cameras has dramatically pushed forward the effort in single-particle imaging cryo-EM (4). In particular, it allows a reduction of the required number of objects for meaningful and high-resolution reconstructions. This technical innovation radically improves the signal-to-noise ratio for the same electron dose in comparison with a conventional charge-coupled–device camera coupled to a scintillator, a crucial aspect when imaging low atomic number and beam-sensitive material. Several research groups are trying to reduce the radiation damage problem by lowering the electron energy. To our knowledge, 20 keV is the lowest energy used in an aberration-corrected TEM (5). However, the radiation damage to biomolecules by electrons with a kinetic energy in the kiloelectronvolt range will possibly never permit imaging of truly single proteins at atomic resolution (3, 6). Staining proteins with heavy metal atoms is unfortunately not a viable alternative, because it is well known that the chemical processes involved alter the protein structure (7). Moreover, heavy metal atoms are highly mobile under high-energy electron beams, which leads to ambiguous images. A recent approach to structural biology is associated with the X-ray free electron laser (XFEL) projects. With this impressive technological development and novel experimental tool, it is now possible to elucidate the structure of proteins brought in the form of crystals of just nanometer size (8⇓⇓–11). This method even appeared as a way of gaining information at the atomic scale from just a single biomolecule. Meanwhile it has become clear that averaging over a large number of molecules will unfortunately not be avoidable (12). Future XFELs with orders of magnitude enhanced brightness and reduced pulse duration might eventually achieve the goal of single-molecule imaging.

In contrast to the radiation damage problem experienced when using high-energy electrons or X-rays, biomolecules, for instance DNA, can withstand prolonged irradiation by electrons with a kinetic energy in the range of 50–250 eV. Even after hours of illumination and the exposure to a total dose of at least five orders of magnitude larger than the permissible dose in X-ray or high-energy electron imaging, biomolecules remain unperturbed as exemplified in a detailed study concerning DNA molecules (13). This, combined with the fact that the de Broglie wavelengths associated with this energy range are between 0.7 Å and 1.7 Å, makes low-energy electron microscopy techniques, especially holography, auspicious candidates for investigations at the truly single-molecule level. In this lens-less microscopy scheme inspired by Gabor’s original idea of holography (14), the samples are presented to a highly coherent beam of low-energy electrons generated by an atomically sharp field emitter (15⇓–17) tip placed as close as 100 nm in front of the sample. The interference pattern formed by the scattered and unscattered electron waves, the so-called hologram, is recorded at an electron detector several centimeters away (for more details, Low-Energy Electron Holography). Because in a hologram, the scattered and unscattered electrons are contributing to the image formation, acquisition times as short as 100 μs are sufficient for high signal-to-noise ratio records (18). Whereas highly coherent sources for low-energy electrons have been available for more than two decades, holography has long suffered from the lack of a substrate transparent to low-energy electrons but still robust enough that nanometer-sized objects can be deposited onto it. Recently, we have shown that ultraclean freestanding graphene fulfills these two requirements (19⇓–21).

In the following, we show how subnanometer resolution images of individual proteins are obtained by means of low-energy electron holography. Although the damage-free radiation of coherent low-energy electrons and the conceptual simplicity of the experimental scheme for holography are appealing, this tool for single-protein imaging critically relies on the sample preparation method. The proteins in their native state must be brought into an ultrahigh vacuum (UHV) environment and fixed in space for an appropriate period to accumulate sufficient structural information on the one hand, while avoiding the emergence of contaminants on the other hand. Here, native protein ions are transferred from aqueous solutions to the gas phase (22⇓⇓⇓⇓–27) and deposited onto ultraclean freestanding graphene in an UHV environment by means of soft-landing electrospray ion beam deposition (ES-IBD) (28⇓–30). The workflow for imaging a single protein involves several steps, as illustrated in Fig. 1. An ultraclean freestanding graphene sample covering 500 × 500-nm2 apertures milled in a 100-nm-thick SiN membrane is prepared following the recently developed platinum metal catalysis method (31) and is characterized in the low-energy electron holographic microscope (Fig. 1, Left). The sample is subsequently transferred to an ES-IBD system (Fig. 1, Center and Fig. S1) under permanent UHV conditions by means of a UHV suitcase operating in the 10−11-mbar regime (UHV Transfer for more details). Native cytochrome C (CytC), BSA, and hemoglobin (HG) ion beams are generated by electrospray ionization and mass filtering. The charge states z = 5–7 are selected for CytC (22, 26) and the charge states z = 15–18 are selected for BSA (32). In the case of HG, the charge states z = 16 or z = 17 of the intact complex are known to be of native conformation (33) and hence the corresponding m/z region is selected (the corresponding mass spectra are displayed in ES-IBD and Fig. S2). In all three cases, the ions are decelerated to a very low kinetic energy of 2–5 eV per charge, which ensures retention of the native state upon deposition onto ultraclean freestanding graphene (26). Preparative mass spectrometry of proteins (28, 34, 35) followed by electron microscopy has already been reported in the literature, but there the deposition was made on thick carbon films and not performed under UHV conditions (36, 37). Furthermore, the imaging by high-energy electrons in a TEM required exposure to atmospheric conditions and negative staining of the proteins.

Fig. 1. Schematic workflow for imaging a single protein. Left to Right: An ultraclean graphene sample is characterized in a low-energy electron holographic microscope. Shown is deposition of proteins onto freestanding graphene in an m/z filtered ES-IBD system. Imaging of the proteins within the previously characterized region by means of low-energy electron holography. An electron point source (EPS) emits a divergent beam of highly coherent low-energy electrons and holograms of the deposited proteins are recorded on the detector. Throughout the experimental workflow, the sample is kept under strict UHV conditions with the help of an UHV suitcase for the transfer between the two experimental chambers (UHV Transfer for more details).

Fig. S1. Illustration of the working principle of the home-built instrument for soft-landing electrospray ion beam deposition of proteins. The proteins are directly deposited from the liquid phase onto a substrate kept at UHV conditions to avoid contaminations.

Fig. S2. Mass spectra of CytC (Top), BSA (Middle), and hemoglobin (Bottom). (Left) The m/z spectra before mass filtering are displayed. (Right) The corresponding mass-filtered spectra.

Conclusion and Outlook The ultimate goal of directly uncovering the structure of unknown proteins or protein complexes and describing their conformations at the atomic level still requires experimental efforts toward a better chemical and conformational selectivity of the deposition process. This could be attained by adding ion-mobility capability to the ES-IBD device. With this more elaborated deposition device, it would be possible to select the objects on the basis of not only their charge state but also their gas phase conformation. By this, an assessment of the relation between the conformation in the gas phase and on the surface, influenced by the charge state (22⇓⇓–25), deposition conditions (49, 56), and surface properties, would be possible. The mapping of the proteins with unknown structure will also require an improved imaging resolution, fundamentally limited solely by the electron wavelength. Furthermore, as 3D information is encoded in a single in-line hologram, improved spatial resolution will already permit to determine the (x,y,z) spatial coordinates of every atom of a protein from this very holographic record. A complementary strategy to reveal the complete 3D structure of a single protein is to add tomographic capability to the experimental setup. At this stage, the comparison of the low-energy electron micrographs with atomic models available at the PDB has the character of a control experiment, proving the feasibility of this methodology. Nevertheless, fundamental questions remain to be addressed. Most crucial is the influence of the environment on the protein’s structure. For proteins the deposition as native gas phase ions onto graphene in vacuum where they are neutralized definitely represents a significantly different environment from the aqueous medium of the cell. There is a significant body of strong evidence, especially from ion-mobility/mass-spectrometry investigations, demonstrating that proteins and protein complexes can be transferred from the liquid phase to a vacuum environment while maintaining their tertiary, respectively quaternary, structures unperturbed (25, 57⇓⇓–60). The low-energy electron micrographs presented here are further strong evidence that proteins in a folded state are stable in UHV. As recently demonstrated it is possible to add water molecules to small peptides (61) directly in an UHV environment. Low-energy electron holography with its ability to image proteins individually will also allow us to study the effects of adding hydration shells to the protein. Furthermore, questions related to transport, such as diffusion of proteins and subsequent association into protein complexes, will be addressed. First observations of the diffusion of folded proteins on freestanding graphene by means of low-energy electron holography are presented in Fig. S5, illustrating that the method described here is also capable of accessing dynamic processes. Fig. S5. Time evolution of the orientation of CytC complexes. The time lapse between subsequent observations amounts to 30 s. From these images it is evident that at least some of the deposited proteins are mobile on freestanding graphene. Thus, low-energy electron holography appears to be a method for also studying diffusion of proteins on surfaces. This observation suggests that a low-energy electron holographic microscope operating at cryogenic temperatures might be needed to fix the protein in space and attain atomic resolution. (Scale bars, 5 nm.) To conclude, we have shown here how to image a single protein by combining ES-IBD technology with low-energy electron holography. This method has led to a tool for revealing structural details of single native proteins and protein complexes without destroying them. With the recent advances in electrospray ionization and mass spectrometry of large protein complexes (62), and in particular membrane proteins (53, 55), even the structure of these biologically important but reluctant to readily crystalize entities may become accessible in the future.

Materials and Methods Ultraclean freestanding graphene is prepared by the Pt-metal catalysis method described in detail elsewhere (31). Before the transfer of the ultraclean substrate from the UHV chamber of the low-energy electron holographic microscope to the UHV chamber of the ES-IBD device, the cleanliness of the substrate is characterized and reference images are recorded for comparing the very same region of freestanding graphene before and after protein deposition. During the whole experimental workflow, the samples are kept under strict UHV conditions with the help of a UHV suitcase for transfer between the two experimental chambers. Details of the ES-IBD procedure and of the low-energy electron holography experimental scheme are described in ES-IBD and Low-Energy Electron Holography.

ES-IBD Soft-landing electrospray ion beam deposition takes place in a home-built instrument (29, 30) (Fig. S1). The ion beam is generated by a nanoelectrospray source with an optimized hydrodynamic vacuum transfer (63) at a flow rate of 20–30 µL/h and an emitter voltage of ∼3 kV. The positive ions enter the vacuum through a heated metal capillary and are collimated in an ion funnel and a collisional collimation quadrupole operated in rf-only mode. In the third pumping stage a mass filtering quadrupole selects the m/z region of interest, which is monitored by the time-of-flight mass spectrometer in the fourth pumping stage. Here, a retarding field energy analyzer measures the kinetic energy of the ions. The beam is then guided by electrostatic lenses toward the target in the sixth pumping stage being at 2 × 10−10 mbar, where the protein deposition takes place. To ensure gentle landing, the collision energy is controlled by applying a bias voltage to the target and hence reducing the kinetic energy of the protein ions to 2–5 eV per charge. To obtain ion beams of native CytC (bovine; Fluka 30398), a solution of 0.15 mg/mL was prepared in aqueous 50 mM ammonium acetate buffer. With a spray flow rate of 25 μL/h, an ion current of 1.1 nA is detected at the TOF-MS. Unfiltered mass spectra (Fig. S2, Top Left) show low charge states of +5 to +7, corresponding to folded CytC. At lower m/z values peaks corresponding to highly charged (z > +8) unfolded CytC and peaks that relate to fragments or contamination are found. Note that, due to the limited dynamic range of the TOF-MS, the detector amplification was set very high, such that the peaks of the native CytC are distorted. The m/z selective quadrupole was tuned to select an m/z window from 1,250 u/e to ∼3,500 u/e, with u being the atomic mass and e the elementary charge, by setting an rf amplitude of 700 V with a differential dc voltage of 5%. This results in a beam of predominantly native CytC ions (Fig. S2, Bottom Left) from which unfolded proteins (low m/z) and undefined heavy aggregates (high m/z) are removed. The current of the m/z filtered ion beam amounts to 20 pA at the substrate. A total charge of ∼20 pAh is deposited while the pressure is maintained in the 10−10-mbar range. Ion beams of native BSA are generated following the same general procedure as for CytC. Solutions are prepared by dissolving BSA in aqueous 50-mM ammonium acetate buffer at a concentration of ∼10−5 M. The unfiltered mass spectrum displays intense peaks around charge state z = 16, which are related to native BSA (32), whereas higher charge states can be expected to be at least partially unfolded. Further, at m/z > 6,000 u/e unresolved intensity may be related to unspecific agglomeration. Therefore, the region between 3,250 u/e and 6,000 u/e is selected for deposition. The current of the m/z-filtered beam amounts to 8 pA at the substrate. A total charge of ∼20 pAh is deposited while the pressure is maintained in the 10−10-mbar range. Hemoglobin (bovine; Sigma H2500) is a protein complex of four myoglobin subunits, two A and two B. Ion beams are generated from solutions of 0.3 mg/mL HG prepared in 50 mM ammonium acetate buffer. A current of 600 pA is detected at the TOF-MS. The mass spectrum is very complex and is resolved only partially due to the limited performance concerning resolution and dynamic range of the home-built linear TOF-MS. Nevertheless, a comparison with literature spectra allows identifying characteristic peaks (Fig. S2, Bottom Right), which become more pronounced after removal of the unspecific agglomeration in the high-m/z range (>5,000 u/e) by mass filtering with the quadrupole. The beam transmitted for deposition contains native intact HG complexes (Q17+, Q16+) along with dimers (D) and monomers (HBA). A total charge of 70 pAh was deposited.

Low-Energy Electron Holography In the low-energy electron holographic setup (15), inspired by Dennis Gabor’s original idea of in-line holography (14, 64), a sharp (111)-oriented tungsten tip acts as an EPS, providing a divergent beam of highly coherent electrons (65, 66). The atomic-sized electron field emitter can be brought as close as 100 nm to the sample with the help of a three-axis nanopositioner. Part of the electron wave is elastically scattered off the object and hence is called the object wave, whereas the unscattered part of the wave represents the reference wave. At a distant detector, the hologram, i.e., the pattern resulting from the interference of these two wave fronts, is recorded. The detector unit comprises a multichannel plate (MCP) for signal enhancement, a phosphor screen for translating the electron signal in an optical image, a fiber-optic plate for guiding this image out of the vacuum system, and a CCD camera for recording. To discriminate secondary electrons, a small retarding potential can be applied to the entrance of the MCP. This is particularly necessary when imaging metallic clusters or metallic thin films producing a lot of secondary electrons when exposed to the low-energy electron beam. Our experience is that, when imaging low atomic number materials such as carbon fibers or biological materials, no substantial signal originating from the production of secondary electrons at those materials can be detected and the discriminator voltage at the MCP is obsolete. These observations confirm also that predominantly elastic scattering occurs at biological objects like proteins. The magnification of the imaging system is given by the ratio between detector-to-source distance Z and sample-to-source distance z. Given that in the present system Z = 70 mm and that z = 100 nm to 3 mm, the magnification can be as high as 105. The electron energy is dictated by the sample-to-source distance and the sharpness of the field emitter tip for a given electron emission current. All holograms presented here were recorded with electron energies between 60 eV and 140 eV and an emission current of typically 50 nA, which corresponds at high magnification to a current density of 5 pA/nm2 or 107 electrons⋅s−1⋅nm−2 for a field of view of 100 × 100 nm2. A typical acquisition time for a hologram amounts to 100–200 ms, corresponding to a total number of emitted electrons of the order of 1010. A hologram, in contrast to a diffraction pattern, contains also the phase information of the object wave, and the object structure can thus be unambiguously reconstructed. After background subtraction, the shape of the objects is revealed by numerical reconstruction from the hologram, which is essentially achieved by back propagation to the object plane, which corresponds to evaluating the Fresnel–Kirchhoff integral transformation. The individual steps related to the numerical hologram reconstruction are listed in Numerical Reconstruction of Holograms below. A detailed description of the reconstruction procedure can be found elsewhere (38⇓–40). To enable an artifact-free reconstruction, the proteins are deposited on graphene, which is known to be highly transparent for low-energy electrons (20). To attain such high transparency, the graphene samples were prepared by the Pt-metal catalysis method described elsewhere (31).

UHV Transfer A vacuum suitcase, Fig. S4A (Ferrovac GmbH), is used to transfer samples between the two UHV-based experiments, electron holography on the one side and electrospray ion beam deposition on the other side. The suitcase is equipped with an SAES getter/sputter ion pump combination operated by a battery-driven power supply. The pressure is kept below 2 × 10−10 mbar at all times, except for the short duration of the transfer (1–2 min) where it rises into the 1 × 10−9-mbar regime. The performance of this suitcase, originally developed for scanning tunneling microscopy experiments, is well known from various surface science studies (67, 68). Each instrument is equipped with a load lock to which the suitcase can be attached. The load lock is pumped by a turbo-molecular pump supported by a cryogenic active charcoal trap at liquid nitrogen temperature. A pressure in the 10−9-mbar range is established within a few hours, ensuring a contamination-free transfer of the samples. Fig. S4 shows low-energy electron projection images of the very same freestanding graphene region before (Fig. S4B) and after (Fig. S4C) the transfer from the low-energy electron holographic microscope located in Zurich to the ES-IBD chamber in Stuttgart and back to the microscope in Zurich. No relevant sign of contamination due to transfer and transport is observed.

Numerical Reconstruction of Holograms The numerical recipes for the reconstruction of digital holograms are described in detail elsewhere (40, 69). The reconstruction code based on the algorithm presented in ref. 40 is available for download on the MATLAB file exchange server (file ID: 59940). The numerical reconstruction of digital holograms consists of two steps: (i) The first step involves the multiplication of the hologram distribution H ( x H , y H ) with the simulated complex-valued reference wave distribution R = e i k r H / r H , U H ( x H , y H ) = R ( x H , y H ) H ( x H , y H ) , [S1]where r → H = ( x H , y H , z H ) denotes the coordinates in the hologram plane and z H is the source-to-detector distance. (ii)The second step involves the back propagation of the complex-valued wavefront U H ( x H , y H ) distribution from the hologram plane to the object plane, whereby the propagation is described by the integral transform based on the Huygens–Fresnel principle, U ( r → O ) ≈ − i λ z ∫ ∫ U ( r → H ) exp ( − i k | r → H − r → O | ) | r → H − r → O | d σ H , [S2]with r → O = ( x O , y O , z O ) being the coordinate in the object plane. Details concerning evaluating the integral transform given by Eq. S2 are described elsewhere (40, 69).

Acknowledgments We thank Frank Sobott and Ester Martin for advice on preparing native protein beams. The work presented here is financially supported by the Swiss National Science Foundation. We also appreciate support by the European Union commission for building the equipment in the frame of the former Structural Information of Biological Molecules at Atomic Resolution project.

Footnotes Author contributions: J.-N.L. had the original idea to combine ES-IBD and low-energy electron holography and further elaborated the concept with K.K. and H.-W.F. J.-N.L. prepared the ultraclean freestanding graphene supports and recorded the holograms. J.-N.L. and S.R. planned the deposition experiments and along with S.A. performed the electrospray deposition of the proteins onto graphene. T.L. performed the hologram reconstructions with her self-developed software package. J.-N.L. and S.R. interpreted the data. H.-W.F. invented the technology of lens-less holography with low-energy electrons based on atomic sized coherent electron point sources. J.-N.L., C.E., T.L., and H.-W.F. further developed the low-energy electron holographic microscope used in this study. S.R. and K.K. developed the ES-IBD technique. J.-N.L, C.E., and H.-W.F. wrote the manuscript main text and with S.R. the supplementary information, in discussions with all remaining authors.

The authors declare no conflict of interest.

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