Structure. Sometimes conventional methods such as NMR, mass spectrometry, or XRD are not sufficient to unambiguously determine a molecular structure. An alternative attempt can be high resolution imaging by non-contact atomic force microscopy (AFM) combined with electrospray ion beam deposition (ES-IBD) to prepare the chemically perfect samples isolating the molecule of interest.
Function. Isolating an individual molecules further allow to probe it properties on the individual molecule level by tunneling or force spectroscopy in AFM or STM. For instance magnetic and electronic properties leave a clear footprint in the tunneling spectra.
Figure 1: Single molecule imaging. Molecular structure, STM image and simulated conformation of a large organic molecule deposited on a Cu(100) surface by ES-IBD.
Large biological molecules, such as proteins, function only in a state of one specific conformation. The information about this structure and how to reach it is encoded in the amino acid sequence of the protein. With ES-IBD coupled to high-resolution imaging methods like electron- or tunneling microscopy, we have the opportunity to investigate the structure of these large molecules at the atomic level under very controlled conditions. We want to learn what changes when a molecule is transferred into the gas phase and decipher the structure of folded proteins and protein complexes.
Figure 2: High-resolution STM micrograph of an unfolded protein (BSA, m=65500 Da) adsorbed on a Cu(100) surface after ES-IBD. The polypeptide chain has a length of approx. 80 nm. Some sections show overlap or partial folding (high regions) in other parts submolecular resolution at the amino-acid level is found.
With ES-IBD the formation of molecular structures on surfaces following the principles of biological self-assembly is possible. Fig. 3 for instance shows an example of two-dimensional folding. This methods hold a great technological promise of designing molecular nanostructures of high chemical specificity following an evolutionary pathway.
In this project we aim to fabricate such nanostructures and understand the mechanics of their formation based on the sequence control given by amino acid polymer. Further, this can be extended to artificially designed polymers.
Figure 3: Two-Dimensional Folding on a Surface. (left) Individual molecules of the peptide Bradykinin deposited on a strongly interacting surface are not able to move and thus display the conformational freedom they have. (right) Once allowed to diffuse, the molecules meet in dimers of identical shape, adopting one single conformation, which is not found for the individuals on the left.
In contrast to conventional deposition methods, ES-IBD has the capacity to control charge state and kinetic energy of the deposited molecule. In particular the kinetic energy of can be used to trigger chemical reactions that would not occur under thermal conditions. This represents an entirely new way of chemistry, occurring away from thermal equilibrium and hence with the possibility to create substances that cannot be reached through conventional means. Fig. 4 illustrates this by showing the covalent modification of graphene occurring at 165 eV kinetic energy at room temperature.
Figure 4: Covalent chemical modification of graphene: (top) Raman spectra reveal the chemisorption at 165 eV, whereas nothing is seen for soft landing at 5 eV. The reaction is reversible by annealing at 200ºC, which shows that not defects were created but actual chemisorption occurred. (bottom) Proposed reaction scheme.
High performance surface science methods, for instance tunneling microscopy, are able to give unprecedented insight into the atomic details of structure and function of molecules, however they require a perfectly prepared environment, typically ultrahigh vacuum(UHV) of 10-10 mbar. At this pressure the preparation of molecular adsorbates proceeds through the sublimation of molecules and their condensation on a solid surface to ensure the chemical purity. For a long time this excluded macromolecules since they do not have significant vapour pressure.
Electrospray ion beam deposition solves this problem by bringing large molecules into the gas phase as molecular ions via electrospray ionization, a ambient, soft ionization method. The ES-IBD source forms a molecular beam and transmits it to a surface in UHV. On its ways, ion optics guide the beam trough several differential pumping stages, reducing the pressure by 13 orders of magnitude. The ions are further mass-filtered, their energy is adjusted, and the ion current can be measured at any point. This allows an unprecedented level of control and precision in handling macromolecules, opening a large number of analytical and synthetic applications.
Transmission Electron Microscopy, namely cryo-EM, can provide high resolution internal structures of large biomolecules such as proteins and protein complexes. Samples are conventionally prepared by plunge freezing to embed molecules in a thin layer of vitreous ice. Thousands of 2D projection images of single particles are recorded at very low dose and combined into a 3D image using projection matching algorithms. A major limitation is the intrinsic complexity of the plunge freezing workflow which can result in sample deterioration as well as inhomogeneous composition and distribution. We explore an alternate, ice-free, sample preparation route based on native mass spectrometry and ion beam deposition and aim to produce chemically selected, high density, homogeneous samples for cryo-EM.
ES-IBD was initially constructed to produce samples of non-volatile molecules on atomically clean substrates for high resolution STM investigation. Scanning Probe Microscopy is the one of the main analysis method for the investigation of molecular nanostructures. Scanning Tunneling and Force Microscopy are ultimately capable of atomic resolution, given a perfectly prepared sample. Providing such samples for macromolecules requires ES-IBD preparation, because thermal sublimation is excluded for large nonvolatile molecules.
Figure: Scheme of Scanning Tunneling Microscopy and Spectroscopy: (a) A piezo motor positions and moves an atomically sharp tip over a surface with subatomic precision. The tunneling current is used to generate a feedback loop to measure the tip-surface distance and create a topography map by (b) scanning of line profiles . (c) Energy diagram of the tip-surface interaction in tunneling microscopy. Ramping the tip-surface voltage will generate a spectrum that is related to the electronic density of states.
(in collaboration with Prof. K. Kern, MPI Stuttgart)
https://www.fkf.mpg.de/6562175/07_Low_Energy_Electron_Holography
(in collaboration with Prof. M Duerr, Univ. of Giessen)
ES-IBD is a new technique and thus commercial solutions are not available. We drive the field by constructing and building this novel instrumentation from scratch, starting from design calculation, simulation, CAD construction, overseeing the fabrication, assembly and test. As a result a unique instrument with novel capabilities is available for our research.
Figure 7: Construction drawing of a electrostatic lens system. Such a lens is a key element in every ES-IBD system. It is used to shape the beam and align it with the sample position.
For the deposition of proteins as native gas phase protein ions we modified a commercial mass spectrometer. The Orbitrap Q-exactive II UHMR (ultrahigh mass range) is optimised for transmission and analysis of heavy ions of low charge state (m/z>10000) as well as controlling their energy along the way.
This instrument is the basis of the ES-IBD/SPM instrumentation at the MPI-Stuttgart (former group of the PI) and was the prototype instrument of ES-IBD for surface science. It was intended for the deposition of non-volatile molecules on atomically clean surfaces in UHV to enable their investigation in a scanning tunneling microscope (STM). Due to several improvement and innovations, it became a very versatile instrument, ultimately demonstrating native protein deposition and hyperthermal surface chemistry.
Figure: Scheme of the electrospray ion beam deposition/scanning probe microscopy experiment (ES-IBD). The ion beam is generated by ESI at ambient pressure (left) and transferred to UHV via a differentially pumped vacuum system (pressures given). The SPM sample is prepared under UHV conditions and is transferred in situ to a deposition stage and finally undergoes SPM analysis. Abbreviations: ESI, electrospray ionization; ES-IBD/SPM, electrospray ion beam deposition/scanning probe microscopy; UHV, ultrahigh vacuum.
Features
Source: online low flow or offline nanospray source
Ion Optics: Ion funnel, 6mm diam. rf-only quadrupole ion guide, 7 electrostatic lenses with beam deflectors.
Mass Filter/analysis: quadrupole mass filter, linear time-of-flight mass spec.
Samples: HV sample holder (6-fold) for AFM and TEM, UHV sample stage for in-situ STM transfer and suitcase connection.
High intensity molecular ion-beam deposition source
for surface modification and molecular imaging
[FIG]
With high intensity ion beams, the coating of macroscopic surfaces by ES-IBD is possible. We currently develop a high intensity ion source for the fabrication of surface coatings, capable of harnessing the full potential of molecular ion beam deposition, i.e. precise mass selection, chemical activation by collisions, and the use of nonvolatile molecules.
The performance of ES-IBD critically depends on the intensity of the molecular ion beams since for the deposition of a layer of molecules on a macroscopic surface we need orders of magnitude more molecules as for a mass spectrum. Conventional analytical ion sources are often highly inefficient. The optimization of the gas flow field has recently been shown to have a major impact on the efficiency. Currently our goal is to reach high efficiency, as well as high absolute current molecular ion sources capable of operating in coating large areas with pure, microamp molecular beams.
Figure 8: Hydrodynamic optimized electrospray ion sources: (left) Scheme of the comparison experiment of conventional vs. funnel ion source. (right) performance of the funnel ion source (transmission current vs. emission current) shows unity transmission up to a space charge limit of up to 40 nA.
High-Voltage rf-amplifier (HV-AMP400)
This is a highly powered rf-generator for our main mass filter quadrupole. This is able to supply 500kHz, 1MHz, and 2MHz (on the Stuttgart instrument), which allows to handle very light ions (e.g. Na+ at m/z=23) or very heavy ions (e.g. GroEL, m=815kDa, m/z=12000).
http://www.cgc-instruments.com/cgi-bin/main.cgi/lang=en/Products/AF-RF/HV-AMP400FN-4+400-D/Main
Radio frequency generator (RFG50-10)
For operating ion funnels and rf-only ion guides.
http://www.cgc-instruments.com/cgi-bin/main.cgi/lang=en/Products/AF-RF/RFG50-10/Main
We use the rbd model 9103 picoampmeter for detection of molecular ion currents impinging on isolated, i.e. floating electrodes (apertures, detector plates, samples) throughout the instrument.
The instrument is controlled via a LabVIEW interface, made based on the library supplied by the vendor. Picoampere resolution is achieved in floating measurements in a setup outlined in the technical note on using the rbd9103.
Right: four rbd9103 picoampmeters connected to the ion optics in the lab.
An application note with in-detail wiring schemes for the rbd 9103 can be found here.