Final Report Summary - FORCETOOL (Multipurpose Force Tool for Quantitative Nanoscale Analysis and Manipulation of Biomolecular, Polymeric and Heterogeneous Materials)
The aim of the 'Multipurpose force tool for quantitative nanoscale analysis and manipulation of biomolecular, polymeric and heterogeneous materials' (FORCETOOL) was to develop a multipurpose tool for quantitative nanoscale analysis and manipulation of biomolecular, polymeric and heterogeneous surfaces. The project had several specific key goals:
- topography, composition analysis and manipulation of biomolecules, polymers and heterogeneous surfaces;
- operation in air or liquids so it will be compatible with industrial environments;
- 1 nm spatial resolution and 1 pN force sensitivity;
- compatible with existing atomic force microscopes, so an additional module could up-grade their capabilities.
Major Impacts:
(1) FORCETOOL developed a new multipurpose tool for quantitative nanoscale analysis and manipulation of heterogeneous surfaces in relevant technological environments
(2) It improved European competitiveness by establishing the technological and scientific foundations of the next generation of advanced scanning probe microscopes.
Executive summary of results
Bimodal Atomic Force Microscopy (AFM)
We have designed, manufactured and tested several bi-modal AFM excitation/detection prototypes. The bimodal AFM have successfully imaged proteins (antibodies) in air and liquid environments. We have also characterised the performance of the instrument under different excitation forces (mechanical and electrostatic), cantilever types, and different samples, such as biomolecules, polymers and layered materials. The force sensitivity of the instrument has been determined about 0.2 pN, i.e. a factor 10 better than conventional tapping mode AFM. In addition, we have demonstrated that the bimodal AFM concept is compatible with existing AFMs and with nanotomography methods. A multilevel theoretical approach has been applied to understand bimodal AFM operation. This approach has involved continuous modelling, finite element simulations and analytical approaches. The analytical model identifies the virial and the energy dissipated by the tip-surface forces as the parameters responsible for the material contrast. The agreement obtained among the theory, experiments and numerical simulations validates the model.
We have also demonstrated that dynamic AFM operation at higher harmonics renders high resolution images of biological membranes and virus capsids in liquids. We have also shown that higher harmonics imaging is also compatible with molecular recognition process.
Multimaterial methodology
We have developed a method to identify the mechanism of energy dissipation at the nanoscale. The method requires the determination of the energy dissipated on the sample surface as a function of the oscillation amplitude while the tip approaches the surface. The representation of the dissipated energy and, in particular, its derivative with respect to the amplitude, dynamic-dissipation curves hereafter, characterizes the dissipation process. Three different non-conservative processes were studied: surface energy hysteresis, viscoelasticity and long-range dissipative interfacial interactions. The method is being applied to characterise the organisation of thin polymer films.
Multiscale theoretical simulations have also provided insight into the relationship between forces, molecular re-orientations and energy dissipation processes. We have performed a combined experimental and multiscale theoretical approach to establish the atomistic origins and hence the contrast, of the dissipative processes occurring in phase-imaging. First-principle simulations show that the configuration space sampled by the tip depends on whether the tip approaches or withdraws from the surface. The quantitative agreement obtained between simulations and experiments demonstrates that the above asymmetry is the origin of the observed contrast. The asymmetry arises because the presence of energy barriers among different bonding configurations.
The development of the bimodal AFM microscope and its applications has involved the participation of several FORCETOOL partners. It comprised bimodal AFM excitation/detection unit (with bimodal AFM imaging based on the simultaneous excitation of two flexural modes of the microcantilever) and bimodal AFM operation. The latter involves a variety of activities such as the design of tailored cantilevers for bimodal AFM operation, simulation of the nonlinear noise of the instrument, optimum ratio between amplitudes or the dependence of the experimental parameters amplitude and phase shifts with the tip-surface distance.
To optimise the performance of bi-modal AFM operation, we have studied the influence on the bi-modal AFM contrast on the first and second mode amplitude ratio.
Comparison between bimodal and tapping mode AFM methods (CSIC)
To asses and compare the performance of the bimodal AFM we have taken images of conjugated molecular materials with the bimodal AFM and a conventional amplitude modulation AFM.
Bi-modal AFM imaging applications
%Bimodal AFM on chlorite allows for a clear distinction between the two different layers of the mineral. While the contrast is already pronounced in common intermittent contact mode imaging, it is enhanced in the detection of the second eigenmode oscillation information.
Imaging antibodies
The general character of the method as well as the spatial resolution of bi-modal AFM in air are characterised by imaging biomolecules (single antibodies). Antibodies are proteins that have well defined structures and binding sites which makes them good candidates to test the sensitivity and resolution of the bi-modal AFM for biomolecular imaging. First, we have imaged small and flexible IgG antibodies deposited on mica.
A direct comparison between amplitude modulation (tapping mode) and bi-modal AFM images illustrates some of the advantages of the latter for high resolution imaging of isolated biomolecules under the application of very small forces. To assure a meaningful comparison, we have used an AFM that enables to perform both amplitude modulation and bi-modal AFM imaging.
Bimodal AFM imaging of antibodies in water
In this period we have applied the bi-modal AFM system to image IgG and IgM antibodies in water. Bimodal imaging AFM in water is more hard than in air because of the difficulty to find the eigenmodes. Here we have located the eigenmodes in liquids by analysing the thermal noise spectrum of the cantilever.
Combination of nanotomography and bimodal AFM imaging
We have demonstrated the compatibility of bimodal AFM imaging with nanotomography imaging of heterogeneous polymers. In this way bimodal AFM could be applied to render three dimensional images. The bimodal concept was applied to image elatomeric polypropylene (ePP) where the first two flexural eigenmodes of the cantilever are mechanically excited and the cantilever deflection signal is analyzed using two lock-in amplifiers realized by the bimodal control unit.
The multimaterial methodology will allow to transform the amplitude, frequency or phase shift changes measured by the instrument into quantitative information about the sample properties.
Transformation between phase shifts and energy dissipation: phase shift between the mechanical excitation of the cantilever and its response as a means to extract information about the sample's properties with nanoscale spatial resolution. We have developed a method to identify the mechanism of energy dissipation at the nanoscale. The method requires the determination of the energy dissipated on the sample surface as a function of the oscillation amplitude while the tip approaches the surface. The representation of the dissipated energy and, in particular, its derivative with respect to the amplitude, dynamic-dissipation curves hereafter, characterises the dissipation process. Three different non-conservative processes are studied: surface energy hysteresis, viscoelasticity and long-range dissipative interfacial interactions. We have performed both simulations and experiments. The quantitative and qualitative agreement obtained between calculations and experiments performed on silicon and polystyrene samples supports the validity of the identification method proposed here.
Molecular dissipation mechanism in sexithiophene monolayers: We have combined experimental measurements of the energy transferred by a silicon dioxide tip into a region of sexithiophene molecules, with continuum modelling and first-principles calculations to identify the molecular processes responsible for the contrast observed in phase-imaging force microscopy.
Nanoscale electrical properties: In an effort to understand the dynamics of the tip-surface interaction under electrostatic interactions, we have studied the sensitivity and contrast in Kelvin probe force microscopy by applying an electrostatic force to the cantilever tuned at the first and second flexural frequencies. The measurements were performed on different test samples such as macroscopic electrodes, small nanocrystals and thin organic layers.
High resolution imaging of biomolecules at higher harmonics: The nonlinear character of the tip-surface forces gives rise to the excitation of higher harmonic components of the driving frequency ω. Those harmonics are multiples of the driving frequency nω. They are very prominent in liquids, where the quality factor of the cantilever is very low.
Exploitable knowledge and its use
Bimodal AFM concept: new concept of operation of an atomic force microscope. The bi-modal AFM concept considers the cantilever as a three dimensional object with several resonance modes, in particular two. The double excitation allows to separate topography from composition contributions in the experimental data. Furthermore, computer simulations show that the bimodal AFM is about two orders of magnitude more sensitive to force variations than state of the art tapping mode AFMs. The concept was developed by CSIC scientists.
Intellectual property rights: Patents WO 2008/003796 and WO 2007/036591 filed.
Contacts have been established with some companies interested in AFM development (air or liquids) such as Asylum Research (USA), Nanotec (Spain) and ScienTec (France). In July 2007 Asylum Research (USA) has acquired the rights to commercialise the CSIC patents on the bi-modal AFM concepts. The product is already available in the market (http://www.asylumresearch.com).
Bimodal AFM cantilevers for operation liquids: Nanoworld services has designed a cantilever with a geometry suitable to amplify the second eigenmode in liquids.
- topography, composition analysis and manipulation of biomolecules, polymers and heterogeneous surfaces;
- operation in air or liquids so it will be compatible with industrial environments;
- 1 nm spatial resolution and 1 pN force sensitivity;
- compatible with existing atomic force microscopes, so an additional module could up-grade their capabilities.
Major Impacts:
(1) FORCETOOL developed a new multipurpose tool for quantitative nanoscale analysis and manipulation of heterogeneous surfaces in relevant technological environments
(2) It improved European competitiveness by establishing the technological and scientific foundations of the next generation of advanced scanning probe microscopes.
Executive summary of results
Bimodal Atomic Force Microscopy (AFM)
We have designed, manufactured and tested several bi-modal AFM excitation/detection prototypes. The bimodal AFM have successfully imaged proteins (antibodies) in air and liquid environments. We have also characterised the performance of the instrument under different excitation forces (mechanical and electrostatic), cantilever types, and different samples, such as biomolecules, polymers and layered materials. The force sensitivity of the instrument has been determined about 0.2 pN, i.e. a factor 10 better than conventional tapping mode AFM. In addition, we have demonstrated that the bimodal AFM concept is compatible with existing AFMs and with nanotomography methods. A multilevel theoretical approach has been applied to understand bimodal AFM operation. This approach has involved continuous modelling, finite element simulations and analytical approaches. The analytical model identifies the virial and the energy dissipated by the tip-surface forces as the parameters responsible for the material contrast. The agreement obtained among the theory, experiments and numerical simulations validates the model.
We have also demonstrated that dynamic AFM operation at higher harmonics renders high resolution images of biological membranes and virus capsids in liquids. We have also shown that higher harmonics imaging is also compatible with molecular recognition process.
Multimaterial methodology
We have developed a method to identify the mechanism of energy dissipation at the nanoscale. The method requires the determination of the energy dissipated on the sample surface as a function of the oscillation amplitude while the tip approaches the surface. The representation of the dissipated energy and, in particular, its derivative with respect to the amplitude, dynamic-dissipation curves hereafter, characterizes the dissipation process. Three different non-conservative processes were studied: surface energy hysteresis, viscoelasticity and long-range dissipative interfacial interactions. The method is being applied to characterise the organisation of thin polymer films.
Multiscale theoretical simulations have also provided insight into the relationship between forces, molecular re-orientations and energy dissipation processes. We have performed a combined experimental and multiscale theoretical approach to establish the atomistic origins and hence the contrast, of the dissipative processes occurring in phase-imaging. First-principle simulations show that the configuration space sampled by the tip depends on whether the tip approaches or withdraws from the surface. The quantitative agreement obtained between simulations and experiments demonstrates that the above asymmetry is the origin of the observed contrast. The asymmetry arises because the presence of energy barriers among different bonding configurations.
The development of the bimodal AFM microscope and its applications has involved the participation of several FORCETOOL partners. It comprised bimodal AFM excitation/detection unit (with bimodal AFM imaging based on the simultaneous excitation of two flexural modes of the microcantilever) and bimodal AFM operation. The latter involves a variety of activities such as the design of tailored cantilevers for bimodal AFM operation, simulation of the nonlinear noise of the instrument, optimum ratio between amplitudes or the dependence of the experimental parameters amplitude and phase shifts with the tip-surface distance.
To optimise the performance of bi-modal AFM operation, we have studied the influence on the bi-modal AFM contrast on the first and second mode amplitude ratio.
Comparison between bimodal and tapping mode AFM methods (CSIC)
To asses and compare the performance of the bimodal AFM we have taken images of conjugated molecular materials with the bimodal AFM and a conventional amplitude modulation AFM.
Bi-modal AFM imaging applications
%Bimodal AFM on chlorite allows for a clear distinction between the two different layers of the mineral. While the contrast is already pronounced in common intermittent contact mode imaging, it is enhanced in the detection of the second eigenmode oscillation information.
Imaging antibodies
The general character of the method as well as the spatial resolution of bi-modal AFM in air are characterised by imaging biomolecules (single antibodies). Antibodies are proteins that have well defined structures and binding sites which makes them good candidates to test the sensitivity and resolution of the bi-modal AFM for biomolecular imaging. First, we have imaged small and flexible IgG antibodies deposited on mica.
A direct comparison between amplitude modulation (tapping mode) and bi-modal AFM images illustrates some of the advantages of the latter for high resolution imaging of isolated biomolecules under the application of very small forces. To assure a meaningful comparison, we have used an AFM that enables to perform both amplitude modulation and bi-modal AFM imaging.
Bimodal AFM imaging of antibodies in water
In this period we have applied the bi-modal AFM system to image IgG and IgM antibodies in water. Bimodal imaging AFM in water is more hard than in air because of the difficulty to find the eigenmodes. Here we have located the eigenmodes in liquids by analysing the thermal noise spectrum of the cantilever.
Combination of nanotomography and bimodal AFM imaging
We have demonstrated the compatibility of bimodal AFM imaging with nanotomography imaging of heterogeneous polymers. In this way bimodal AFM could be applied to render three dimensional images. The bimodal concept was applied to image elatomeric polypropylene (ePP) where the first two flexural eigenmodes of the cantilever are mechanically excited and the cantilever deflection signal is analyzed using two lock-in amplifiers realized by the bimodal control unit.
The multimaterial methodology will allow to transform the amplitude, frequency or phase shift changes measured by the instrument into quantitative information about the sample properties.
Transformation between phase shifts and energy dissipation: phase shift between the mechanical excitation of the cantilever and its response as a means to extract information about the sample's properties with nanoscale spatial resolution. We have developed a method to identify the mechanism of energy dissipation at the nanoscale. The method requires the determination of the energy dissipated on the sample surface as a function of the oscillation amplitude while the tip approaches the surface. The representation of the dissipated energy and, in particular, its derivative with respect to the amplitude, dynamic-dissipation curves hereafter, characterises the dissipation process. Three different non-conservative processes are studied: surface energy hysteresis, viscoelasticity and long-range dissipative interfacial interactions. We have performed both simulations and experiments. The quantitative and qualitative agreement obtained between calculations and experiments performed on silicon and polystyrene samples supports the validity of the identification method proposed here.
Molecular dissipation mechanism in sexithiophene monolayers: We have combined experimental measurements of the energy transferred by a silicon dioxide tip into a region of sexithiophene molecules, with continuum modelling and first-principles calculations to identify the molecular processes responsible for the contrast observed in phase-imaging force microscopy.
Nanoscale electrical properties: In an effort to understand the dynamics of the tip-surface interaction under electrostatic interactions, we have studied the sensitivity and contrast in Kelvin probe force microscopy by applying an electrostatic force to the cantilever tuned at the first and second flexural frequencies. The measurements were performed on different test samples such as macroscopic electrodes, small nanocrystals and thin organic layers.
High resolution imaging of biomolecules at higher harmonics: The nonlinear character of the tip-surface forces gives rise to the excitation of higher harmonic components of the driving frequency ω. Those harmonics are multiples of the driving frequency nω. They are very prominent in liquids, where the quality factor of the cantilever is very low.
Exploitable knowledge and its use
Bimodal AFM concept: new concept of operation of an atomic force microscope. The bi-modal AFM concept considers the cantilever as a three dimensional object with several resonance modes, in particular two. The double excitation allows to separate topography from composition contributions in the experimental data. Furthermore, computer simulations show that the bimodal AFM is about two orders of magnitude more sensitive to force variations than state of the art tapping mode AFMs. The concept was developed by CSIC scientists.
Intellectual property rights: Patents WO 2008/003796 and WO 2007/036591 filed.
Contacts have been established with some companies interested in AFM development (air or liquids) such as Asylum Research (USA), Nanotec (Spain) and ScienTec (France). In July 2007 Asylum Research (USA) has acquired the rights to commercialise the CSIC patents on the bi-modal AFM concepts. The product is already available in the market (http://www.asylumresearch.com).
Bimodal AFM cantilevers for operation liquids: Nanoworld services has designed a cantilever with a geometry suitable to amplify the second eigenmode in liquids.
