Structured Water Molecules on Membrane Proteins Resolved by Atomic Force Microscopy.

Water structuring on the outer surface of protein molecules called the hydration shell is essential as well as the internal water structures for higher-order structuring of protein molecules and their biological activities in vivo. We now show the molecular-scale hydration structure measurements of native purple membrane patches composed of proton pump proteins by a noninvasive three-dimensional force mapping technique based on frequency modulation atomic force microscopy. We successfully resolved the ordered water molecules localized near the proton uptake channels on the cytoplasmic side of the individual bacteriorhodopsin proteins in the purple membrane. We demonstrate that the three-dimensional force mapping can be widely applicable for molecular-scale investigations of the solid-liquid interfaces of various soft nanomaterials.

Molecular-scale quantitative charge density measurement of biological molecule by frequency modulation atomic force microscopy in aqueous solutions.

Surface charge distributions on biological molecules in aqueous solutions are essential for the interactions between biomolecules, such as DNA condensation, antibody-antigen interactions, and enzyme reactions. There has been a significant demand for a molecular-scale charge density measurement technique for better understanding such interactions. In this paper, we present the local electric double layer (EDL) force measurements on DNA molecules in aqueous solutions using frequency modulation atomic force microscopy (FM-AFM) with a three-dimensional force mapping technique. The EDL forces measured in a 100 mM KCl solution well agreed with the theoretical EDL forces calculated using reasonable parameters, suggesting that FM-AFM can be used for molecular-scale quantitative charge density measurements on biological molecules especially in a highly concentrated electrolyte.

Molecular-scale investigations of structures and surface charge distribution of surfactant aggregates by three-dimensional force mapping.

Surface charges on nanoscale structures in liquids, such as biomolecules and nano-micelles, play an essentially important role in their structural stability as well as their chemical activities. These structures interact with each other through electric double layers (EDLs) formed by the counter ions in electrolyte solution. Although static-mode atomic force microscopy (AFM) including colloidal-probe AFM is a powerful technique for surface charge density measurements and EDL analysis on a submicron scale in liquids, precise surface charge density analysis with single-nanometer resolution has not been made because of its limitation of the resolution and the detection sensitivity. Here we demonstrate molecular-scale surface charge measurements of self-assembled micellar structures, molecular hemicylinders of sodium dodecyl sulfate (SDS), by three-dimensional (3D) force mapping based on frequency modulation AFM. The SDS hemicylindrical structures with a diameter of 4.8 nm on a graphite surface were clearly imaged. We have succeeded in visualizing 3D EDL forces on the SDS hemicylinder surfaces and obtaining the molecular-scale charge density for the first time. The results showed that the surface charge on the trench regions between the hemicylinders was much smaller than that on the hemicylinder tops. The method can be applied to a wide variety of local charge distribution studies, such as spatial charge variation on a single protein molecule.

Immunoactive two-dimensional self-assembly of monoclonal antibodies in aqueous solution revealed by atomic force microscopy.

The conformational flexibility of antibodies in solution directly affects their immune function. Namely, the flexible hinge regions of immunoglobulin G (IgG) antibodies are essential in epitope-specific antigen recognition and biological effector function. The antibody structure, which is strongly related to its functions, has been partially revealed by electron microscopy and X-ray crystallography, but only under non-physiological conditions. Here we observed monoclonal IgG antibodies in aqueous solution by high-resolution frequency modulation atomic force microscopy (FM-AFM). We found that monoclonal antibodies self-assemble into hexamers, which form two-dimensional crystals in aqueous solution. Furthermore, by directly observing antibody-antigen interactions using FM-AFM, we revealed that IgG molecules in the crystal retain immunoactivity. As the self-assembled monolayer crystal of antibodies retains immunoactivity at a neutral pH and is functionally stable at a wide range of pH and temperature, the antibody crystal is applicable to new biotechnological platforms for biosensors or bioassays.

Development of multi-environment dual-probe atomic force microscopy system using optical beam deflection sensors with vertically incident laser beams.

We developed a dual-probe atomic force microscopy (DP-AFM) system with two cantilever probes that can be operated in various environments such as in air, vacuum, and liquid. The system employs the optical beam deflection method for measuring the deflection of each cantilever mounted on a probe scanner. The cantilever probes mounted on the probe scanners are attached to inertia sliders, which allow independent control of the probe positions. We constructed three types of probe scanners (tube, shear-piezo, and tripod types) and characterized their performance. We demonstrated AFM imaging in ambient air, vacuum, and ultrapure water, and also performed electrical measurement and pick-up manipulation of a Au nanorod using the DP-AFM system.

Imaging of Au nanoparticles deeply buried in polymer matrix by various atomic force microscopy techniques.

Recently, some papers reported successful imaging of subsurface features using atomic force microscopy (AFM). Some theoretical studies have also been presented, however the imaging mechanisms are not fully understood yet. In the preceeding papers, imaging of deeply buried nanometer-scale features has been successful only if they were buried in a soft matrix. In this paper, subsurface features (Au nanoparticles) buried in a soft polymer matrix were visualized. To elucidate the imaging mechanisms, various AFM techniques; heterodyne force microscopy, ultrasonic atomic force microscopy (UAFM), 2nd-harmonic UAFM and force modulation microscopy (FMM) were employed. The particles buried under 960 nm from the surface were successfully visualized which has never been achieved. The results elucidated that it is important for subsurface imaging to choose a cantilever with a suitable stiffness range for a matrix. In case of using the most suitable cantilever, the nanoparticles were visualized using every technique shown above except for FMM. The experimental results suggest that the subsurface features buried in a soft matrix with a depth of at least 1 µm can affect the local viscoelasticity (mainly viscosity) detected as the variation of the amplitude and phase of the tip oscillation on the surface. This phenomenon presumably makes it possible to visualize such deeply buried nanometer-scale features in a soft matrix.

Beyond the helix pitch: direct visualization of native DNA in aqueous solution.

The DNA double helix was first elucidated by J.D. Watson and F.H.C. Crick over a half century ago. However, no one could actually "see" the well-known structure ever. Among all real-space observation methods, only atomic force microscopy (AFM) enables us to visualize the biologically active structure of natural DNA in water. However, conventional AFM measurements often caused the structural deformation of DNA because of the strong interaction forces acting on DNA. Moreover, large contact area between the AFM probe and DNA hindered us from imaging sub-molecular-scale features smaller than helical periodicity of DNA. Here, we show the direct observation of native plasmid DNA in water using an ultra-low-noise AFM with the highly sensitive force detection method (frequency modulation AFM: FM-AFM). Our micrographs of DNA vividly exhibited not only overall structure of the B-form double helix in water but also local structures which deviate from the crystallographic structures of DNA without any damage. Moreover, the interaction force area in the FM-AFM was small enough to clearly discern individual functional groups within DNA. The technique was also applied to explore the synthesized DNA nanostructures toward the current nanobiotechnology. This work will be essential for considering the structure-function relationship of biomolecular systems in vivo and for in situ analysis of DNA-based nanodevices.

Preparation and photophysical and photoelectrochemical properties of a covalently fixed porphyrin-chemically converted graphene composite.

Chemically converted graphene (CCG) covalently linked with porphyrins has been prepared by a Suzuki coupling reaction between iodophenyl-functionalized CCG and porphyrin boronic ester. The covalently linked CCG-porphyrin composite was designed to possess a short, rigid phenylene spacer between the porphyrin and the CCG. The composite material formed stable dispersions in DMF and the structure was characterized by spectroscopic, thermal, and microscopic measurements. In steady-state photoluminescence spectra, the emission from the porphyrin linked to the CCG was quenched strongly relative to that of the porphyrin reference. Fluorescence lifetime and femtosecond transient absorption measurements of the porphyrin-linked CCG revealed a short-lived porphyrin singlet excited state (38 ps) without yielding the porphyrin radical cation, thereby substantiating the occurrence of energy transfer from the porphyrin excited state to the CCG and subsequent rapid decay of the CCG excited state to the ground state. Consistently, the photocurrent action spectrum of a photoelectrochemical device with a SnO(2) electrode coated with the porphyrin-linked CCG exhibited no photocurrent response from the porphyrin absorption. The results obtained here provide deep insight into the interaction between graphenes and π-conjugated systems in the excited and ground states.

A Photoconductive, Thiophene-Fullerene Double-Cable Polymer, Nanorod Device.

Gold/double-cable copolymer/gold multisegmented nanorods were prepared electrochemically via a template-based method. These "bulk heterojunction" nanorods showed photoconductivity providing us with a platform to study photoinduced charge separation/transport at the nanointerface and begin to think about the rational design of nanoscale solar cells based on such structures.

Reduction of frequency noise and frequency shift by phase shifting elements in frequency modulation atomic force microscopy.

We recently reported the analysis of the frequency noise in the frequency modulation atomic force microscopy (FM-AFM) both in high-Q and low-Q environments [Rev. Sci. Instrum. 80, 043708 (2009)]. We showed in the paper that the oscillator noise, the frequency fluctuation of the oscillator, becomes prominent in the modulation frequency lower than f(0)∕2Q, where f(0) and Q are the resonance frequency and Q-factor. The magnitude of the oscillator noise is determined by the slope of the phase versus frequency curve of the cantilever at f(0). However, in actual FM-AFM in liquids, the phase versus frequency curve may not be always ideal because of the existence of various phase shifting elements (PSEs). For example, the spurious resonance peaks caused by the acoustic excitation and a band-pass filter in the self-oscillation loop increase the slope of the phase versus frequency curve. Due to those PSEs, the effective Q-factor is often increased from the intrinsic Q-factor of the cantilever. In this article, the frequency noise in the FM-AFM system with the PSEs in the self-oscillation loop is analyzed to show that the oscillator noise is reduced by the increase of the effective Q-factor. It is also shown that the oscillation frequency deviates from the resonance frequency due to the increase of the effective Q-factor, thereby causing the reduction in the frequency shift signal with the same factor. Therefore the increase of the effective Q-factor does not affect the signal-to-noise ratio in the frequency shift measurement, but it does affect the quantitativeness of the measured force in the FM-AFM. Furthermore, the reduction of the frequency noise and frequency shift by the increase of the effective Q-factor were confirmed by the experiments.

Development of dual-probe atomic force microscopy system using optical beam deflection sensors with obliquely incident laser beams.

We developed a dual-probe (DP) atomic force microscopy (AFM) system that has two independently controlled probes. The deflection of each cantilever is measured by the optical beam deflection (OBD) method. In order to keep a large space over the two probes for an objective lens with a large numerical aperture, we employed the OBD sensors with obliquely incident laser beams. In this paper, we describe the details of our developed DP-AFM system, including analysis of the sensitivity of the OBD sensor for detection of the cantilever deflection. We also describe a method to eliminate the crosstalk caused by the vertical translation of the cantilever. In addition, we demonstrate simultaneous topographic imaging of a test sample by the two probes and surface potential measurement on an α-sexithiophene (α-6T) thin film by one probe while electrical charges were injected by the other probe.

Nanoscale liquid droplet deposition using the ultrasmall aperture on a dynamic mode AFM tip.

We have developed a novel method for nanometer-sized droplet deposition of electrically conductive liquids based on the dynamic mode atomic force microscopy (AFM). We succeeded in depositing liquid droplets with a volume of ten zeptoliters or smaller and in making arrays of the droplets with a spacing of several tens of nanometers using the AFM cantilever with a small aperture fabricated by a focused ion beam. The liquid droplets were deposited through the aperture by applying an electric field between the liquid and a conductive surface.

A procedure to determine the optimum imaging parameters for atomic/molecular resolution frequency modulation atomic force microscopy.

We propose a general procedure to determine the optimum imaging parameters (spring constant and oscillation amplitude) to obtain the optimum resolution in frequency modulation atomic force microscopy. We calculated the effective signal-to-noise ratio for various spring constants and oscillation amplitudes, based on the measurement of frequency shift and energy dissipation versus tip-sample distance curves, to find the optimum. We applied this procedure for imaging a lead phthalocyanine (PbPc) thin film on a MoS(2)(0001) substrate, and found that the optimum parameters were about 5 N/m and 20 nm, respectively. An improved signal-to-noise ratio was attained in a preliminary experiment using parameters which were close to the calculated optimum.

Dispersion of carbon nanotubes by photo- and thermal-responsive polymers containing azobenzene unit in the backbone.

Photo- and thermal-responsive polymers containing azobenzene units in the main chain have been utilized as removable dispersants for single-walled carbon nanotubes (SWNTs) in organic solvents. Intermolecular interactions between SWNTs and the polymers are reversibly controllable by tuning the trans-cis composition.

Fabrication of ionic liquid thin film by nano-inkjet printing method using atomic force microscope cantilever tip.

We demonstrate the fabrication of thin films of ionic liquid (IL), 1-butyl-3-methyl-imidazolium tetrafluoborate, by nano-inkjet printing method using an atomic force microscope (AFM) cantilever. The IL filled in a pyramidal hollow of the AFM cantilever tip was extracted from an aperture at the bottom of the hollow and deposited onto a Pt substrate when the bias voltage was applied between the cantilever and the substrate. We succeeded in fabricating IL thin films with a thickness of 4nm. The areas and thicknesses of IL thin films were controlled by the fabrication conditions in this method, which is also useful for the investigations of nanometer-scale properties of ionic liquid.

Process parameters for fast production of ultra-thin polymer film with electrospray deposition under ambient conditions.

Poly(vinylidene fluoride) films between 60 and 120nm have been prepared with electrostatic spray deposition (ESD) in 25-45s. The films are robust and exhibit a strong adhesion to the substrate surface. The important electrospray parameters for ultra-thin film formation are droplet size, initial polymer concentration, shear rate at impact, and volume flux. The latter can be understood as a measure for the solvent balance between deposition and evaporation; it affects overall film quality. The droplet size determines the minimum film thickness at which continuous film forms without voids. Polymer concentration affects thin-film smoothness and below a fixed concentration threshold, films cease to appear. For the very first droplets, wetting behavior on the substrate is most important. Subsequently, shear rate determines how voids are filled up and it determines final film smoothness. In addition to the electrospray conditions, substrates that favor wetting and have a capability to conduct charges away from the surface contribute to the formation of well-defined, ultra-thin films.

Frequency noise in frequency modulation atomic force microscopy.

Atomic force microscopy (AFM) using the frequency modulation (FM) detection method has been widely used for atomic/molecular-scale investigations of various materials. Recently, it has been shown that high-resolution imaging in liquids by the FM-AFM is also possible by reducing the noise-equivalent displacement in the cantilever displacement sensor and by oscillating the cantilever at a small amplitude even with the extremely reduced Q-factor due to the hydrodynamic interaction between the cantilever and the liquid. However, it has not been clarified how the noise reduction of the displacement sensor contributes to the reduction in the frequency noise in the FM-AFM in low-Q environments. In this article, the contribution of the displacement sensor noise to the frequency noise in the FM-AFM is described in detail to show how it is important to reduce the noise-equivalent displacement in the displacement sensor especially in low-Q environments.

Alkyl and alkoxyl monolayers directly attached to silicon: chemical durability in aqueous solutions.

For practical application of self-assembled monolayers (SAMs), knowledge of their chemical durability in acidic or basic solutions is important. In the present work, a series of SAMs directly immobilized on a silicon (111) surface through Si-C or Si-O-C covalent bonds without a native oxide layer were prepared by thermally activated chemical reactions of a hydrogen-terminated Si(111) substrate with linear molecules, i.e., 1-hexadecene, 1-hexadecanol, 1-dodecanol, and n-dodecanal, to investigate the durability of the SAMs to HF and Na2CO3 solutions. While grazing incidence X-ray reflectivity measurements showed that all the as-prepared SAMs had almost the same film density and molecular coverage, keeping the original step and terrace structure of Si(111) as is observed by atomic force microscopy, they gave different degradation behaviors, i.e., pitting and concomitant surface roughening in both solutions. 1-hexadecene SAM was stable against immersion in both solutions, while the other SAMs were damaged within 60 min, most likely due to the difference in chemical bonding modes at the SAM/Si interface, i.e., Si-C and Si-O-C.

Submolecular resolution viscoelastic imaging of a poly(p-toluene-sulfonate) single crystal using force modulation microscopy.

Submolecular resolution viscoelastic imaging of a poly(2,4-hexadiyne-1,6-diyl bis(p-toluene-sulfonate)) single crystal was achieved using force modulation microscopy under ambient conditions. The elastic image clearly visualized the structure of p-toluene-sulfonate side chains. The viscotic image visualized that the phase delay on the main chains was smallest, while it became largest on the toluene rings in the side chains. The result is considered to be closely related to the molecular dynamics of the crystal.

The effect of local polarized domains of ferroelectric P(VDF/TrFE) copolymer thin film on a carbon nanotube field-effect transistor.

We produced local polarized domains of ferroelectric P(VDF/TrFE) copolymer thin films on a carbon nanotube field-effect transistor (CN-FET) channel by atomic force microscopy (AFM). The drain current versus gate voltage (I(d)-V(g)) curves measured after forming the local polarized domains showed a shift in the threshold voltages. We also found that the amount of the shifts in the threshold voltages gradually decreased during the measurement of this characteristic over 100 h after forming the polarized domains. The mechanisms of the shifts in the threshold voltages and their decreasing behaviour were explained in terms of the excessive charges that were induced upon the formation of the polarized domains.