A current challenge in the life sciences is to understand how biological systems change their structural, biophysical and chemical properties to adjust functionality. Addressing this issue has been severely hampered by the lack of methods capable of imaging biosystems at high resolution while simultaneously mapping their multiple properties. Recent developments in force-distance (FD) curve–based atomic force microscopy (AFM) now enable researchers to combine (sub)molecular imaging with quantitative mapping of physical, chemical and biological interactions. Here we discuss the principles and applications of advanced FD-based AFM tools for the quantitative multiparametric characterization of complex cellular and biomolecular systems under physiological conditions.

Multiparametric imaging of biological systems by force-distance curve-based AFM.

Mussels use a variety of 3, 4-dihydroxyphenyl-l-alanine (DOPA) rich proteins specifically tailored to adhering to wet surfaces. Synthetic polypeptide analogues of adhesive mussel foot proteins (specifically mfp-3) are used to study the role of DOPA in adhesion. The mussel-inspired peptide is a random copolymer of DOPA and N(5) -(2-hydroxyethyl)-l-glutamine synthesized with DOPA concentrations of 0-27 mol% and molecular weights of 5.9-7.1 kDa. Thin films (3-5 nm thick) of the mussel-inspired peptide are used in the surface forces apparatus (SFA) to measure the force-distance profiles and adhesion and cohesion energies of the films in an acetate buffer. The adhesion energies of the mussel-inspired peptide films to mica and TiO(2) surfaces increase with DOPA concentration. The adhesion energy to mica is 0.09 μJ m(-2) mol(DOPA) (-1) and does not depend on contact time or load. The adhesion energy to TiO(2) is 0.29 μJ m(-2) mol(DOPA) (-1) for short contact times and increases to 0.51 μJ m(-2) mol(DOPA) (-1) for contact times >60 min in a way suggestive of a phase transition within the film. Oxidation of DOPA to the quinone form, either by addition of periodate or by increasing the pH, increases the thickness and reduces the cohesion of the films. Adding thiol containing polymers between the oxidized films recovers some of the cohesion strength. Comparison of the mussel-inspired peptide films to previous studies on mfp-3 thin films show that the strong adhesion and cohesion in mfp-3 films can be attributed to DOPA groups favorably oriented within or at the interface of these films.

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The Contribution of DOPA to Substrate-Peptide Adhesion and Internal Cohesion of Mussel-Inspired Synthetic Peptide Films.

Mussel foot proteins (mfps) have been investigated as a source of inspiration for the design of underwater coatings and adhesives. Recent analysis of various mfps by a surface forces apparatus (SFA) revealed that mfp-1 functions as a coating, whereas mfp-3 and mfp-5 resemble adhesive primers on mica surfaces. To further refine and elaborate the surface properties of mfps, the force–distance profiles of the interactions between thin mfp (i.e. mfp-1, mfp-3 or mfp-5) films and four different surface chemistries, namely mica, silicon dioxide, polymethylmethacrylate and polystyrene, were measured by an SFA. The results indicate that the adhesion was exquisitely dependent on the mfp tested, the substrate surface chemistry and the contact time. Such studies are essential for understanding the adhesive versatility of mfps and related/similar adhesion proteins, and for translating this versatility into a new generation of coatings and (including in vivo) adhesive materials.

https://royalsocietypublishing.org/doi/pdf/10.1098/rsif.2012.0759

Adhesion of mussel foot proteins to different substrate surfaces

The current status and future prospects of non-contact atomic force microscopy (nc-AFM) and Kelvin probe force microscopy (KPFM) for studying insulating surfaces and thin insulating films in high resolution are discussed. The rapid development of these techniques and their use in combination with other scanning probe microscopy methods over the last few years has made them increasingly relevant for studying, controlling, and functionalizing the surfaces of many key materials. After introducing the instruments and the basic terminology associated with them, state-of-the-art experimental and theoretical studies of insulating surfaces and thin films are discussed, with specific focus on defects, atomic and molecular adsorbates, doping, and metallic nanoclusters. The latest achievements in atomic site-specific force spectroscopy and the identification of defects by crystal doping, work function, and surface charge imaging are reviewed and recent progress being made in high-resolution imaging in air and liquids is detailed. Finally, some of the key challenges for the future development of the considered fields are identified.

Recent trends in surface characterization and chemistry with high-resolution scanning force methods.

A method that combines high spatial resolution, quantitative and non-destructive mapping of surfaces and interfaces is a long standing goal in nanoscale microscopy. The method would facilitate the development of hybrid devices and materials made up of nanostructures of different properties. Here we develop a multifrequency force microscopy method that enables simultaneous mapping of nanomechanical spectra of soft matter surfaces with nanoscale spatial resolution. The properties include the Young's modulus and the viscous or damping coefficients. In addition, it provides the peak force and the indentation. The method does not limit the data acquisition speed nor the spatial resolution of the force microscope. It is non-invasive and minimizes the influence of the tip radius on the measurements. The same tip is used to measure in air heterogeneous interfaces with near four orders of magnitude variations in the elastic modulus, from 1 MPa to 3 GPa.

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Fast nanomechanical spectroscopy of soft matter

We have developed a method referred to as three-dimensional scanning force microscopy (3D-SFM) which enables us to visualize water distribution at a solid-liquid interface with an atomic-scale resolution in less than 1 min. The 3D-SFM image obtained at a mica-water interface visualizes 3D distributions of adsorbed water molecules above the center of hexagonal cavities and the laterally distributed hydration layer. The atomically resolved 3D-SFM image showing mirror symmetry suggests the existence of surface relaxation of the cleaved mica surface next to the aqueous environment.

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Atomic-Scale Distribution of Water Molecules at the Mica-Water Interface Visualized by Three-Dimensional Scanning Force Microscopy

At biological interfaces, flexible surface structures and mobile water interact with each other to present non-uniform three-dimensional (3D) distributions. In spite of their impact on biological functions, molecular-scale understanding of such phenomena has remained elusive. Here we show direct visualization of such interfacial structures with subnanometer-scale resolution by 3D scanning force microscopy (3D-SFM). We measured a 3D force distribution at an interface between a model biological membrane and buffer solution by scanning a sharp tip within the 3D interfacial space. We found that vertical cross sections of the 3D image taken along a specific lateral direction show characteristic molecular-scale contrasts tilted at 30° to the membrane surface. Detailed analysis of the 3D image reveals that the tilted contrast corresponds to the time-averaged conformation of fluctuating lipid headgroups. On the basis of the obtained results, we discuss the relationships among the hydration structure, headgroup fl...

Spatial distribution of lipid headgroups and water molecules at membrane/water interfaces visualized by three-dimensional scanning force microscopy.

We have developed a wideband phase-locked loop (PLL) circuit with real-time phase correction for high-speed and accurate force measurements by frequency modulation atomic force microscopy (FM-AFM) in liquid. A high-speed operation of FM-AFM requires the use of a high frequency cantilever which, however, increases frequency-dependent phase delay caused by the signal delay within the cantilever excitation loop. Such phase delay leads to an error in the force measurements by FM-AFM especially with a low Q factor. Here, we present a method to compensate this phase delay in real time. Combined with a wideband PLL using a subtraction-based phase comparator, the method allows to perform an accurate and high-speed force measurement by FM-AFM. We demonstrate the improved performance by applying the developed PLL to three-dimensional force measurements at a mica/water interface.

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Shunsuke Yoshioka

Papers

Atomic-Scale Distribution of Water Molecules at the Mica-Water Interface Visualized by Three-Dimensional Scanning Force Microscopy

Spatial distribution of lipid headgroups and water molecules at membrane/water interfaces visualized by three-dimensional scanning force microscopy.

Wideband phase-locked loop circuit with real-time phase correction for frequency modulation atomic force microscopy.

Mp49-16 impaired urothelial barrier function induced lower urinary tract dysfunction by protease-activated recepter 2 activation