Measurement of nanomechanical properties of biomolecules using atomic force microscopy

The capabilities of atomic force microscopy (AFM) have been rapidly expanding beyond topographical imaging to now allow for the analysis of a wide range of properties of diverse materials. The technique of nanoindentation, traditionally performed via dedicated indenters can now be reliably achieved using AFM instrumentation, enabling mechanical property determination at the nanoscale using the high spatial and force resolutions of the AFM. In the study of biological systems, from biomolecules to complexes, this technique provides insight into how mesoscale properties and functions may arise from a myriad of single biomolecules. In vivo and in situ analyses of native structures under physiological conditions as well as the rapid analysis of molecular species under a variety of experimental treatments are made possible with this technique. As a result, AFM nanoindentation has emerged as a critical tool for the study of biological systems in their natural state, further contributing to both biomaterial design and pharmacological research. In this review, we detail the theory and progression of AFM-based nanoindentation, and present several applications of this technique as it has been used to probe biomolecules and biological nanostructures from single proteins to complex assemblies. We further detail the many challenges associated with mechanical models and required assumptions for model validity. AFM nanoindentation capabilities have provided an excellent improvement over conventional nanomechanical tools and by integration of topographical data from imaging, enabled the rapid extraction and presentation of mechanical data for biological samples.     

Nanoscale laser processing and diagnostics

The article summarizes research activities of the Laser Thermal Laboratory on pulsed nanosecond and femtosecond laser-based processing of materials and diagnostics at the nanoscale using optical-near-field processing. Both apertureless and apertured near-field probes can deliver highly confined irradiation at sufficiently high intensities to impart morphological and structural changes in materials at the nanometric level. Processing examples include nanoscale selective subtractive (ablation), additive (chemical vapor deposition), crystallization, and electric, magnetic activation. In the context of nanoscale diagnostics, optical-near-field-ablation-induced plasma emission was utilized for chemical species analysis by laser-induced breakdown spectroscopy. Furthermore, optical-near-field irradiation greatly improved sensitivity and reliability of electrical conductance atomic force microscopy enabling characterization of electron tunneling through the oxide shell on silicon nanowires. Efficient in-situ monitoring greatly benefits optical-near-field processing. Due to close proximity of the probe tip with respect to the sample under processing, frequent degradation of the probe end occurs leading to unstable processing conditions. Optical-fiber-based probes have been coupled to a dual-beam (scanning electron microscopy and focused ion beam) system in order to achieve in-situ monitoring and probe repair.     

Multifunctional universal SPM nanoprobe fabrication with laser technology

Scanning probe microscopy (SPM) is a high spatial resolution method of surface topography visualization and measurement of its local properties. The detecting of interaction arising between the sharp solid-state probe and the sample surface is the foundation of SPM. In dependence from nature of this interaction the scanning tunneling microscopy (STM), scanning force microscopy (SFM), scanning near field optical microscopy (SNOM), etc. are distinguished. The spatial resolution of all types of probe microscopy determines both sharpness of increasing of interaction between a probe and a sample at their approach, and shape and size of a top of a solid-state probe. So, the progress in SPM information capabilities is highly depends on probe properties and first of all on properly fabricated aperture size. Fabrication procedures are rather complicated because of nanometric scale size of aperture and hard requirements to reproducibility and need to be improved. The way how to do it by laser-assisted drawing-out is involving of feed-back in a processing procedure-results in two types of feedback for the process of drawing-out has been suggested, tested and installed into the technological set-up. Different probes have been fabricated by above mentioned laser-assisted stretching during this work: SNOM types from optical fibers, micropipettes from quartz glass capillaries, micropipettes with microwires inside and with metallic covers outside. Some examples of application of above mentioned combined probes for cell membrane technology are described. Most important from them are topographical studying of cells and bacteria in living condition (in liquid) and studying of the mechanical properties of cell (rigidity of cell membrane) using the nanopipette as a tip of a force sensor. Except for that using the model sample the measurement of ion current that runs through nanopipette which also carries out a role of a tip of a force sensor have been done. Thus it is shown, that using a probe as a nanopipette, it is possible to combine SPM method with well-known patch-clamp method.     

Nanoscale depth-resolved cathodoluminescence spectroscopy of ZnO surfaces and metal interfaces

The electronic properties of ZnO surfaces and interfaces has until recently been relatively unexplored. We have used a complement of ultrahigh vacuum scanning electron microscope (SEM)-based, depth-resolved cathodoluminescence spectroscopy (DRCLS), temperature-dependent charge transport, trap spectroscopy, and surface science techniques to probe the electronic and chemical properties of clean surfaces and interfaces on a nanometer scale. DRCLS reveals remarkable nanoscale correlations of native point defect distributions with surface and sub-surface defects calibrated with capacitance trap spectroscopies, atomic force microscopy, and Kelvin probe force microscopy. The measurement of these near-surface states associated with native point defects in the ZnO bulk and those induced by interface chemical bonding is a powerful extension of cathodoluminescence spectroscopy that provides a guide to understanding and controlling ZnO electronic contacts.     

Towards a nanoscale view of lactic acid bacteria

Probiotic bacteria have a strong potential in biomedicine owing to their ability to induce various beneficial health effects. Bacterial cell surface constituents play a key role in establishing tight interactions between probiotics and their host. Yet, little is known about the spatial organization and biophysical properties of the individual molecules. In this paper, we discuss how we have been using atomic force microscopy imaging and force spectroscopy to probe the nanoscale surface properties of gram-positive lactic acid bacteria, with an emphasis on probiotic strains. Topographic imaging has enabled us to visualize bacterial cell surface structures (peptidoglycan, teichoic acids, pili, polysaccharides) under physiological conditions and with unprecedented resolution. In parallel, single-molecule force spectroscopy has been used to localize and force probe single cell surface constituents, providing novel insights into their spatial distribution and molecular elasticity.     

Study of Perylenetetracarboxylic Acid Dimethylimide Films by Cyclic Thermal Desorption and Scanning Probe Microscopy

Some results of studying the direct-current (DC) conductivity of perylenetetracarboxylic acid dimethylimide films by cyclic oxygen thermal desorption are presented. The microscopic parameters of hopping electron transport over localized impurity and intrinsic states were determined. The bandgap width and the sign of major current carriers were determined by scanning probe microscopy methods (atomic force microscopy, scanning probe spectroscopy, and photoassisted Kelvin probe force microscopy). The possibility of the application of photoassisted scanning tunneling microscopy for the nanoscale phase analysis of photoconductive films is discussed.     

Probing Chemical and Mechanical Nanodomains in Copolymer Nanorods with Correlative Atomic Force Microscopy—Nano‐correscopy

The interplay between size, shape, mechanical properties, and surface chemistry of nanoparticles orchestrates cellular internalization, toxicity, circulation time, and biodistribution. Therefore, the safety of nanoparticles hinges on our ability to quantify nanoscale physicochemical characteristics. Current characterization tools, due to their limited resolution, are unable to map these properties correlatively at nanoscale. An innovative use of atomic force microscopy‐based techniques, namely nano‐correscopy, overcomes this limitation and offers multiprobe capability to map mechanical (viscous and elastic) and chemical domains of nanoparticles correlatively. The strengths of this approach are demonstrated using polymer composite nanorods: m‐PEG‐PLGA ((m‐PEG–methoxy‐poly (ethylene glycol)‐b‐poly (lactic‐co‐glycolic) acid). Precise distribution of PLGA (monomers of lactide and glycolide) and poly(ethylene glycol) (PEG) polymer across nanorods is identified. The hydrophobic lactide component is found predominantly at the apex, while hydrophilic glycolide and PEG assembled at the body of the nanorods and correlate with a gradient of nanomechanical properties. New knowledge of how both nanochemical domains and nanomechanical properties are distributed across the nanorod will allow elucidating the interactions of nanorods with the proteins and biomolecules in the future, which will directly influence the fate of nanorods in vivo and will guide new synthesis methods.     

Quantitative determination of contact stiffness using atomic force acoustic microscopy

Atomic force acoustic microscopy is a near-field technique which combines the ability of ultrasonics to image elastic properties with the high lateral resolution of scanning probe microscopes. We present a technique to measure the contact stiffness and the Young's modulus of sample surfaces quantitatively, with a resolution of approximately 20 nm, exploiting the contact resonance frequencies of standard cantilevers used in atomic force microscopy. The Young's modulus of nanocrystalline ferrite films has been measured as a function of oxidation temperature. Furthermore, images showing the domain structure of piezoelectric lead zirconate titanate ceramics have been taken.     

Highly reproducible technique for three-dimensional nanostructure fabrication via anodization scanning probe lithography

The effects of coated materials for Si probes on the sizes of line structure by using anodization scanning probe lithography (SPL) are investigated by using atomic force microscopy (AFM). Anodization SPL was carried out on the 1-decane monolayer directly attached to hydrogen-terminated silicon. Gold-coated, diamond-coated, and uncoated Si probes were used as the SPL probes in order to fabricate line structures of silicon oxide. In the cases of Au-coated and uncoated Si probes, the widths of line structures are widely influenced by the changes of scanning rate and applied bias voltage. However, the line width fabricated by use of the diamond-coated probe maintained 15 nm under any condition of scanning rate and applied bias voltage. One of the most narrow and three-dimensional nanostructures is found to be successfully fabricated on the hydrogen-terminated Si substrates when the diamond-coated probe was used as a probe of anodization SPL. In addition, this result indicated that high reproducibility of oxide nanostructures is attainable by anodization SPL used the diamond-coated probe.     

Nanomechanical properties of single amyloid fibrils

Amyloid fibrils are traditionally associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease or Creutzfeldt-Jakob disease. However, the ability to form amyloid fibrils appears to be a more generic property of proteins. While disease-related, or pathological, amyloid fibrils are relevant for understanding the pathology and course of the disease, functional amyloids are involved, for example, in the exceptionally strong adhesive properties of natural adhesives. Amyloid fibrils are thus becoming increasingly interesting as versatile nanobiomaterials for applications in biotechnology. In the last decade a number of studies have reported on the intriguing mechanical characteristics of amyloid fibrils. In most of these studies atomic force microscopy (AFM) and atomic force spectroscopy play a central role. AFM techniques make it possible to probe, at nanometer length scales, and with exquisite control over the applied forces, biological samples in different environmental conditions. In this review we describe the different AFM techniques used for probing mechanical properties of single amyloid fibrils on the nanoscale. An overview is given of the existing mechanical studies on amyloid. We discuss the difficulties encountered with respect to the small fibril sizes and polymorphic behavior of amyloid fibrils. In particular, the different conformational packing of monomers within the fibrils leads to a heterogeneity in mechanical properties. We conclude with a brief outlook on how our knowledge of these mechanical properties of the amyloid fibrils can be exploited in the construction of nanomaterials from amyloid fibrils.     

Atomic force microscopy of swelling and hardening of intact erythrocytes fixed on substrate

Peak force measurements with the aid of atomic force microscopy are used to quantitatively map nanomechanical properties of intact erythrocytes of rats under conditions that are close to physiological conditions. Erythrocytes that are immobilized on the substrate preliminary processed using poly-L-lysine predominantly exhibit plane shape. However, cells may also exhibit stepwise transformation to semispherical objects with an increase in volume and hardening. Possible reasons for such transformations are discussed.     

Identification of surface nanobubbles and resolving their size-dependent stiffness

We report a comparative investigation of the topographic features and nanomechanical responses of surface nanobubbles,polymeric nanodrops, and solid microparticles submerged in water and probed by atomic force microscopy in different operating modes. We show that these microscopic objects exhibit similar topographies, either hemispherical or hemiellipsoidal, in the standard tapping mode, and thus are difficult to distinguish. However, distinct differences, caused not only by their different mechanical properties but also by different cantilever tip-sample mechanical interactions that are affected by tip wettability, were observed in successive topographic imaging with controlled scanning forces and the nanoindentation tests, allowing for the identification of surface nanobubbles. Based on the indentation force-distance curves, we further extrapolated the stiffness of surface nanobubbles spanning a wide range of sizes and then developed a simple theoretical model to explain this size dependence. We also demonstrate how size-dependent stiffness can be used to determine the surface tension of nanobubbles,which was found to be much lower than the bulk value of water.     

Quantitative atomic force microscopy with carbon monoxide terminated tips

Noncontact atomic force microscopy (AFM) has recently progressed tremendously in achieving atomic resolution imaging through the use of small oscillation amplitudes and well-defined modification of the tip apex. In particular, it has been shown that picking up simple inorganic molecules (such as CO) by the AFM tip leads to a well-defined tip apex and to enhanced image resolution. Here, we use the same approach to study the three-dimensional intermolecular interaction potential between two molecules and focus on the implications of using molecule-modified AFM tips for microscopy and force spectroscopy experiments. The flexibility of the CO at the tip apex complicates the measurement of the intermolecular interaction energy between two CO molecules. Our work establishes the physical limits of measuring intermolecular interactions with scanning probes.     

X-ray and electron microscopy of actinide materials

Actinide materials demonstrate a wide variety of interesting physical properties in both bulk and nanoscale form. To better understand these materials, a broad array of microscopy techniques have been employed, including transmission electron microscopy (TEM), electron energy-loss spectroscopy (EELS), energy dispersive X-ray spectroscopy (EDXS), high-angle annular dark-field imaging (HAADF), scanning electron microscopy (SEM), wavelength dispersive X-ray spectroscopy (WDXS), electron back scattered diffraction (EBSD), scanning tunneling microscopy (STM), atomic force microscopy (AFM), and scanning transmission X-ray microscopy (STXM). Here these techniques will be reviewed, highlighting advances made in the physics, materials science, chemistry, and biology of actinide materials through microscopy. Construction of a spin-polarized TEM will be discussed, considering its potential for examining the nanoscale magnetic structure of actinides as well as broader materials and devices, such as those for computational magnetic memory.     

Mapping of mechanical properties of the surface by utilization of torsional oscillation of the cantilever in atomic force microscopy

The measurement of the surface topography in dynamic mode (intermittent contact mode) is one of the most popular ways of imaging surfaces at nanoscale with atomic force microscopy. It also allows obtaining so called phase images which reveal the viscous-elastic non-homogeneities of the surface, therefore can be used for detecting the presence of different materials. It is, however, very difficult to interpret the phase map due to the origin of phenomena, method of signal detection and processing. Therefore one cannot determine whether the observed feature is caused by increase or decrease of any of specific mechanical properties of the surface. In this article we present the modified setup of commercially available AFM, where detection of torsional oscillation of the cantilever is used for the determination of mechanical properties such as: elasticity, adhesion, peak force and energy dissipation. By advanced signal processing, the reconstruction of the force spectroscopy curve and the calculation of mentioned parameters are performed. All the operations are done in real time regime. The developed method allows one to obtain much more complex information about measured surface. Test measurement results are also presented.     

Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.     

Structure and function in native and pathological erythrocytes: A quantitative view from the nanoscale

The red blood cells (RBCs) are among the most simple and less expensive cells to purify; for this reason and for their physiological relevance, they have been extensively studied with a variety of techniques. The picture that results is that these cells have several peculiarities including extreme mechanical performances, relatively simple architecture, biological relevance and predictable behavior that make them a perfect laboratory of testing for novel techniques, methodologies and ideas. These include the re-evaluation of old concepts, such as the relationship between structure and function (which is one of the guideline of this report) but considered at the cellular level. The studies reported on this paper, indeed, exploit the full potential of an high resolution quantitative microscopy such as the atomic force microscopy (AFM) to investigate different aspect of the erythrocytes' life, death and interaction with the environment. Indeed, the erythrocytes have a special relationship with the environment that is able to deeply influence their morphology as consequence of alteration of their biochemical or biophysical status. In this context the conditions under which the erythrocytes can be considered as biochemically programmable systems have been investigated by analyzing different environmentally induced alteration of the cell's morphology and comparing the results with naturally occurring pathological morphologies. This class of studies takes great advantage by the additional consideration of the nanomechanical properties of the cells. These latter are particularly important for the cell functionality and are shown to be of practical usefulness to discriminate and partition environmental effects charging different cellular structure (e.g. membrane or membrane-skeleton). Moreover, the development of novel morphological parameter can be important to push the level of investigation on the RBCs' status towards the molecular level. In particular, we describe the introduction and use of the plasma membrane roughness as a morphometric parameter of simple derivation from the AFM images and that results sensitive to the structural integrity of the cells' membrane-skeleton. This offer a remarkable opportunity to investigate the relationship between structure and function in normal and pathological cells by using a morphometric parameter that probes the cell surface at the nanoscale level. At last, a complex but physio-pathologically important phenomenon such as the erythrocytes aging was considered. To properly analyze the many variation that the cells experience during the whole aging path we used all the parameters that the AFM can provides: quantitative imaging, analysis of the membrane roughness and local measure of the nanomechanical properties analyzed together with biochemical parameter such as the ATP content. The picture that emerged is that the aging path is triggered by the ATP intracellular concentration that influence the membrane-skeleton structure and the support exerted on the plasma membrane. The consequences of the membrane-skeleton involvement can be monitored by AFM and showed the occurrence of peculiar morphologies and morphological defects that appear in the very place where the membrane-skeleton contact with the membrane became loose. As a whole, the collected data enable to describe the entire phenomenon as a sequence of morphological intermediates following one another along the aging path.     

Nanoindentation properties and the microstructure of grain boundary precipitate-free zones (PFZs) in an AlCuSiGe alloy

We have characterized the nanoscale mechanical properties of grain boundary precipitate-free zones (PFZ's) in an AlCuSiGe alloy, using combined nanoindentation and in-situ atomic force microscopy (AFM). These mechanical properties were then correlated to the composition, precipitate distribution and, indirectly, to the vacancy concentration within these regions, as analyzed by transmission electron microscopy and spectroscopy. Using these results we constructed a structure-zone map of the area adjacent to the grain boundary, which relates the reduced elastic modulus and nanoindentation hardness of the alloy to its graded microstructure. Our analysis indicates that the lowest hardness was found in the region where no precipitates are present at all, regardless of solute concentration. In regions where precipitation is different from that of the bulk, somewhat inferior mechanical properties are achieved.     

New development of atmospheric pressure plasma polishing

Atmospheric pressure plasma polishing （APPP） high quMity optical surfaces. The changes of is a precision machining technology used for manufacturing surface modulus and hardness after machining prove the distinct improvement of surface mechanical properties. The demonstrated decrease of surface residual stresses testifies the removal of the former deformation layer. And the surface topographies under atomic force microscope （AFM） and scanning electron microscope （SEM） indicate obvious amelioration of the surface status, showing that the 0.926-nm average surface roughness has been achieved.