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The concept of biomolecule release from co-assembled PS-b-PEO films (left) is depicted. The main findings (right) display an analysis of protein stability, a release comparison depending on cargo size, and tuning release by adjusting film thickness.

Biominerals, such as nacreous bivalve shells, are important archives of environmental information. The processes explaining how the organisms code this information into the structure and composition of their shells are yet unknown. Most marine calcifiers form their shells from amorphous calcium carbonate by particle attachment and stepwise crystallisation of metastable precursor phases. However, the mechanism of this transformation including its incorporation of trace metal ions used for environmental reconstructions are poorly constrained. Using the nacreous shell of the Mediterranean mussel, we explore the formation of nacre from the meso- to the atomic scale. We use a novel combination of strontium pulse-chase labelling in aquaculture experiments and correlative micro- to sub-nanoscale analysis to show that nacre grows in a two-step process and crystallizes via localised dissolution and reprecipitation within nanogranules. Our findings elucidate how stepwise crystallization pathways affect trace element incorporation in natural biominerals, while preserving their intricate hierarchical structure.

The challenges of patterning next-generation integrated circuits have driven the semiconductor industry to look outside of traditional lithographic methods in order to continue cost-effective size ...

The challenges of patterning next generation integrated circuits have driven the semiconductor industry to look outside of traditional lithographic methods in order to continue cost effective size scaling. The directed self-assembly (DSA) of block copolymers (BCPs) is a nanofabrication technique used to reduce the periodicity of patterns prepared with traditional optical methods. BCPs with large interaction parameters (χ(eff)), provide access to smaller pitches and reduced interface widths. Larger χ(eff) is also expected to be correlated with reduced line edge roughness (LER), a critical performance parameter in integrated circuits. One approach to increasing χ(eff) is blending the BCP with a phase selective additive, such as an Ionic liquid (IL). The IL does not impact the etching rates of either phase, and this enables a direct interrogation of whether the change in interface width driven by higher χ(eff) translates into lower LER. The effect of the IL on the layer thickness and interface width of a BCP are examined, along with the corresponding changes in LER in a DSA patterned sample. The results demonstrate that increased χ(eff) through additive blending will not necessarily translate to a lower LER, clarifying an important design criterion for future material systems.

International audience; All-semiconductor plasmonic gratings are investigated by spectroscopic nanoimaging in the vicinity of the plasma frequency, where the material behaves as an epsilon near-zero (ENZ) material. Both phase-sensitive scattering type nanoscopy (s-SNOM) and photoinduced force microscopy (PiFM) are carried out on this structure. The obtained data and models reveal that PiFM, as for s-SNOM, can have a mostly dispersive line shape, in contrast with recent near-field spectra obtained with photothermal AFM nanoscopic imaging on ENZ material where absorption maxima are observed. On the obtained result, PiFM signal exhibited better sensitivity to the dielectric function variation while interferometric s-SNOM can provide additional phase information. Localized surface plasmon resonances (LSPR), highly confined on the structure edges were also observed with both techniques. A higher sensitivity was observed with PiFM for both dielectric contrast imaging and LSPR observation. In addition, for both microscopies, the near-field response is phenomenologically described using a similar formalism based on dipole-image dipole approach. In this model, the sensitivity difference between both techniques is mostly accounted for by probes having different polarizabilities.

Developing a sensor that identifies and quantifies trace amounts of analyte molecules is crucially important for widespread applications, especially in the areas of chemical and biological detection. By non-invasively identifying the vibrational signatures of the target molecules, surface-enhanced Raman scattering (SERS) has been widely employed as a tool for molecular detection. Here, we report on the reproducible fabrication of wafer-scale dense SERS arrays and single-nanogap level near-field imaging of these dense arrays under ambient conditions. Plasmonic nanogaps densely populated the spaces among globular Ag nanoparticles with an areal density of 120 particles per μm2 upon application of a nanolithography-free simple process consisting of the Ar plasma treatment of a polyethylene terephthalate substrate and subsequent Ag sputter deposition. The compact nanogaps produced a high SERS enhancement factor of 3.3 × 107 and homogeneous (coefficient of variation of 8.1%) SERS response. The local near fields at these nanogaps were visualized using photo-induced force microscopy that simultaneously enabled near-field excitation and near-field force detection under ambient conditions. A high spatial resolution of 3.1 nm was achieved. Taken together, the generation of a large-area SERS array with dense plasmonic nanogaps and the subsequent single-nanogap level characterization of the local near field have profound implications in the nanoplasmonic imaging and sensing applications.

In the twenty-first century, scanning probe microscopy characterization techniques have seen significant progress and are capable of probing complex structures and devices for a variety of near-surface features and phenomena with nanometer scale resolution. With modest customization, we can deploy these techniques for industrial metrology purposes in a simultaneous and multimethod system capable of shedding light on device function and failure modes, as well as determining the most efficient methods for data collection. To demonstrate this concept with a current, complex industrial device under development, several scanning probe microscopy techniques advantageous to the progress of heat-assisted magnetic recording heads were selected. This work describes simultaneous and multimethod approaches for performing heat-assisted magnetic recording head characterization using atomic force microscopy with scattering scanning near-field optical microscopy simultaneously performed with magnetic force microscopy or photo-induced force microscopy that could be extended to applications of other complex nanoscale devices. We demonstrate that the optical and magnetic fields are overlapping for fabricated heads, which is necessary for performing heat-assisted magnetic recording. We also observed that the multimethod atomic force microscopy methods show strong agreement between the measured optical and magnetic fields and the locale of their associated parts on the head.

The organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Furthermore, the stabilization of enzyme activity has many practical applications in biotechnology and medicine. Here we describe the symmetry-directed design of an extremely stable, enzymatically active, hollow protein cage of Mr ≈ 2.1 MDa with dimensions similar to those of a small icosahedral virus. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain, and cryo-EM and by atomic force and IR-photoinduced force microscopy established that Ico8 assembles into a flexible hollow cage comprising 20 copies of the esterase trimer, 60 protein subunits in total, with overall icosahedral geometry. Notably, the cages formed by Ico8 proved to be extremely stable toward thermal and chemical denaturation: whereas TriEst was unfolded by heating ( Tm ≈ 75 °C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 °C or in 8 M guanidine hydrochloride. The increased stability of the cages is a new property that emerges from the higher-order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.

Nanoscale microscopy is an important technique in analyzing current semiconductor processes and devices. Many of the current microscopy techniques can render high resolution images of morphology and, in some cases, elemental information. However, techniques are still needed to give definitive nanoscale mapping of compound materials utilized in semiconductor processes such as Si3N4, SiO2, SiGe, and low-k materials. Photo-induced force microscopy (PiFM) combines IR spectroscopy with atomic force microscopy (AFM) to provide concurrent information on topography and chemical mapping. PiFM measures the attractive dipole-dipole photo-response between the tip and the sample and does not rely on repulsive force arising from absorption-based sample expansion. As such, PiFM works well with many of the inorganic semiconductor compounds (with low thermal expansion coefficients) as well as organic materials (with high thermal expansion coefficients) [1]. In this study, various examples of nanoscale chemical mapping of semiconductor samples (surfaces processed via directed self-assembly (DSA), strain in SiGe/SiO2 structure, photoresist, etc.) will be presented, all demonstrating ~ 10 nm spatial resolution

The amount and distribution of water in nominally anhydrous minerals (NAMs) are usually determined by Fourier-transform infrared spectroscopy. This method is limited by the spot size of the beam to the study of samples with dimensions greater than a few micrometers. Here, we demonstrate the potential of using photoinduced force microscopy for the measurement of water in NAMs with samples sizes down to the nanometer scale with a study of water concentration across grain boundaries in forsterite. This development will enable the study of water speciation and diffusion in small-grained rock matrixes and allow a determination of the influence of nanoscale heterogeneity on the incorporation of water to NAMs.

The organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Here we describe the symmetry-directed design of a hollow protein cage with dimensions similar to those of many icosahedral viruses. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain and cryo EM, and by atomic force and IR-photo-induced force microscopy established that Ico8 assembles into a flexible hollow cage with comprising 60-subunits with overall icosahedral geometry. Unexpectedly, the cages were found to encapsulate DNA, even though neither protein component binds nucleic acids on its own. Notably, the cages formed by Ico8 proved to be extremely stable towards thermal and chemical denaturation: whereas TriEst was unfolded by heating (Tm ~75 °C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 °C or in 8 M guanidine hydrochloride. The encapsulation of DNA and increased stability of the cages are new properties that emerge from the higher order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.

Carrier mobility enhancement through local strain in silicon is a means of improving transistor performance. Among the scanning probe microscopy based techniques, tip-enhanced Raman spectroscopy (TERS) has shown some promising results in measuring strain. However, TERS is known to depend critically on the quality of the plasmonic tip, which is difficult to control. In this study, a test structure is used to demonstrate the capability of photo-induced force microscopy with infrared excitation (IR PiFM) in direct measurement of strain with approximately 10 nm spatial resolution. For SiGe pitch less than about 800 nm, the region between the SiGe lines should maintain residual strain. For a region with SiGe pitch of 1000 nm, it is verified that the strain between the SiGe lines is fully relaxed. PiFM promises to be a powerful tool for studying nanoscale strain in diverse material.

For Tip Enhanced Near-field Optical Microscopy (TENOM) utilizing detection of fluorescence or Raman emission, signal to noise amplification is highly desirable for higher resolution imaging. This goal may be achieved by amplifying the signal produced by the probe at the sample through a highly resonant geometry and/or by filtering out the unwanted signal of the excitation source through the addition of an aperture in the collection optical pathway. Making highly resonant tip geometries via nanofabrication can be a difficult process, while the aperture method is a much easier method. With this technique, even tips with undesirably low resonance can be utilized for imaging. We demonstrate the concept through the use of a low field enhancement probe by showing the spatial separation of the excitation and field enhancement locations. We also are able to predict this effect using finite difference time domain modeling of the potential geometries for a desired wavelength.

Photoinduced force microscopy resolves nanometer-scale topology with chemical recognition based on material absorption., Correlating spatial chemical information with the morphology of closely packed nanostructures remains a challenge for the scientific community. For example, supramolecular self-assembly, which provides a powerful and low-cost way to create nanoscale patterns and engineered nanostructures, is not easily interrogated in real space via existing nondestructive techniques based on optics or electrons. A novel scanning probe technique called infrared photoinduced force microscopy (IR PiFM) directly measures the photoinduced polarizability of the sample in the near field by detecting the time-integrated force between the tip and the sample. By imaging at multiple IR wavelengths corresponding to absorption peaks of different chemical species, PiFM has demonstrated the ability to spatially map nm-scale patterns of the individual chemical components of two different types of self-assembled block copolymer films. With chemical-specific nanometer-scale imaging, PiFM provides a powerful new analytical method for deepening our understanding of nanomaterials.

Collagen is the major structural protein of bone, dentine and the extracellular matrix and can template the nucleation and growth of numerous mineral phases. Collagen meso-scale architecture on surfaces, which is critical for its function, is controlled by the relative magnitude of collagen-substrate (C-S) and collagen-collagen (C-C) interactions. Thus, understanding the nature of these interactions and the mechanisms of assembly on surfaces may enable us to manufacture complex 2D protein assemblies for tissue engineering.Infrared Photo-induced Force Microscopy (IR PiFM) is based on an atomic force microscopy (AFM) platform that is coupled to a widely tunable mid-IR laser. PiFM measures the sample's polarizability by detecting the dipole-dipole force that exists between the light induced dipole in the sample and the mirror image dipole in the metallic AFM tip. This interaction is strongly affected by the IR absorption of the sample. Due to its AFM heritage, PiFM acquires both the topography and spectral images concurrently and provides information on the relationship between local chemistry and topology with spatial resolution of ∼ 10nm.PiFM studies on various stages of fibril formation of collagen molecules deposited onto muscovite mica will be presented. The results consist of PiFM spectral images associated with Amide I absorption band. Hyperspectral images, where each pixel consists of PiFM spectrum centered about the Amide I band, are used to study C-C and C-S interactions by tracking the peak shape and position. By enabling imaging at the nm-scale with chemical specificity, PiFM provides a powerful new analytical method for deepening our understanding of bio-materials and facilitating technological applications of such materials.

Summary With increasing interest in nanometer scale studies, a common research issue is the need to use different analytical systems with a universal substrate to relocate objects on the nanometer scale. Our paper addresses this need. Using the delicate milling capability of a focused ion beam (FIB) system, a region of interest (ROI) on a sample is labelled via a milled reference grid. FIB technology allows for milling and deposition of material at the sub 20-nm level, in a similar user environment as a standard scanning electron microscope (SEM). Presently commercially available transmission electron microscope (TEM) grids have spacings on the order 100 μm on average; this technique can extend this dimension down to the submicrometre level. With a grid on the order of a few micrometres optical, FIBs, TEMs, scanning electron microscopes (SEMs), and atomic force microscopes (AFM) are able to image the ROI, without special chemical processes or conductive coatings required. To demonstrate, Au nanoparticles of ∼ 25 nm in size were placed on a commercial Formvar®- and carbon-coated TEM grid and later milled with a grid pattern. Demonstration of this technique is also extended to bulk glass substrates for the purpose of sample location. This process is explained and demonstrated using all of the aforementioned analytical techniques.

The enormous advances made in nanotechnology have also intensified the need for tools that can characterize newly synthesized nanoaterials with high sensitivity and with high spatial resolution. Many existing tools with nanoscopic resolution or better, including scanning electron microscopy (SEM), atomic force microscopy (AFM), and scanning tunneling microscopy (STM) methods, can generate highly detailed maps of nanoscopic structures. However, while these approaches provide great views of the morphological properties of nanomaterials, it has proven more challenging to derive chemical information from the corresponding images. To address this issue, attempts have been made to dress existing nanoscopy methods with spectroscopic sensitivity. A powerful approach in this direction is the combination of scan probe techniques with optical illumination, which aims to marry the nanoscopic resolution provided by a sharp tip with the chemical selectivity provided by optical spectroscopy. Examples of this approach include existing techniques such as scattering-type scanning near-field optical microscopy and tip-enhanced Raman spectroscopy. A new and emerging technique in this direction is photoinduced force microscopy (PiFM), which enables spectroscopic probing of materials with a spatial resolution well under 10 nm. In PiFM, the sample is optically excited and the response of the material is probed directly in the near-field by reading out the time-integrated force between the tip and the sample. Because the magnitude of the force is dependent on the photoinduced polarization in the sample, PiFM exhibits spectroscopic sensitivity. The photoinduced forces measured in PiFM are spatially confined on the nanometer scale, which translates into a very high spatial resolution even under ambient conditions. The PiFM approach is compatible with a wide range optical excitation frequencies, from the visible to the mid-infrared, enabling nanoscale imaging contrast based on either electronic or vibrational transitions in the sample. These properties make PiFM an attractive method for the visualization and spectroscopic characterization of a vast variety of nano materials, from semiconducting nanoparticles to polymer thin films to sensitive measurements of single molecules. In this Account, we review the principles of the PiFM technique and discuss the basic components of the photoinduced force microscope. We highlight the imaging properties of the PiFM instrument and demonstrate the inherent spectroscopic sensitivity of the technique. Furthermore, we show that the PiFM approach can be used to probe both the linear and nonlinear optical properties of nano materials. In addition, we provide several examples of PiFM imaging applications.

© 2015 AIP Publishing LLC. We perform time-resolved pump-probe microscopy measurements by recording the local force between a sharp tip and the photo-excited sample as a readout mechanism for the material's nonlinear polarization. We show that the photo-induced force is sensitive to the same excited state dynamics as measured in an optical pump-probe experiment. Ultrafast pump-probe force microscopy constitutes a non-optical detection technique with nanoscale resolution that pushes pump-probe sensitivities close to the realm of single molecule studies.

Advances in nanotechnology have intensified the need for tools that can characterize newly synthesized nanomaterials. A variety of techniques has recently been shown which combines atomic force microscopy (AFM) with optical illumination including tip-enhanced Raman spectroscopy (TERS), scattering-type scanning near-field optical microscopy (sSNOM), and photothermal induced resonance microscopy (PTIR). To varying degrees, these existing techniques enable optical spectroscopy with the nanoscale spatial resolution inherent to AFM, thereby providing nanochemical interrogation of a specimen. Here we discuss photoinduced force microscopy (PiFM), a recently developed technique for nanoscale optical spectroscopy that exploits image forces acting between an AFM tip and sample to detect wavelength-dependent polarization within the sample to generate absorption spectra. This approach enables ~10 nm spatial resolution with spectra that show correlation with macroscopic optical absorption spectra. Unlike other techniques, PiFM achieves this high resolution with virtually no constraints on sample or substrate properties. The applicability of PiFM to a variety of archetypal systems is reported here, highlighting the potential of PiFM as a useful tool for a wide variety of industrial and academic investigations, including semiconducting nanoparticles, nanocellulose, block copolymers, and low dimensional systems, as well as chemical and morphological mixing at interfaces.

We report on stimulated Raman spectroscopy and nanoscopy of molecules, excited without resonant electronic enhancement gain, and recorded using near field photon induced forces. Photon-induced interaction forces between the sharp metal coated silicon tip of an Atomic Force Microscope (AFM) and a sample resulting from stimulated Raman excitation were detected. We controlled the tip to sample spacing using the higher order flexural eigenmodes of the AFM cantilever, enabling the tip to come very close to the sample. As a result, the detection sensitivity was increased compared with previous work on Raman force microscopy. Raman vibrational spectra of azobenzene thiol and l-phenylalanine were measured and found to agree well with published results. Near-field force detection eliminates the need for far-field optical spectrometer detection. Recorded images show spatial resolution far below the optical diffraction limit. Further optimization and use of ultrafast pulsed lasers could push the detection sensitivity towards the single molecule limit.

Presented is a microscope design that employs two-photon non-linear excitation to allow the imaging of the fluorescence from almost any visible fluorophore at resolutions below 30 nm without changing filters or excitation wavelength. The ability of the microscope to image samples at atmospheric pressure, room temperature, and in solution makes it a very promising tool for the biological and materials science communities. The microscope demonstrates the ability to image topographical, far-field and near-field optical responses from the sample of interest. A single computer, simple custom control circuits, field programmable gate array (FPGA) data acquisition, and a simplified custom optical system are used. This versatility enables the end user to custom-design experiments from confocal far-field single molecule imaging to high resolution scanning probe microscopy imaging. Demonstrated is the far-field, topographic and near-field imaging using two-photon excitation of Bovine Pulmonary Artery Endothelial (BPAE) cells and J-aggregates (PVS Poly vinyl Sulfate) and PIC (Pseudo-Isocyanine) dye.

Scanning near-field optical microscopy (SNOM) employs many different forms of optical probes to achieve sub-diffraction limited imaging. The first commonly used probes utilized optical fibers pulled or etched to a small end diameter. This technique has successfully demonstrated spatial optical resolution better than 100 nm. These original near-field probes utilized a coating of aluminum on the sidewalls to achieve field confinement. The fabrication process had problems with irreproducible coatings (leading to blockage or leakage of light), insensitive scanning surface interaction mechanisms, or taper issues leading to low throughput. To overcome these issues, a probe design which involved illumination of sharp metals with optimal polarization was developed to achieve higher topographic and optical spatial resolution. This technique has been termed tip enhanced near-field scanning optical microscopy (TENOM). Although this technique overcomes many of the issues with using fibers, it introduces other issues. This work will cover how one overcome some of the issues with metal probes as well as presenting show our latest results.

We present the development of a versatile spectroscopic imaging tool to allow for imaging with single-molecule sensitivity and high spatial resolution. The microscope allows for near-field and subdiffraction-limited far-field imaging by integrating a shear-force microscope on top of a custom inverted microscope design. The instrument has the ability to image in ambient conditions with optical resolutions on the order of tens of nanometers in the near field. A single low-cost computer controls the microscope with a field programmable gate array data acquisition card. High spatial resolution imaging is achieved with an inexpensive CW multiphoton excitation source, using an apertureless probe and simplified optical pathways. The high-resolution, combined with high collection efficiency and single-molecule sensitive optical capabilities of the microscope, are demonstrated with a low-cost CW laser source as well as a mode-locked laser source.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

The increasing popularity of nanometrology and nanospectroscopy has pushed researchers to develop complex new analytical systems. This paper describes the development of a platform on which to build a microscopy tool that will allow for flexibility of customization to suit research needs. The novelty of the described system lies in its versatility of capabilities. So far, one version of this microscope has allowed for successful near-field and far-field fluorescence imaging with single molecule detection sensitivity. This system is easily adapted for reflection, polarization (Kerr magneto-optical (MO)), Raman, super-resolution techniques, and other novel scanning probe imaging and spectroscopic designs. While collecting a variety of forms of optical images, the system can simultaneously monitor topographic information of a sample with an integrated tuning fork based shear force system. The instrument has the ability to image at room temperature and atmospheric pressure or under liquid. The core of the design is a field programmable gate array (FPGA) data acquisition card and a single, low cost computer to control the microscope with analog control circuitry using off-the-shelf available components. A detailed description of electronics, mechanical requirements, and software algorithms as well as examples of some different forms of the microscope developed so far are discussed.