The extraction and upgrading of bitumen from oil sands have long been based on conventional methods. One of the major challenges associated with bitumen extraction is the extremely high viscosity caused by asphaltenes during its underground recovery. Asphaltenes are the higher molecular weight component of oil sands and responsible for organic deposits which cause major problems associated in the transport and upgrading of oil containing asphaltene. The aggregation dynamics of asphaltenes can be influenced by various external factors such as temperature, pressure, electrical fields, etc. In the present study, we investigate the physical properties of asphaltenes in bitumen and how temperature affects the morphology and mechanical properties of these components within the oil sands.
Atomic force microscopy is employed to study the structural changes in the morphology and physical characteristics of asphaltene aggregates as a function of temperature. The exotic fractal structure obtained by evaporation-driven asphaltene aggregates shows an interesting dynamics for a large range of temperatures from 25⁰C to 80⁰C. The changes in the topography, surface potential and adhesion are unnoticeable until 70⁰C. However, a significant change in the dynamics and material properties is displayed in the range of 70⁰C - 80⁰C, during which the aspahltene aggregates acquire ‘liquid-like&’ mobility and fuse together. This behaviour is attributed to the transition from the pure amorphous phase to a crystalline liquid phase which occurs at approximately 70⁰C as shown by using Differential Scanning Calorimetry (DSC). Additionally, the charged nature of asphaltenes and bitumen is also explored using kelvin probe microscopy. Such observations can lead to the development of a rational approach to the fundamental understanding of asphaltene aggregation dynamics and may help in devising novel techniques for the handling and separation of asphaltene aggregates using dielectrophoretic methods.

Oil sands are a mixture of sand particles, bitumen, and water that exhibit viscoelastic properties. The complex interplay of forces between the fluids (water and bitumen) and the solid particles (sand and clay) controls the recovery of bitumen from the oil sands, the subsequent removal of water from the tailings, and the overall energy efficiency of oil sands processing. However, the nanoscale structure-property relationships, reactivity, and transport properties of the interfaces are not well understood. Functional scanning probe microscopy offers an ideal tool to understand the fundamental processes occurring at the interfaces between fluids and solids in the oil sands. For example, the most polar components in oil sands bitumen form aggregated structures at a length scale of 5-10 nm, and these in turn attach to sand, clay, and water interfaces to influence all aspects of oil sands processing. Here we present scanning probe-based techniques which can provide in-situ information with nanoscale spatial resolution and with chemical specificity - which cannot be obtained using currently available techniques.

Photosystem I (PS I), the nano-scaled photosynthetic membrane protein complex, undergoes light activated (lambda;=680 nm) charge separation that drives unidirectional electron transfer. The highly efficient photo-electrochemical activity of PS I (100% quantum efficiency over 54% of solar spectrum) inspired our recent studies on tailoring uniform PS I monolayer on alkanethiolate self-assembled (SAM)/Au surfaces as the first step towards PS I incorporation into hybrid photovoltaic devices. We have shown the use of detergent-mediated colloidal chemistry and electric field assisted assembly to alter the surface attachment dynamics of PS I deposited from aqueous buffer suspensions. Furthermore, we use direct electrochemical characterization to elucidate photoactivated electron transfer by surface immobilized PS I monolayer on SAM (thiol)/Au surfaces for varying thiol chain lengths. The results reveal the effect of interfacial thiol brush density and chain length on limiting the electron transfer rates at the donor side from Au surface to oxidized reaction center of PS I. Our findings shine light, for the first time, on the critical role of dissolved O₂ in mediating the electron uptake rate at the acceptor side from reduced terminals of PS I to the solution, via soluble electron acceptor (Methyl Viologen, MV²⁺). Such studies provide valuable insight into the true bottleneck for photoactivated electron mediation through PS I monolayer, by analyzing the interfacial electron transfer at both donor and acceptor sides of PS I that was heretofore not clearly explained. Motivated by our promising results, we have directed recent research efforts in soft matter based bio-electronic devices towards understanding PS I-proteoliposome formation in lipid bilayer systems that mimic natural membrane based housing of PS I. To this end, we study detergent-mediated protein reconstitution in driving unidirectional arrangements of PS I trimeric complexes within lipid vesicles. We investigate the interaction of two nonionic detergents n-dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX-100) with two phospholipids, namely DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) and DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), that commonly constitute the natural thylakoid membrane housing of PS I. Optical density, dynamic light scattering (DLS) and advanced transmission electron microscopy (TEM) measurements (negative staining and cryo-TEM), in conjunction with site-specific tagging with quantum dots, reveal the mechanistic picture behind systematic, directional assembly of PS I-proteoliposomes as dictated by complex structural evolution that membrane bilayers undergo during detergent-mediated solubilization. The aforementioned studies provide insight into mimicking natural protein-phospholipid interactions for future fabrication of bio-hybrid devices.

Lignocellulosic biomass (LB) is a complex network of polymers that constitute plant cell walls (PCWs). It comes from various sources: residues from agriculture and forest or dedicated plants. Since LB is composed of various polymers such as cellulose, hemicellulose (polysaccharides) and lignin (polyphenols), LB transformation can potentially produce chemicals, materials and biofuels in dedicated biorefineries. Consequently, LB exploitation is considered as a way to limit greenhouse gas emission and a sustainable alternative to fossil carbon-derived products.
However, the architectural and chemical complexity of LB is also a bottleneck to its cost-effective industrial conversion. Today, to achieve this goal, not only the cellulosic part of LB but also the hemicellulosic and lignin parts must be retrieved and the relationships between the structure and the properties of the polymers should be well understood from the macroscopic to the nanoscopic scales.
The goal of this talk is to show the potentialities of atomic force microscopy (AFM) in different modes to realize the nanoscale characterization of plant cell walls and of bioinspired polymer films.
Thanks to the use of adhesion measurements via tip functionalization with lignocellulosic polymers, of nanoInfrared absorption, nanomechanical and nanothermal measurements, and by comparison between real plant cell walls and lignocellulosic bioinspired films, we will try to understand the relationship between the composition and the supramolecular organization of lignocellulosic polymers and their nanoscale properties.

Man-made nanomaterials could potentially pose a serious environmental concerns once they are released to the environment. Fluorescent Quantum dots (Qdots) are a class of ultra-small size (~ 1.5 nm - 12 nm size range) semi-conductor nanomaterials which find different applications in electronic and opto-electronic industries. Qdots are also used as probes for live cell imaging, tissue imaging, stem cell tracking and diagnostics applications. Photostability, high-quantum efficiency and size-tunable optical properties are unique characteristics of Qdots. While Qdot nanotechnology has been succesfully used in electronic and biomedical industries, very limited studies have been reported on the environmental transport and fate of these nanomaterials. There is a strong possibility that water-soluble Qdots could be readily uptaken by the plant and distribute systemically. Qdots could accumulate in plant tissue systems and might impact plant physiology. In this paper we studied the effect of ultra-small size (<5.0 nm) N-Acetyl cysteine coated fluorescent quantum dots (Qdots) on seed germination and seedling growth processes using a snow pea (Pisum sativum L.) model system. Qdot uptake by the seed and root systems were confirmed by the confocal and Raman imaging studies. Fluorescence confocal and Raman imaging results revealed that Qdots were localized on the surface seed coat, epidermis and intercellular regions. Interestingly, no acute Cd metal toxicity was observed upto Qdot concentration of 100 ppm. Interestingly, below 40 ppm concentration Qdots promoted seed germination and seedling growth processes.

In-vitro functional and structural studies of membrane proteins rely on suitable choice of synthetic membrane mimics (e.g., detergents and liposomes) that maintain the protein function. To this end, we investigate the interaction of two nonionic detergents n-dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX-100) with two phospholipids, namely DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) and DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), that commonly constitute the natural thylakoid membrane housing of Photosystem I (PS I), the photosynthetic protein complex. PS I, a supramolecular protein complex,functions as a biological photodiode and undergoes light-activated (lambda; = 680 nm) photochemical charge separation that results in unidirectional electron transfer .The highly efficient photo-electrochemical properties (100% quantum efficiency) of PS I motivates our on-going research towards successful incorporation of individually oriented PS I into their natural membrane bound structures in an effort to fabricate soft matter based solid-state bio-electronic or bio-hybrid photovoltaic devices. We use optical density and dynamic light scattering (DLS) measurements in conjunction with advanced transmission electron microscopy (TEM) techniques (namely, negative staining and cryo-TEM) to study the detailed mechanistic picture behind processes that drive membrane bilayers to undergo complex structural arrangements during different stages of detergent-mediated solubilization. Specifically, negative staining and cryo-TEM images are used to investigate the solution-phase morphological arrangement of PS I-proteoliposomes. These characterizations provide a fundamental understanding of the role of detergent-mediated protein reconstitution in driving unidirectional arrangements of PS I trimeric complexes within lipid vesicles during PS I-lipid interactions in colloidal solutions. Furthermore, orientations of PSI within the liposomes are investigated by fluorescence microscopy. In turn, the aforementioned studies provide valuable insight into mimicking the naturally occurring protein-phospholipid interactions in cell membranes. In future, these bio-mimetic systems shall facilitate easy incorporation of membrane protein-lipid complexes into novel bio-hybrid devices.

Cellulose, the basic structural component of plants, is of fundamental importance in plant biology, bioenergy production and engineering of nanocomposite materials. Critical to cellulose function are its hydration and mechanical properties. We systematically investigate how cellulose rigidity and dynamics vary with temperature and hydration and find cellulose nanocrystals to stiffen when hydrated to ~20% by weight. This finding is explained by cellulose interaction with the solvent, rendering chains at its core less dynamic and more ordered than surface chains. New insights are thus obtained on how cellulose provides rigidity to plants and suggest that inclusion of modest amount of water may enhance the mechanical properties of cellulose nanocomposite materials.

Single-molecule biophysical tools are making it possible to measure the response of individual biomolecules to external force - in real time and with unprecedented resolution - revealing information that is typically lost when studied though traditional “bulk” methods. I will present a general analytical theory of force-induced molecular transitions. The theoretical procedures of interpreting experimental data will be illustrated with (i) unfolding of riboswitches and (ii) unbinding of a receptor-ligand complex involved in blood clot formation with optical tweezers, (iii) unzipping of individual DNA hairpins with nanopores, and (iv) unfolding of proteins with an atomic force microscope.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy is a prevalent tool for the exploration of individual (bio)molecules, providing exquisite information on many molecular-level processes.¹ For example, proteins, DNA, polysaccharides, supramolecular polymers and polyelectrolytes have been investigated, revealing details about the strength of intramolecular interactions, folding and unfolding pathways, mechanics, conformational changes, reactivity, kinetics, etc.
For each particular system under investigation, the experimental design is a decisive phase that often involves a multistep chemical protocol, including grafting, derivatization, coupling, (de-)protection, and other functionalization reactions. Procedures of sample preparation are often complex and time-consuming. Hence, there is a need for new general platforms allowing for straightforward sample preparation adapted to single-molecule studies, i.e. a tight attachment to both the substrate and the tip, and a low density to favor single-molecule detection.
We report here on the use of poly(ethylene oxide) (PEO) as a tether to probe various properties of individual molecules. The polymeric linker acts as a handle that stably binds to the AFM tip. The simple adsorption of poly(ethylene oxide) to the tip is versatile and provides an appropriate system configuration for the investigation of many different biological and synthetic molecular systems. To attest for this versatility and adequacy with advanced single-molecule investigation, we present different examples of PEO-mediated studies about the unfolding of a synthetic peptide, the mechanochemical behavior of a molecular machine and finally the stability of a metallo-supramolecular complexed polymer.
More generally, this method based on non-covalent sorption of PEO on an AFM tip, can be implemented in a wide range of solvents, for the study of many intra- or intermolecular phenomena at the single-molecule level over orders of magnitude of force loading rates. Connecting PEO tethers to a very broad variety of (bio)molecules is a facile and versatile route. The commercial availability of many different functional PEOs makes this tethering strategy even more accessible.
1. A.-S. Duwez and N. Willet, Molecular Manipulation with Atomic Force Microscopy, CRC Press, Boca Raton, 2011.

A recent development which adds nanoscale chemical analysis to Atomic Force Microscopy (AFM) is the integration of a tunable infrared source with an AFM [1], referred to as AFM-IR. The AFM-IR technique makes use of the nanosecond pulsed IR source to induce a rapid expansion of the sample caused by the IR absorption. This expansion causes the AFM probe to briefly oscillate at its resonant modes. The measured amplitude directly correlates to the local absorption coefficient of the sample in proximity to the AFM probe. The amplitude, for thin materials, is an integration of the absorption through the thickness of the material allowing analysis of subsurface components as demonstrated by measurements of structures internal to cells and bacteria [2].
In addition to the chemical information and the topography information provided by the AFM, the technique can also provide simultaneous mechanical information about the sample. This is accomplished by analyzing the frequency of the mechanical resonances of the cantilever generated by the IR pulse, in a mode typically called Contact Resonance AFM [3, 4]. The contact resonance of an AFM cantilever has been shown to correlate well with the storage modulus of the sample [5]. The IR light produces a clean excitation of the cantilever allowing a more accurate measurement of the frequency as well as inducing oscillation of a number of modes of the cantilever providing for measurements over a broad range of sample stiffness.
The IR source can tune over a broad range of the mid IR (900 - 3600 cm^-1) producing a spectrum which correlates well with traditional spectra from transmission FTIR and allowing the analysis of a number of different absorption bands important to characterizing biological materials. The AFM-IR technique has been used to analyze a large number of biological as well as polymeric materials. Recent applications showing the benefit of the nanoscale chemical measurements, in addition to the simultaneous multimodal analysis will be discussed. These applications include measurements of bacterial components, lipid distribution in hair and skin [6] as well as protein structure in membranes.
[1] Dazzi, A., et al., Optics Letters 30, 2388-2390 (2005).
[2] Deniset-Besseau, A., et al., The Journal of Physical Chemistry Letters, 5 (4) 654-658 (2014).
[3] Yamanaka, K. and S. Nakano, Applied Physics A: Materials Science & Processing 66(0): S313-S317, (1998).
[4] Rabe, U., et al., Applied Physics A: Materials Science & Processing 66(0): S277-S282, (1998).
[5] Yuya, P.A., et al., Journal of Applied Physics 104(7): 074916-7, (2008).
[6] Marcott, C., et al., Applied Spectroscopy, 68 (5) 564-569 (2014).

LRP-1 is a transmembrane receptor mediating the clearance of proteinases which degrade the extracellular matrix in cancer progression. As such, it was considered as a target against cell invasion. In addition to this role, LRP-1 is known to regulate the cell-to-matrix adhesion-deadhesion balance.
How this latter role could affect the widely acknowledged anticancer potential of LRP-1 remains unclear. LRP-1 silencing has indeed been shown to increase pericellular proteolytic activities, which are correlated with an increase in tumor cell invasion. However, our recent data involving a human thyroid carcinoma FTC-133 cell line have provided evidence that cell invasion is decreased by LRP-1 silencing.
In this work, we have monitored the effects of LRP-1 silencing by a short hairpin-RNA (shRNA) strategy in a FTC-133 cell line using optical microscopy, live cell videomicroscopy and atomic force microscopy (AFM). We found that dramatic modifications in the morphological phenotype of LRP-1-silenced cells exhibited by high-speed AFM are correlated with an increased Young&’s modulus, when a low-stiffness phenotype is usually observed for cancer cells with greater migration capabilities. This mechanical phenotype is associated with a decreased cell velocity and altered migration patterns. By using transfection of FTC133 cells with talin and integrin subunits fused with fluorescent proteins, we have investigated the changes in elastic modulus measured by AFM at sites where talin/integrin clustering takes place - visualized by epifluorescence microscopy.
These analysis were complemented bymolecular recognition measurements thanks to tip functionalization with antibodies directed against integrins subunits, and with specific substrat peptides.

Atomic force microscopy (AFM) is a powerful tool capable of imaging and chemical characterization of bio-samples at molecular resolution in physiologically relevant environments. However, the localized tip-sample interactions limit high-resolution images to the topmost layer of samples, which hinder the characterization of the three-dimensional (3-D) inner structures of biomolecules. We have developed an AFM based approach for 3-D localization of chemical groups within a single protein complex. We use short DNA sequences to label specific chemical regions within a protein complex. T-shaped cantilevers with complementary DNA probes allow locating each label with sequence specificity and sub-nanometer resolution. We then convert measured pairwise distances between labels into the 3-D loci of the target chemical groups using simple geometric calculations. Imaging of biotin-streptavidin complexes showed that the 3-D loci of carboxylic acid moieties of biotins are within close proximity of their respective 3-D loci in the corresponding crystal structure.

We investigate the thermomechanical properties of Poly(p-phenylene vinylene) (PPV). Conjugated PPV films were prepared by thermolytic conversion of poly[p-phenylene (tetrahydrothiophenium)ethylene chloride] precursor films at different temperatures. The reaction kinetics was investigated by isothermal thermogravimetry and the reaction was found to follow first order kinetics with a low activation energy of about 59 kJ mol^minus;1. Film processing conditions that result in conjugated polymer films free of carbonyl defects, were found. Mechanical properties of the films showed a dependence on the extent of conversion and chemical composition of the films. Storage modulus (Eprime;) and plasticity index (Psi;) decreased with increase in conversion, whereas opposite trend was observed in loss modulus (EPrime;). Fully converted PPV polymer was found to have storage and loss moduli of 4.4 GPa and 0.2 GPa (room temperature), respectively. Temperature dependent mechanical properties of PPV on the nanometer scale were investigated using high temperature nanoindentation. High temperature results revealed significant changes in mechanical properties near glass transition. Sharp drops in reduced modulus, and storage modulus of the conjugated PPV polymer films were observed in the glass transition region. In addition to viscoelastic properties such as storage modulus, loss modulus, and loss tangent, in-depth analysis of time dependent behavior, i.e. indentation stress relaxation, was also performed at different temperatures with the use of appropriate viscoelastic model. Stress relaxation measurements show that the normalized equilibrium modulus increased with temperature.

The elegance of biological systems lies in their hierarchical organization; programmed at the molecular scale but its manifestation occurs across varied length-scales with diverse functional characteristics. This hierarchical architecture is often affected by the environment & local perturbations; the overall system functions akin to a well-conducted melodious symphony. This elegance of complexity & inter-connected length-scales & functionalities of biological systems makes it exceedingly difficult to characterize the various components & their functional connectivity.
Various tools & techniques have evolved in the past couple decades that are able to address the nature & function of isolated components; e.g. a particular protein structure or structural folding pattern or GFP imaging of binding or other functional characteristics of biomolecules. Because the various components occur at varied length-scales & have diverse characteristics, we have been developing correlative & fluidic-cell microscopy/characterization as an integrative approach to address the structural complexities & functional characterization of biological systems.
We have developed fluidic-cells for imaging biomolecules & their assembly under physiologically viable conditions with AFM & electron microscopy. For example, utilizing fluidic-cell AFM, we are able to image biomolecules such as A-beta oligomers; believed to be responsible for early onset of Alzheimer&’s disease (AD). The specific cluster formation of A-beta oligomers is imaged directly in their fluidic state to understand the assembly processes. At the same time, we use magnetic nanostructures (MNS) as “tags” for enhanced MR imaging (T2 contrast) of the same A-beta oligomers in-vitro (& in-vivo) to investigate their spatio-temporal evolution & localization in the brain. Collectively, microscopy & characterization provide some of the critical spatio-temporal links necessary for form-function relationships.
In another system, we have investigated endothelial cells (EC) which form the lining of the pulmonary vascular system. It acts as a semi-permeable barrier between blood & the interstitium of the lung, & regulates a variety of functions. Probing the human pulmonary artery endothelial cells (HPAEC) in terms of coupled adhesion & deformation properties using AFM force mapping; the wider distribution & finer details of cytoskeletal structural re-arrangements of the same sample were imaged using fluorescence microscopy & scanning transmission electron microscopy (STEM). This hierarchical structural organization was examined as a function of their response to the EC barrier disrupting agent, thrombin, & barrier enhancing agent, sphingosine 1-phosphate (S1P). We directly link, for the first time, the EC cell remodeling & cytoskeletal mechanical properties spatially & temporally using high-resolution correlative multi-microscopy methods; provides novel insights into the biomechanical function of the endothelial barrier.

Recent work on the physical-chemistry of polyelectrolyte brushes is presented. The water content of “ grafted from” brushes synthesized by the Atomic Radical Transfer Polymerization, is determined combining elipsometry and quartz crystal microbalance techniques. Water content was measured for brushes of poly[2-(methacryloyloxy)ethyl] trimethylammonium chloride (PMETAC) and poly(potassium sulfopropyl methacrylate) (PSPM) , varying initator density. Ion-paired collapsed PMETAC brushes in NaClO₄ result in compact stiff structures with less amount of entrapped water. Highly charged dense poly(sulfopropyl methacrylate) polyelectrolyte brushes were indented with an atomic force microscopy tip as well as with an 8 mu;m silica colloidal probe at different ionic strengths ranging from Millipore water to 1 M NaCl. The force response during indentation was fitted to a phenomenological equation analogous to the equation of state of a compressible fluid. In this way, internal energy and brush thickness were obtained as a function of ionic strength. Long-range forces decayed exponentially with distance with characteristic decay lengths much larger than the Debye screening lengths at the respective ionic strengths. It was therefore concluded that long-range repulsion was due to compression of a loose corona of polymers in front of the dense part of the brush. Molecular transport in brushes was studied by electrochemical and fluorescence techniques. Combined use of electrochemical impedance spectroscopy, cyclic voltammetry and quartz crystal microbalance with dissipation allowed to resolve separately the thermal effects on diffusion and electron-transfer steps of the electrochemical reaction of the [Fe(CN) ₆]^3-/4- redox couple at a Au electrode modified with PMETAC brushes. Arrhenius-type dependences of the kinetic constant and the diffusion coefficient with temperature were observed in different electrolytes. A thermal transition for PMETAC brushes in the presence of ClO₄^- ions at near-ambient temperature (sim;17 C). Activation energies for electron-transfer and diffusion processes become twice as large as those for temperatures above the thermal transition. The zeta;-potential of spherical brushes of PMETAC and PSPM was measured as a function of ionic strength. The zeta;-potential of PMETAC brushes varies from +30 mV at 10 mM NaCl to +20 mV at 200 mM, while for PSPM brushes changes from -30 mV to -25 mV, at their respective ionic strengths. This unusual weak dependence of the zeta;-potential on ionic strength is quantitatively explained on the basis of the responsiveness of brushes toward changes in the ionic strength as well as taking into account the specific hydrodynamics of the hairy brush solution interface.

We measured the shape change of periodic ridge surface profiles in gelatin organogels resulting from deformation driven by their solid-vapor surface stress. Gelatin organogel was molded onto Poly-dimethylsiloxane (PDMS) masters having ridge heights of 1.7 and 2.7 µm and several periodicities. Gel replicas were found to have a shape deformed significantly compared to their PDMS master. Systematically larger deformations in gels were measured for lower elastic moduli. Measuring elastic modulus independently, we estimate a surface stress of 107±7 mN/m for the organogels in solvent composed of 70 wt% glycerol and 30 wt% water. Shape changes are in agreement with a small strain linear elastic theory. We also measured the deformation of deeper ridges (with height 13 µm), and analysed the resulting large surface strains using finite element analysis.

Proteins and virus nano particles can be seen also as important building blocks for the construction of hierarchical materials. The behavior of these biomolecular systems in crystallization, self-association and molecular recognition, is determined by the long range forces between them. They include electrostatic interaction, which arises from the net charge on the particle, polar interaction that depends on the dipolar and multipolar distribution of charges on the surface of the body, and the van der Waals-London interaction, which arises from the dielectric material response properties. Understanding and tracking the variation of long range interactions under different solution conditions facilitates the manipulation and design of novel hierarchical structures.
Among many methodologies that are used to determine intermolecular long range interactions, composition-gradient multi-angle static light scattering (CG-MALS) turns out to be a promising emerging experimental method. Equipped with the automated fluctuation-free sample mixing and delivery system, CG-MALS is able to effectively characterize the osmotic second virial coefficient of any molecular solution, which directly relates to the long range interactions between molecules via the standard statistical mechanical theory.
Here we first use CG-MALS system with Calypso and Dawn Heleos II tools to measure the second virial coefficient of bovine serum albumin (BSA), a standard protein for the interpretation of second virial coefficient values. It was measured as a function of pH (3.07-6.68) and ionic strength (25 - 150 mM NaCl) in Na₂HPO₄-citric acid buffer. Proteins such as BSA are prone to form aggregates, therefore Fast Protein Liquid Chromatography was utilized to characterize the percentage of monomers and aggregates in the biomolecular solution. In addition, the variation of the hydrodynamic size and zeta potential versus solution environments were characterized by dynamic light scattering. Comparison of CG-MALS with traditional experimental techniques indicates that CG-MALS is an effective and precise method for determining the A2 of molecules. The systematic variation of the second virial coefficient under the various solution conditions reveals changes in the net charge and the isoelectric point of BSA.
We next target also larger biomolecular aggregates such as the cowpea mosaic virus (CPMV) and PEGylated CPMV (CPMV-PEG2000) capsids. CPMV serves as a promising nanoparticle for the application in biomedicine and energy conversion/storage devices, and PEGylation has been proved to be an effective strategy to change the properties of bio-nanoparticles. The second virial coefficient of CPMV and CPMV-PEG2000 were again measured under a variety of pH and ionic strength conditions. We observe that PEGylation has a significant influence on the solubility and long range interactions between CPMV particles.

Imaging techniques for the features deeply buried under the surface are significantly important in various fields. Ultrasonic pulse echo imaging, magnetic resonance imaging and X-ray computed tomography have been widely applied to medical diagnosis. Atomic force microscopy (AFM) is a powerful technique not only for obtaining surface topography, but also for investigating various surface properties, including viscoelasticity, with nanometer-scale resolution. Moreover, it has recently demonstrated by several researchers that the AFM techniques are also applicable for imaging subsurface features. Among these studies, we visualized Au particles of 50 nm in diameter, which were deeply buried in a polymer matrix (up to 950 nm underneath the surface)[1], using techniques that have been used for viscoelastic imaging, such as heterodyne force microscopy (HFM) and ultrasonic atomic force microscopy (UAFM). The Au particles dispersed on a thick polyimide film (125 micro-meter in thickness), covered with photo-polymer film were clearly visualized. As far as our experiments, subsurface visualization as mentioned above has been successful only when the Au particles were buried in a soft matrix. We presume that such visualization was brought possible because of the difference in the viscosity or elasticity between the Au particle and polymer matrix. However, it has been difficult to clarify which of these differences plays a role in the imaging mechanisms by HFM and UAFM studies.
In this study, we utilized resonance tracking ultrasonic AFM (RT-UAFM), where both the contact resonance frequency shift and amplitude variation are simultaneously mapped over the scanning area using phase locked loop circuit[2,3], to independently characterize contact elasticity and viscosity. Applying RT-UAFM is a new approach to the subsurface visualization.
In this presentation, we will show subsurface images of above-mentioned Au particles buried in a soft polymer matrix obtained by RT-UAFM. The Au particles buried in the polymer matrix with a depth of 900 nm from the surface were clearly imaged by RT-UAFM. We will discuss the origin of subsurface visualization in the soft matrix by comparing the images obtained by RT-UAFM and UAFM on the same areas.
References
[1] K. Kimura, K. Kobayashi, K. Matsushige, and H. Yamada, Ultramicroscopy 133 (2013) 41-49.
[2] K. Yamanaka, Y. Maruyama, T. Tsuji and K. Nakamoto, Appl. Phys. Lett., 78 (2001) 1939-1941.
[3] K. Kobayashi, H. Yamada and K. Matsushige, Surf. Interf. Anal., 33 (2002) 89-91.

Scanning probe microscopy (SPM) may be the best way to image the surfaces with ultimate near-atomic lateral resolution, but its ability to look under the immediate sample surface is inevitably limited. At the same time, ultrasound is well known for its ability to penetrate objects and imaging internal body organs and defects in semiconductor wafers on millimiter to tens of micrometer length scales. Whereas Ultrasonic and Heterodyne Force Microscopies (UFM and HFM) [1,2] and other ultrasonic SPM methods [1,2] use ultrasound to achieve nanoscale resolution down to 10^-9 m while preserving subsurface imaging capabilities [3-5], questions still remain on
a) how deep one can observe the nanoscale features using these methods,
b) what is the achievable lateral resolution for subsurface features, and
c) what are the physical mechanisms and the role of phase and amplitude detection in subsurface UFM/HFM imaging.
Moreover, the true subsurface imaging in solid state nanostructures composed of stiff materials has yet to be reliably demonstrated, and misconceptions exist as to how the propagation of ultrasonic waves with wavelength 10⁵ -10⁶ times larger than the imaged features can contribute to subsurface imaging.
Here we use UFM images to produce unambiguous subsurface images with 5 nm lateral resolution of internal morphology of high stiffness solid state nanostructures - iii-v semiconductor quantum dots hidden under atomically flat capping layer. We then explore stacks of atomically layered two-dimensional (2D) materials such as graphene, MoS₂, Bi₂Se₃ of varied thickness using wide range of ultrasonic frequencies from kHz to several MHz. This reveals effects of residual stresses in supported graphene layers, and explores nanomechanical behaviour of few layer graphene, MoS₂ and Bi₂Se₃ films as well as visualizes nanoelectromechanical phenomena in these 2D materials [6]. By directly observing the transition of for few layer graphene sheets deformation from plate to stretched membrane behaviour, we create nanoscale maps of shell instability, providing insight to the stresses in the free standing 2D films.
Finally, by analysing the UFM and HFM imaging process, we show that subsurface imaging mechanisms in both are indeed linked to the elastic field produced by the indention of dynamically stiffened cantilever-tip system coupled with the detection of vibrations via nonlinear tip-surface interactions, with phase information providing much less significant contribution. Further expansion of this methodology, challenges and potential applications are also discussed.
References.
[1] Kolosov, O. V. and Yamanaka, K. JJAP, 32, (8A), L1095-8, (1993).
[2] M T Cuberes et al J. Phys. D: Appl. Phys. 33 2347, (2000).
[3] Yamanaka, K., H. Ogiso and O. Kolosov, APL 64(2) :178-80, (1994);
[4] Diebold, A. C., Science 310 (5745): 61-62, (2005).
[5] Tetard, L. et al, Nature Nanotechnology 5 (2), 105-9 (2010).
[6] Kay, N. D. et al, Nano Letters 14, 3400-4 (2014).

Understanding solid-liquid thermal interactions is crucial to a multitude of applications in modern science
and technology. However, the fundamental interactions at the interface between a solid and a liquid are difficult to quantify due to the nanoscale spatial scales coupled with strong influences from structure and chemistry. To glean novel insight into the fundamental interfacial physics behind these interactions, we use picosecond acoustic analysis, in which a short optical pulse is absorbed by a sample resulting in rapid thermal expansion and strain wave propagation. The strain wave reflects off varying interfaces, and the resulting pump-probe signal is related to the transmission of the strain wave across an interface along with dissipation of this wave in the sample. In this study, we use picosecond acoustic analyses to measure the transmission of this thermally-induced strain wave across various solid/liquid interfaces, giving insight into nanoscale wetting of these systems. Our picosecond acoustic measurements indicate that the thermo- mechanical interactions of the acoustic vibrations in the solid are limited by nearly 60% in certain liquid systems. The a solid confined by a highly wetted liquid also exhibits damage at a much lower threshold; allowing permanent lattice distortion at fluence levels ranging from 2.5-10 times less than that of a free solid- air surface. Furthermore, comparison of exemplary liquids with varying contact angles - water and fluorine- based fluids - indicate that despite phenomenological classification of being highly wetting, there is actually decreased thermo-mechanical interaction between a solid-fluorofluid as compared to a solid-water system. These findings not only help to further understand the nano-scale dynamics of energy transfer at solid-liquid interfaces, but begin to justify and guide a discussion of a redefinition of conventional wetting metrics such as contact angles in order to define how liquids meet a solid surface, particularly on the nanoscale.

Imaging subsurface structures with nanometer resolution has been a long#8208;standing desire in science and industry in particular and microscopy in general. To obtain subsurface information one usually applies ultrasound, like e.g. in echocardiography. The implementation of ultrasound in an Atomic Force Microscope (AFM) gives access to additional information of the sample, which enables, under certain conditions, the imaging of subsurface structures with nanometer resolution. The most promising candidate for imaging deeply buried objects or structures with nanometer resolution is based on a special excitation scheme, which makes use of two ultrasound excitations: one through the sample and the other through the cantilever. This technique is called Heterodyne Force Microscopy (HFM). Despite some reported subsurface observations that clearly demonstrate the power of this technique, a decent quantitative understanding of the physical contrast mechanism was (until now) still missing.
This talk focuses on the poorly understood elements in Heterodyne Force Microscopy. We studied the ultrasound propagation in the sample [1], the dynamics of an ultrasonically excited cantilever near a sample that is vibrating at a slightly different frequency [2, 3], and the generation of the heterodyne signal [4]. This recent insight in the basic working principles of HFM [1#8208;4] enabled us to perform a quantitative analysis of our measurements on a well#8208;characterized sample, which contained 20nm large gold nanoparticles buried more than 100nm deep underneath a soft polymer matrix. We demonstrate not only the subsurface imaging capability, but we also determine the exact physical contrast mechanism. Totally unexpected, the contrast is neither related to ultrasonic Rayleigh scattering nor elasticity variations in the sample, but to the rattling motion (and the involved friction) of shaking nanoparticles [5].
[1] G.J. Verbiest, J.N. Simon, T.H. Oosterkamp, and M.J. Rost, Nanotechnology 23, 145704 (2012)
[2] G.J. Verbiest, T.H. Oosterkamp, and M.J. Rost, Ultramicroscopy 135, 113 (2013)
[3] G.J. Verbiest, T.H. Oosterkamp, and M.J. Rost, Nanotechnology 24, 365701 (2013)
[4] G.J. Verbiest and M.J. Rost, Nature Physics submitted
[5] G.J. Verbiest and M.J. Rost, http://arXiv/abs/1307.1292

Membranes of all cells contain a significant fraction of phospholipids arranged in a self-assembled bilayer. This lipid bilayer forms a robust barrier to ions, proteins and other molecules, and separates the inside of the cell from the outside, or compartmentalizes the cells. Embedded in the lipid bilayers are a large variety of glycoproteins, globular proteins, and membrane channels[1]. The way these molecules perform their functions is strongly dependent on their location and the mechanical and chemical properties of the lipid environment around them. In cellular membranes, it is believed that lipid rafts (small lipid domains enriched in cholesterol) act to hold membrane proteins together in a dynamic fashion[2,3]. The difference in the stability between the lipid rafts and the fluid bilayer is believed to be very small.
The stability and rigidity of lipid bilayers depends on the type of lipid, temperature, and the presence of cholesterol amongst other factors. Many naturally occurring lipids exhibit phase transition around room temperature, which drastically alters the membrane&’s stability. In nature, lipid membranes consist of mixtures of many different types of lipids, proteins, and other molecules. The inhomogeneity of these membranes result in spatially distributed differences in stability[4-6] . Here we present a method to measure this inhomogeneity in the stability of lipid membranes with nanometer resolution, by using atomic force microscopy induced phase transition.
We use a combination of global heating and local mechanical work to induce local phase transitions in model lipid membranes. In a modified AFM, we control the local temperature using laser heating, and locally apply mechanical energy using Peak Force Quantitative Nanoscale Mapping. By varying these two parameters we can locally induce phase transitions.
Using this method, we have revealed a region of increased stability at the domain boundaries in a binary mixed system of phospholipids, DLPC and DPPC, in the absence of cholesterol. In the presence of cholesterol, additional nanoscale domains exist inside the predominantly DPPC patches of the mixed lipid bilayer. These domains have increased stability and sizes comparable to those postulated for lipid rafts in cell membranes.
This technique provides the first direct method to image and investigate stable domains on the length scale of lipid rafts. We believe that this technique will provide complementary information to fluorescence and NMR techniques which have been the only indirect methods available to date.
[1] K. Simons, W.L.C. Vaz, An. Rev. Biophys. Biomol. Struct. 33,2004 269
[2] D. Lingwood, H.-J. Kaiser, I. Levental, K. Simons, Biochem. Soc. Trans. 37,2009 955
[3] D. Lingwood, K. Simons, Science 327,2010 46
[4] G.W. Feigenson, J.T. Buboltz, Biophys. J. 80,2001 2775
[5] T. Baumgart, S.T. Hess, W.W. Webb, Nature 425,2003 821
[6] S.L. Veatch, S.L. Keller, Biophys. J. 85,2003 3074

This talk describes nanomechanical property measurements based on Loretz force actuation of heated atomic force microscope (AFM) cantilever tips. Electrical current passing through a U-shaped cantilever in the presence of a magnetic field induces a Lorentz force on the cantilever, resulting in cantilever actuation. This same current flowing through a resistive heater induces a controllable temperature rise. When the tip is in contact with a surface, it is possible to control the temperature at the nanometer-scale contact between the heated tip and the surface. When a periodic force is applied to the tip, the periodic substrate response can indicate the elastic and viscous properties of the substrate material. While quantiative measurements of mechanical properties using AFM still remains a challenge, we present progress on the control of the force and temperature at the AFM tip. We present cantilevers designed for significantly higher actuation forces, compared to previously available self-heating cantilevers. Because of the unique cantilever design, the magnetic force acts on the cantilever free end, near the cantilever tip. The Lorentz actuation cantilever supplies clean, wide bandwidth actuation that is free of parasitic resonances. We demonstrate this actuation mechanism applied to local measurements of temperature-dependent material softening. Our ultimate goal is to measure nanometer-scale elastic modulus and viscosity as a function of temperuture, over a wide range of temperature and actuation frequency.

Streptomyces, filamentous soil bacteria, are well known for their ability to produce antibiotics and other molecules useful in medicine or agriculture. Under specific growth conditions some strains can store an excess of carbon into TriAcylGlycerols (TAGs), a direct precursor for Bio-diesel ^{1, 2}. Streptomyces is thus an interesting canditate to generate bio-oils by fermentation ³.
Our goal is to evaluate, at the subcellular scale, the size/shape and localization of storage lipid inclusions in different Streptomyces strains by using a combination of atomic force microscopy and infrared spectroscopy (AFM-IR).
AFM-IR technique is a user-friendly benchtop technique that enables infrared spectroscopy with a spatial resolution well below conventional optical diffraction limits. It acquires IR absorption imaging spectrally resolved with lateral resolution down to 100 nm ^{4, 5}.
For the study of the local repartition of TAGs inside the cells, AFM-IR was employed to create sub-cellular chemical maps that allows label-free identification of TAGs inclusions in Streptomyces cytoplasm. This was possible since TAGs molecules show a specific response in the mid-infrared region, quite distinct from that of the other cellular constituants (C=O stretching of the esters at 1741 cm^-1).
Hence, the AFM-IR technique is likely to provide new insights into the constitution of the fatty inclusions and the role of TAGs in the morphological and metabolic differentiation processes that characterize Streptomyces developmental cell cycle.
1. Holmbäck, M.; Lehestö, M.; Koskinen, P.; Selin, J. Process and Microorganisms for Production of Lipids. WO2011/148056A1, 2011.
2. Packter, N. M.; Olukoshi, E. R.; Tag, A. Ultrastructural Studies of Neutral Lipid Localisation in Streptomyces. Arch. Microbiol. 1995, 164, 420-427.
3. Deniset-Besseau, A.; Prater, C.; Virolle, M-J. and Dazzi, A. Monitoring TriAcylGlycerols Accumulation by Atomic Force Microscopy Based Infrared Spectroscopy in Streptomyces Species for Biodiesel Applications. J. Phys. Chem. Lett., 2014, 5 (4), pp 654-658
4. Dazzi, A.; Glotin, F.; Carminati, R. Theory of Infrared Nanospectroscopy by Photothermal Induced Resonance. J. Appl. Phys. 2010, 107, 1-7.
5. Lahiri, B.; Holland, G.; Centrone, A. Chemical Imaging beyond the Diffraction Limit: Experimental Validation of the PTIR Technique. Small 2013, 9, 439-45.

Neaspec&’s near-field optical microscopy systems (NeaSNOM) allow to overcome the diffraction limit of classical optical microscopy and spectroscopy enabling optical measurements at a spatial resolution of 10nm not only at visible frequencies but also in the infrared or terahertz spectral range.
Scattering-type Scanning Near-field Optical Microscopy (s-SNOM) [1] employs an externally-illuminated sharp metallic AFM tip to create a nanoscale hot-spot at its apex. The optical tip-sample near-field interaction is determined by the local dielectric properties (refractive index) of the sample material and detection of the elastically tip-scattered light yields nanoscale resolved near-field images simultaneous to topography.
Development of a dedicated Fourier-transform detection module for analyzing light scattered from the tip which is illuminated by a broadband laser source enabled IR spectroscopy of complex nanostructures (nano-FTIR) [2]. Identification of polymers [2], analysis of embedded structural phases in biominerals [3], or investigation of the secondary structure of individual protein complexes [4] demonstrated the successful application of nano-FTIR as an material characterization technology.
Equipping NeaSNOM systems with cw-light sources near-field imaging can be performed at time scales of 30-300s per image. Patented signal detection and analysis technology allows i.e. investigation of phase change materials, analysis of Graphene nanostructures [5], or to study energy storage materials in near-field amplitude and phase at unprecedented scanning speed and signal quality.
The patented modular system design enables tailored system configurations where the ultimate spectral coverage can be achieved by using synchrotron-based broadband IR light sources [6]. Based on reflective optics design of the system novel time-resolved near-field measurements [7] or the integration of THz-TDS systems have been realized.
[1] F. Keilmann, and R. Hillenbrand, Phil. Trans. R. Soc. Lond. A362, 787 (2004)
[2] F. Huth, et al., Nano Lett.12, 3973 (2012)
[3] S. Amarie, et al., Beilstein J. of Nanotech.3, 312 (2012)
[4] I. Amenabar et al., Nature Comm. 4, 2890 (2013)
[5] J. Chen et al., Nature 487, 77 (2012); and Z. Fei et al., Nature 487, 82 (2012)
[6] P. Herrmann, et al., Optics Expr. 21, 2913 (2013)
[7] M. Wagner, et al., Nano Lett. 14, 894 (2014)

NaugaNeedles has developed a set of nanofabrication tools to selectively grow inter-metallic ordered phases of silver-gallium (Ag₂Ga) nanoneedle at any selected location (e.g. atomic force microscope probe) [1]. We have found that single Ag2Ga nanostructures can be induced to grow from silver coated surfaces if contacted with gallium. These needles can be fabricated between 1 and 100 µm in length and from 50 to 500 nm in diameter [1,2]. The orientation of the growth direction can be controlled within a few degrees. We have been able to grow Ag₂Ga nanoneedles on several different types of substrate including AFM probes, tungsten probes, and tuning forks.
Due to several desirable qualities such as their precise and uniform nanoscale dimensions, excellent mechanical [3] and chemical stability, and high electrical conductivity, NeedleProbes are already used for several applications including; Imaging high aspect ratio structures, electrical characterization [4], liquid probing [2], nanoindentation on soft materials [5], scanning electrochemical microscopy (SECM) with AFM [5] , scanning tunneling microscopy (STM), nano-probing inside SEM, tuning fork based AFM, mass sensing using ultra sensitive nanocantilevers [7,8].
In addition, NaugaNeedles successfully demonstrated the feasibility of the batch fabrication method for these types of probes [9]. In this presentation, the technology platform and some of the applications of the NeedleProbes mentioned above will be presented
[1] M. Yazdanpanah, et. al., Journal of Applied Physics. 98(7), (2005), 073510
[2] M. Yazdanpanah, et. al., Langmuir, 24, (2008)
[3] V. Dobrokhotov, et. al., Nanotechnology 19 (2007), 035502
[4] R. Jalilian, et. al., Nanotechnology, 22 (2011), 295705.
[5] C. Rein, et. al. Langmuir, 27(2), (2011)
[6]A. Wain, et. al. Electrochemistry Communications (2010)
[7] L. Biedermann, et. al., Nanotechnology 21, (2010), 305701
[8] D. Kiracofe, et. al. Nanotechnology, 22 (2011), 295504
[9] R. Jalilian, et. al., Nanotechnology, 22 (2011), 295601

Current mass spectrometry imaging methods are limited in spatial resolution when analyzing large biomolecules. The goal of this project is to use atomic force microscope tip enhanced laser ablation to ablate material from cells and tissue and capture it for subsequent mass spectrometry analysis. The laser ablation sample transfer system uses an AFM stage to hold the metal tip at a distance of approximately 10 nm from a sample surface. The metal tip acts as an antenna for the electromagnetic radiation and enables the ablation of the sample with a spot size much smaller than a laser focused with a conventional lens system. A pulsed nanosecond UV or IR is focused onto the gold-coated silicon needle at an angle nearly parallel with the surface, which results in the removal of material from a spot between 500 nm and 1 µm in diameter and 200 and 500 nm deep. This corresponds a few ng of ablated material, which can be captured on a metal surface for MALDI analysis or at the tip of a nanocapillary for electrospray analysis. We have used this approach to transfer small peptides and proteins from a thin film for analysis by mass spectrometry. We are concurrently developing methods that introduce liquid separations following capture and before mass spectrometry analysis. The small-scale laser ablation sampling developed in this project has applications both in mass spectrometry as well as antibody or DNA/RNA methods as well as general sampling method for microfluidic systems.

Mechanical interactions between cells and the extracellular matrix (ECM) are critical to the metastasis of cancer cells. To investigate the mechanical interplay between the cells and ECM during invasion, we created a model using 80-200 µm thick bovine collagen I hydrogels ranging from 0.1-5 kPa in Young&’s modulus that were seeded with highly metastatic MDA-MB-231 breast cancer cells. Significant population fractions invaded the matrices either partially or fully within 24 h. We then combined confocal fluorescence microscopy and AFM indentation to determine the Young&’s moduli of individual embedded cells and the pericellular matrix using novel analysis methods for heterogeneous samples. In partially embedded cells, we observe a statistically significant correlation between the degree of invasion and the Young&’s modulus, which was up to an order of magnitude greater than that of the same cells measured in 2D. ROCK inhibition returned the cells&’ Young&’s moduli to values similar to 2D and diminished but did not abrogate invasion. This provides evidence that Rho/ROCK-dependent acto-myosin contractility is employed for matrix reorganization during initial invasion, and suggests the observed cell stiffening is due to an attendant increase in actin stress fibers.

The recognition of target molecules by DNA probe molecules tethered to surfaces is at the heart of a wide range of sensors and microarrays. Yet, although established models can predict the thermodynamics and kinetics of DNA hybridization in the solution phase, molecular level understanding of the complex dynamics of DNA probes on surfaces remains in its infancy. A major challenge is that little is known about the spatial arrangement and conformations of these probe molecules, which may have a profound impact on molecular recognition on surfaces. Among the current techniques, atomic force microscopy (AFM) is the only one that is potentially capable of visualizing the individual DNA molecules on biosensor surfaces in situ and with nanometer resolution. Yet, existing AFM studies on DNA probes have only achieved low spatial resolution because of the fluctuation of DNA molecules on surfaces.
By exploiting the transient electrostatic pinning enabled by an applied electrochemical potential, we have enabled in situ AFM to visualize the conformational changes of single DNA molecules to gold. Our study has revealed an extreme sensitivity to the nanoscale environment: the electrostatic interaction of the DNA with the surface is dominated by defects in the passivating self-assembled monolayer (SAM) and that the SAM, often regarded as a static structure, is not only high mobile but is actively remodeled by the DNA at different applied potentials. Moreover, by directly visualizing single hybridization events, we have provided nanoscale and single molecule level evidence that the hybridization efficiency is impacted by the presence of neighboring probe molecules. Such molecular level insights into hybridization on surfaces may inform new strategies to engineer more robust and reliable DNA sensors.

Large amplitude fluctuations and significant conformational changes are characteristic features of all Soft Materials. As a result, dynamics play crucial role in their macroscopic properties and understanding the dynamic behavior is the key in design of soft materials with desired properties. Microscopic understanding of relaxation processes and conformational fluctuations, especially on the mesocopic length scale, requires advanced experimental and computational techniques that can cover extremely broad time (or frequency) range. Among the experimental methods, scattering techniques play the important role, because they provide not only characteristic time and energy of the motions, but also their geometry. We will present an overview of scattering techniques (neutrons, X-ray and light scattering) and emphasize their advantages in studies of dynamics in glass-forming liquids, polymers and biological systems. Special focus will be on collective dynamics and dynamic heterogeneities. At the end of the talk we will emphasize new developments in scattering techniques that might provide significant advances in our understanding of dynamics in soft materials at the mesoscale.

The diffusion properties of the components in polymer nanocomposites are largely unknown and depend intimately on the dispersion of the nanoparticles throughout the polymer matrix and the individual dynamics of the nanoparticle and the polymer matrix. Unfortunately, the rational control of nanoparticle dispersion in a polymer matrix is difficult. To circumvent the difficulty of dispersion, we have found that soft, organic nanoparticles offer opportunities to enhance dispersion of the nanoparticle in a polymer matrix due to the interpenetration of polymer chains and particles and the reduction in the depletion of entropy in the system. The impact of the presence of the soft nanoparticles on the diffusion coefficient of polystyrene was recently determined with neutron reflectivity by monitoring the interdiffusion of deuterated and protonated polystyrene bilayers, with and without the soft nanoparticles dispersed throughout both layers. We have extended the neutron reflectivity method to bilayer systems with only the soft nanoparticles as one of the layers and a linear polystyrene analogue as an adjacent layer. The new development of this method has allowed us to extract the tracer diffusion coefficient of the soft polystyrene nanoparticles for the first time.

Synthetic polypeptides are a kind of polymers which have a significant role due to their close relation to proteins so as their ability to assemble hierarchically stable ordered conformations. Even though there are many applications of polypeptides, there are few reports ^1-4 which indicate diameter size dependence on their structural and dynamic behaviors. Since macromolecules in nanoporous alumina (AAO) might have different segmental dynamics according to bulk, herein we investigated the segmental dynamics of poly (Z-L-lysine) (PZLL) confined in self-ordered AAO containing aligned and straight cylindrical nanopores as a function of pore size by differential scanning calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR), Solid-state NMR ( ¹³C CPMAS NMR), Wide-angle X-ray scattering (WAXS), Dielectric spectroscopy (DS). The structural characterization of the nanoconfined PZLL revealed nanorods mostly consist of alpha-helix structure independent of pore diameter. Bulk PZLL exhibits a glass transition temperature (T_g) at about 301 K, while PZLL nanorods showed slightly lower T_g at around 294 K. The dynamic investigation by DS also revealed a small decrease (~4 K) in T_g between bulk and nanoconfined PZLL samples. Subsequently, DS have been employed to study the effect of confinement on the local and global polypeptide dynamics. DS is a very sensitive probe of the local and global (alpha-helical) secondary structure relaxation through the large dipole. The results revealed that the local segmental dynamics, associated with broken hydrogen bonds, speed-up on confinement. The results of this study is of paramount importance, since the understanding the self-assembly, thermodynamics and dynamics of soft materials under confinement will allow for their rational design as functional devices with tunable mechanical strength, processability, electronic and optical properties.
References
1. Duran H., Gitsas A., Floudas G., Mondeshki M., Steinhart M., Knoll W., Macromolecules, Vol. 42, No. 8, 2009.
2. Suzuki Y., Duran H., Steinhart M., Butt H. J., Floudas G., Soft Matter, 2013, 9, 2621-2628.
3. Suzuki Y., Duran H., Akram W., Steinhart M., Floudas G., Butt H. J., Soft Matter,2013, 9, 9189-9198.
4. Tuncel, E.; Suzuki, Y.; Iossifidis, A.; Steinhart, M.; Duran, H.; Butt, H. -J. and Floudas, G. Manuscript in preparation, 2014.

An important goal of nano-material characterization is exploring the correlation of the physical and chemical properties. The aim of this presentation is to show how the confocal Raman - AFM - SNOM can contribute to such studies. In the past two decades, AFM (Atomic Force Microscopy) was one of the main techniques used to characterize the morphology of nano-materials. From such images it is possible to gain information about the physical dimensions of the material, but not their chemical composition, crystallinity or stress state. On the other hand, Raman spectroscopy is known to be used to unequivocally determine the chemical composition of a material. By combining the chemical sensitive Raman spectroscopy with high resolution confocal optical microscopy, it is possible to acquire diffraction limited resolution Raman images. Furthermore, using SNOM (Scanning Near-field Optical Microscopy) technology, additional optical information may be obtained, i.e. it will be shown how the transparency of different graphene sheets is changing as a function of the number of layers. The combination of confocal Raman microscopy with AFM and SNOM is a breakthrough in microscopy. Using such a combination, the topographic information obtained with an AFM can be directly linked to the chemical information provided by confocal Raman and optical properties obtained with SNOM.

Soft Matter/Complex fluid systems are ubiquitous across a range of industrial and consumer sectors, with common examples being inks, paints, drilling fluids, cosmetics, personal care products and foodstuffs. In many instances the final product format of these complex fluids are gels or soft solids and the processing and product functionality attributes of such materials are often dependent on their rheological response and viscosity. The rheology evolution in such complex fluid systems as a function of formulation parameters (e.g. pH, ionic strength) is intimately connected to corresponding changes in micro/mesostructure and intermolecular and intramolecular associations and interactions. Most insights developed into understanding the self-assembly and rheology evolution process in such systems has primarily focused on elucidating the associated micro/mesostructural changes through various scattering (light, x-ray, neutron) and imaging techniques (cryo-TEM, SEM, AFM). Further more detailed insights into the associated chemical conformational/structural changes and various non-covalent interactions (e.g. H-bonds, hydrophobic interactions) leading to the self-assembly process has been very limited. In this talk we provide new structural/interaction insights into the self-assembly and gelation mechanism of complex fluids through combination of a number of well-established analytical techniques namely dynamic light scattering (DLS), Raman spectroscopy and Optical Microrheology. The talk will illustrate the utility of the combination of mesoscale structure-property elucidation techniques such as DLS/microrheology with the high resolution chemical structure/conformation elucidation techniques such as Raman Spectroscopy in generating novel mechanistic insights that will allow the performance engineering of complex fluids and soft matter systems. This will be exemplified through studies into the self-assembly/gelation mechanisms in two very different complex fluids-mixed anionic/zwitterionic surfactant wormlike micelles and a thermo-reversible gel forming agarose biopolymer.

The relationship between microscopic structure and macroscopic flow properties can be probed in real time by mounting the head of a rheometer on the body of high-speed confocal microscope [1]. This capability, called rheo-imaging, has only just begun to be exploited for the study of soft matter. Here, we have used rheo-imaging to investigate the shear flow of particle-stabilized (Pickering) emulsions with different interactions between droplets [2]. The image information is essential for interpreting bulk moduli when the sample response is highly non-linear.
In our experiments, to minimize creaming, we have worked with arrested networks of droplets. We observe three different yielding mechanisms as well as shear thickening at high strain. Pickering emulsions comprised of repulsive droplets respond to small strains initially by reversible movement, then, as the strain is increased, yielding occurs via cage breaking. At large strains there is a peak in both moduli and correlations between droplet trajectories indicative of shear thickening and jamming. Pickering emulsions comprised of attractive droplets share similar features and additionally have a loss modulus peak at low strain associated with the breakage and rearrangement of the attractive bonds. We observe that small attractive droplets already start to move irreversibly at small strains even though yielding has not yet occurred. As the strain is increased, the percolating network of larger droplets is eventually completely broken and the sample yields as a whole. Finally, we explore what happens when a highly compressed emulsion or bi-liquid foam is subjected to shear flow: we find that at high strain the particles fail to stabilize the interface and the emulsion is destroyed.
References:
[1] R. Besseling, L. Isa, E.R. Weeks and W.C.K. Poon, Adv. Colloid Interface Sci. 146, 1, (2009).
[2] M. Hermes and P.S. Clegg, Soft Matter, 9, 7568 (2013).

Bacterial biofilms are essentially surface associated bacterial colonies, where bacterial cells are embedded in a matrix of self-produced extracellular polymeric substances (EPS). EPS acts as natural glue providing mechanical integrity to the biofilm structure. This social form of growth offers resilience to external stresses and thus provides biofilm bacteria with an existential advantage over the planktonic (free swimming) form of living. Once a biofilm forms at an interface it can be very difficult to remove. Due to this reason biofilms have been implicated for their role in biofouling and surface degradation.
Biofilms play a critical role in many defense related applications. For example, biofilm formation on surfaces exposed to environment may degrade their optimal performance. Surfaces of submerged sensors can be inhibited by biofilms. Biofilm formation on implanted sensors prevents the target analytes from reaching the sensing layer resulting in false readings. Biofilm formation also prevents the deployment of unattended, left behind sensors from operating continuously. In some other cases biofims play a positive role, for example water filtration. Our need to control biofilm growth, as well as possess the ability to remove undesirable biofilms, necessitates a proper understanding of the material characteristics of this biological soft matter.
Biofilms are now recognized to belong to the class of viscoelastic materials; this property dictates deformation behavior of biofilms under external forces. In order to better understand the viscoelastic properties of biofilms, we investigate using two-point correlation microrheology the complex shear modulus of the biofilms. Two-point correlation microrheology is a technique that uses information regarding Brownian tracers dispersed in a soft matter to yield information regarding its viscoelastic properties. The use of Brownian tracers provides a local probe of complex modulus in miniscule sample volumes. Two-point microrheology also has other advantages, in addition to its ability to probe inhomogeneous media. Utilizing these measurements, we interpret the deformation of biofilms at long time scales even in presence of very weak creeping flows. We explain that biofilms being viscoelastic ‘liquids&’ are characterized by a viscoelastic relaxation time scale. When these biofilms are subject to very weak shear stresses for time scales much longer than the viscoelastic relaxation time scale, then the viscous behavior dominates and the biofilms ‘flow&’. Under such conditions, biofilms can form long slender bodies called streamers. These streamers can serve as precursors to mature biofilm formation in complex systems and can lead to catastrophic clogging of fluidic channels.