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Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.

Single molecule experiments have demonstrated a progressive transition from a B-to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B-to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit∗. For these sequences available studies cover ensemble and single-molecule analyses, although for kit∗ the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit∗ are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype.

Background: DNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. Methods: We apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. Results: In the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. Conclusions: We observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. General Significance: Our data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Two‐photon laser writing is used here to fabricate 3D proteinaceous microstructures with photothermal functionality in the near‐infrared spectral region and tunable elasticity. The photo‐cross‐linking is initiated in bovine serum albumin (BSA) by rose bengal or methylene blue and the photo‐thermal effect arises from gold non‐spherically symmetric nanoparticles dispersed in the ink. Massive energy transfer of the plasmonic resonances of the gold nanoparticles to methylene blue prevents effective photo‐crosslinking of BSA. However, stable microstructures with photo‐thermal functionality can be fabricated in the rose bengal proteinaceous inks. On these microstructures, with a gold atom concentration as low as 1% w/w, a highly localized temperature increase can be quickly (≅1 s) reached and maintained under continuous wave laser irradiation at 800 nm. The photothermal efficiency under continuous wave laser irradiation depends on the thickness of the microstructure and can reach 12.2 ± 0.4 °C W−1 These proteinaceous microstructures represent therefore a promising platform for future applications in the fields like physical stimulation of cells for regenerative nanomedicine.

The cell microenvironment plays a pivotal role in mediating cell adhesion, survival, and proliferation in physiological and pathological states. The relevance of extracellular matrix (ECM) proteins in cell fate control is an important issue to take into consideration for both tissue engineering and cell biology studies. The glycosylation of ECM proteins remains, however, largely unexplored. In order to investigate the physio-pathological effects of differential ECM glycosylation, the design of affordable chemoselective methods for ECM components glycosylation is desirable. We will describe a new chemoselective glycosylation approach exploitable in aqueous media and on non-protected substrates, allowing rapid access to glyco-functionalized biomaterials.

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensi

The fibrotic tissue and the stroma adjacent to cancer cells are characterised by the presence of activated fibroblasts (myofibroblasts) which play a role in creating a supportive tissue charac-terised by abundant extracellular matrix (ECM) secretion. The myofibroblasts remodel this tissue through secreted molecules and modulation of their cytoskeleton and specialized con-tractile structures. The non-receptor protein tyrosine kinase Arg (also called Abl2) has the unique ability to bind directly to the actin cytoskeleton, transducing diverse extracellular sig-nals into cytoskeletal rearrangements. In this study we analysed the 1ALCTL and 1BLCTL Arg isoforms in Arg-/- murine embryonal fibroblasts (MEF) cell line, focusing on their capac-ity to activate fibroblasts and to remodel ECM. The results obtained showed that Arg isoform 1BLCTL has a major role in proliferation, migration/invasion of MEF and in inducing a milieu able to modulate tumour cell morphology, while 1ALCTL isoform has a role in MEF adhesion maintaining active focal adhesions. On the whole, the presence of Arg in MEF supports the proliferation, activa-tion, adhesion, ECM contraction and stiffness, while the absence of Arg affected these myofibroblast features

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with a supportive microenvironment. These processes are affected by the mechanical forces present in the environment and by the capability of the cells to sense the forces. In order to migrate from a fluid environment like the blood to a significantly more viscous surrounding, B lymphocytes need to modify their cytoskeleton and consequently their rigidity. Those processes are known to be regulated by Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein, which promotes the homing processes and is involved in cell-cell communication and focal adhesions formation. From a nanomechanical point of view, cytoskeleton rearrangements can be observed as a difference in the force distribution along the cell and, ultimately, as a change of the overall rigidity of the cell. To measure this change, we used Atomic Force Microscopy (AFM) as an external pressure stimulus and we observed the cell deformation, which finally determines the value of its stiffness. By AFM we measured primary B lymphocytes from selected CLL patients and healthy donors, and we found a significant decrease of stiffness in leukaemia cells compared to healthy ones. We also performed Real-Time Deformability Cytometry (RT-DC) to test the rigidity of cells in flow condition. Finally, we tested the effect of a first line drug (Ibrutinib) on the mechanical properties of leukemic and healthy cells with both techniques in order to understand its effect on the mechanics of cells along their path to development of the disease.

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with the microenvironment. These processes are affected by the mechanical-forces present in the environment and by the capability of the cells to sense the forces. In this context we demonstrated that Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein is a cytoskeletal regulator and a prognostic factor in CLL. We proved that interfering with HS1 expression impacts on the progression and homing of CLL cells. To further study HS1 role in CLL we knocked-down HS1 expression by CRISPR/CAS9 technology in a CLL cells line (MEC1). By RNAseq and network analysis on MEC1HS1ko vs MEC1UT we found that HS1-KO significantly affects the expression of molecules involved in: cell motility, adhesion, cell-cell communication, focal adhesion formation. In particular, we found LEF1, FAK and Cortactin genes are up-regulated in MEC1HS1ko, suggesting their involvement in the mechano-signalling pathway. To study the nano-mechanical properties of those cells we are performing AFM measurements of single cell. The results provide an evaluation of the cell stiffness that is related to its deformation in response to an externally applied force. By AFM we found that MEC1HS1ko cells are less stiff if compared with MEC1UT cells. This results demonstrates a putative role for HS1 in regulating the mechanical properties of CLL cells. Due to the prognostic value of HS1 we are currently performing AFM measurement on selected patients and healthy B cells. We are planning to study in depth the role of HS1 in regulating the mechanical properties of the leukemic cells and how this contributes to leukemia development, progression and resistance to therapy.

Chronic lymphocytic leukaemia (CLL) is one of the most common and incurable B cell leukaemia. CLL cells traffic between peripheral blood, bone marrow and secondary lymphatic tissues where interact with the microenvironment. These processes are affected by the mechanical-forces present in the environment and by the capability of the cells to sense the forces. In this context we demonstrated that Hematopoietic-cell-specific Lyn-substrate-1 (HS1) protein is a cytoskeletal regulator and a prognostic factor in CLL. We proved that interfering with HS1 expression impacts on the progression and homing of CLL cells. To further study HS1 role in CLL we knocked-down HS1 expression by CRISPR/CAS9 technology in a CLL cells line (MEC1). By RNAseq and network analysis on MEC1HS1ko vs MEC1UT we found that HS1-KO significantly affects the expression of molecules involved in: cell motility, adhesion, cell-cell communication, focal adhesion formation. In particular, we found LEF1, FAK and Cortactin genes are up-regulated in MEC1HS1ko, suggesting their involvement in the mechano-signalling pathway. To study the nano-mechanical properties of those cells we are performing AFM measurements of single cell. The results provide an evaluation of the cell stiffness that is related to its deformation in response to an externally applied force. By AFM we found that MEC1HS1ko cells are less stiff if compared with MEC1UT cells. This results demonstrates a putative role for HS1 in regulating the mechanical properties of CLL cells. Due to the prognostic value of HS1 we are currently performing AFM measurement on selected patients and healthy B cells.

Methylation is one of the most important epigenetic mechanisms in eukaryotes. As a consequence of cytosine methylation, the binding of proteins that are implicated in transcription to gene promoters is severely hindered, which results in gene regulation and, eventually, gene silencing. To date, the mechanisms by which methylation biases the binding affinities of proteins to DNA are not fully understood; however, it has been proposed that changes in double-strand conformations, such as stretching, bending, and over-twisting, as well as local variations in DNA stiffness/flexibility may play a role. The present work investigates, at the single molecule level, the morphological consequences of DNA methylation in vitro. By tracking the atomic force microscopy images of single DNA molecules, we characterize DNA conformations pertaining to two different degrees of methylation. In particular, we observe that methylation induces no relevant variations in DNA contour lengths, but produces measurable incremental changes in persistence lengths. Furthermore, we observe that for the methylated chains, the statistical distribution of angles along the DNA coordinate length is characterized by a double exponential decay, in agreement with what is predicted for polyelectrolytes. The results reported herein support the claim that the biological consequences of the methylation process, specifically difficulties in protein-DNA binding, are at least partially due to DNA conformation modifications.

Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.

Cytosine methylation is a widespread epigenetic regulation mechanism. In healthy mature cells, methylation occurs at CpG dinucleotides within promoters, where it primarily silences gene expression by modifying the binding affinity of transcription factors to the promoters. Conversely, a recent study showed that in stem cells and cancer cell precursors, methylation also occurs at non-CpG pairs and involves introns and even gene bodies. The epigenetic role of such methylations and the molecular mechanisms by which they induce gene regulation remain elusive. The topology of both physiological and aberrant non-CpG methylation patterns still has to be detailed and could be revealed by using the differential stability of the duplexes formed between site-specific oligonucleotide probes and the corresponding methylated regions of genomic DNA. Here, we present a systematic study of the thermal stability of a DNA oligonucleotide sequence as a function of the number and position of non-CpG methylation sites. The melting temperatures were determined by monitoring the fluorescence of donor-acceptor duallabelled oligonucleotides at various temperatures. An empirical model that estimates the methylationinduced variations in the standard values of hybridization entropy and enthalpy was developed.

The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA) system. The polymorphisms of the trait of the DQB1 gene including codons 52–57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52–57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52–57 trait of DQB1 (8 homozygous and 28 heterozygous). We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes.

Recently, we demonstrated that TLQP-21 triggers lipolysis and induces resistance to obesity by reducing fat accumulation [1]. TLQP-21 is a 21 amino acid peptide cleavage product of the neuroprotein VGF and was first identified in rat brain. Although TLQP-21 biological activity and its molecular signaling is under active investigation, a receptor for TLQP-21 has not yet been characterized. We now demonstrate that TLQP-21 stimulates intracellular calcium mobilization in CHO cells. Furthermore, using Atomic Force Microscopy (AFM), we also provide evidence of TLQP-21 binding-site characteristics in CHO cells. AFM was used in force mapping mode equipped with a cantilever suitably functionalized with TLQP-21. Attraction of this functionalized probe to the cell surface was specific and consistent with the biological activity of TLQP-21; by contrast, there was no attraction of a probe functionalized with biologically inactive analogues. We detected interaction of the peptide with the binding-site by scanning the cell surface with the cantilever tip. The attractive force between TLQP-21 and its binding site was measured, statistically analyzed and quantified at approximately 40 pN on average, indicating a single class of binding sites. Furthermore we observed that the distribution of these binding sites on the surface was relatively uniform. © 2012 Elsevier B.V. All rights reserved.

The opening of DNA double strands is extremely relevant to several biological functions, such as replication and transcription or binding of specific proteins. Such opening phenomenon is particularly sensitive to the aqueous solvent conditions in which the DNA molecule is dispersed, as it can be observed by considering the classical dependence of DNA melting temperature on pH and salt concentration. In the present work, we report a single-molecule study of the stability of DNA against denaturation when subjected to changes in solvent. We investigated the appearance of DNA instability under specific external applied force and imposed twist values, which was revealed by an increase in the temporal fluctuations in the DNA extension. These fluctuations occur in the presence of a continuous interval of equilibrium states, ranging from a plectonemic state to a state characterized by denaturation bubbles. In particular, we observe the fluctuations only around a characteristic force value. Moreover, this characteristic force is demonstrated to be notably sensitive to variations in the pH and ionic strength. Finally, an extension of a theoretical model of plectoneme formation is used to estimate the average denaturation energy, which is found to be linearly correlated to the melting temperature of the DNA double strands.

We use the "magnetic tweezers" technique to show the structural transitions that the DNA undergoes in the force-torsion space. In particular, we focus on the regions corresponding to negative supercoiling. These regions are characterized by the formation of the so-called denaturation bubbles, which play an essential role in the replication and transcription of DNA. We experimentally map the region of the force-torsion space where the denaturation takes place. We observe that large fluctuations in DNA extension occur at one of the boundaries of this region, i.e., when the formation of denaturation bubbles and of plectonemes compete. To describe the experiments, we introduce a suitable extension of the classical model. The model correctly describes the position of the denaturation regions, the transition boundaries, and the measured values of the DNA extension fluctuations. © 2012 American Physical Society.

Binding of ligands to DNA gives rise to several relevant biological and biomedical effects. Here, through the use of atomic force microscopy (AFM), we studied the consequences of drug binding on the morphology of single DNA molecules. In particular, we quantitatively analyzed the effects of three different DNA-binding molecules (doxorubicin, ethidium bromide, and netropsin) that exert various pharmacologic and therapeutic effects. The results of this study show the consequences of intercalation and groove molecular binding on DNA conformation. These single-molecule measurements demonstrate morphological features that reflect the specific modes of drug-DNA interaction. This experimental approach may have implications in the design of therapeutically effective agents.

Herein, we study the nanomechanical characteristics of single DNA molecules in the presence of DNA binders, including intercalating agents (ethidium bromide and doxorubicin), a minor groove binder (netropsin) and a typical alkylating damaging agent (cisplatin). We have used magnetic tweezers manipulation techniques, which allow us to measure the contour and persistence lengths together with the bending and torsional properties of DNA. For each drug, the specific variations of the nanomechanical properties induced in the DNA have been compared. We observed that the presence of drugs causes a specific variation in the DNA extension, a shift in the natural twist and a modification of bending dependence on the imposed twist. By introducing a naive model, we have justified an anomalous correlation of torsion data observed in the presence of intercalators. Finally, a data analysis criterion for discriminating between different molecular interactions among DNA and drugs has been suggested.

Light scattering techniques are widely used in many fields of condensed and soft matter physics. Usually these methods are based on the study of the scattered light in the far field. Recently, a new family of near field detection schemes has been developed, mainly for the study of small angle light scattering. These techniques are based on the detection of the light intensity near to the sample, where light scattered at different directions overlaps but can be distinguished by Fourier transform analysis. Here we report for the first time data obtained with a dynamic near field scattering instrument, measuring both polarized and depolarized scattered light. Advantages of this procedure over the traditional far field detection include the immunity to stray light problems and the possibility to obtain a large number of statistical samples for many different wave vectors in a single instantaneous measurement. By using the proposed technique we have measured the translational and rotational diffusion coefficients of rod-like colloidal particles. The obtained data are in very good agreement with the data acquired with a traditional light scattering apparatus. © 2009 Optical Society of America.

The electrical properties of cluster-assembled nanostructured palladium oxide (ns-PdOx) thin films grown by supersonic cluster beam deposition have been characterized by means of a customized ac current-sensing atomic force microscope. Scanning impedance microscopy is shown to provide a deep picture of the electrical properties of thin nanostructured interfaces even in the case of very soft and poorly adherent films. In particular, the dielectric constant of ns-PdOx can be quantitatively determined as well as its I-V characteristics. Moreover, the measurement of the tip-sample parasitic capacitance can be exploited to probe the overall mesoscale conductive character of thin films and to give a complementary and more precise view of the oxidation of ns-PdOx obtained by x-ray photoemission spectroscopy. © 2009 The American Physical Society.

By using scattering in near field techniques, a microscope can be easily turned into a device measuring static and dynamic light scattering, very useful for the characterization of nanoparticle dispersions. Up to now, microscopy based techniques have been limited to forward scattering, up to a maximum of 30°. In this paper we present a novel optical scheme that overcomes this limitation, extending the detection range to angles larger than 90° (back-scattering). Our optical scheme is based on a microscope, a wide numerical aperture objective, and a laser illumination, with the collimated beam positioned at a large angle with respect to the optical axis of the objective (Tilted Laser Microscopy, TLM). We present here an extension of the theory of near field scattering, which usually applies only to paraxial scattering, to our strongly out-of-axis situation. We tested our instrument and our calculations with calibrated spherical nanoparticles of several different diameters, performing static and dynamic scattering measurements up to 110°. The measured static spectra and decay times are compatible with the Mie theory and the diffusion coefficients provided by the Stokes-Einstein equation. The ability of performing backscattering measurements with this modified microscope opens the way to new applications of scattering in near field techniques to the measurement of systems with strongly angle dependent scattering. © 2009 Optical Society of America.

Light scattering detection in the near field, a rapidly expanding family of scattering techniques, has recently proved to be an appropriate procedure for performing dynamic measurements. Here we report an algorithm, first suggested by Oh et al. (Phys. Rev. E 69, 21106 (2004)), based on the evaluation of the Exposure Time Dependent Spectrum (ETDS), which makes it possible to measure the fast dynamics of a colloidal suspension with the aid of a simple near field scattering apparatus and a CCD camera. The algorithm consists in acquiring static spectra in the near field at different exposure times, so that the measured decay times are limited only by the exposure time of the camera and not by its frame rate. The experimental set-up is based on a modified microscope, where the light scattered in the near field is collected by a commercial objective, but (unlike in standard microscopes) the light source is a He-Ne laser which increases the instrument sensitivity. The apparatus and the algorithm have been validated by considering model systems of standard spherical nano-particle.

We investigated the electrical transport properties of nanostructured carbon and carbon/titanium oxide nanocomposite films produced by supersonic cluster beam deposition and irradiated by highly focused vacuum UV photon beam. We have observed a relevant increase of the density of states at Fermi level, suggesting that the films acquire a ¿metallic¿ character. This is confirmed by the increment of the conductivity of four orders of magnitude for pure nanostructured carbon films and at least eight orders of magnitude for films containing 9 at. % of titanium. A partial reversibility of the process is observed by exposing the modified films to molecular oxygen or directly to air. We demonstrate the capability of writing micrometric conductive strips (2-3 µm width and 60 µm length) and controlling the variation of the conductivity as a function of the titanium concentration.

We report the structural and tribological characterization of nanostructured CNx thin films produced by the deposition of a supersonic carbon cluster beam assisted by nitrogen ion bombardment. The influence of the deposition parameters on the chemical composition and structure of the films has been systematically studied by X-ray photoelectron spectroscopy, elastic recoil detection analysis, transmission electron microscopy and atomic force microscopy. Depending on the deposition parameters, the films show a structure ranging from amorphous to disordered graphitic with interlinked planes. Nitrogen content depends on the nitrogen ion kinetic energy. The films have a very low density with a high surface roughness. Friction measurements at the nanoscale show a correlation between nitrogen content and mechanical properties of the system.

We have investigated the influence on adhesion and proliferation of NIH 3T3 fibroblasts of the surface morphology of cluster assembled carbon films deposited by Supersonic Cluster Beam Deposition. Nanostructured carbon films exhibit a multi-scale morphology, which resembles the surface structure of the extracellular matrix, and possess a high specific area, while being relatively smooth at all scales. Correlations between measured morphological parameters and adaptive cell response have been brought out. High specific area and smoothness appear to conceivably favour both the early attachment of plated cells and the long-term survival of adherent cells. Moreover, nanostructured carbon films affect the cells morphology as well as the extension and the number of the focal contacts.