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ABSTRACT: The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat PDMS substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell-substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
Biotechnology and Bioengineering 02/2013; · 3.95 Impact Factor
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ABSTRACT: A DNA-origami actuator capable of autonomous internal motion in accord to an external chemical signal was designed, built, operated and imaged. The functional DNA nanostructure consists of a disk connected to an external ring in two, diametrically opposite points. A single stranded DNA, named probe, was connected to two edges of the disk perpendicularly to the axis of constrain. In the presence of a hybridizing target molecule, the probe coiled into a double helix that stretched the inner disk forcing the edges to move toward each other. The addition of a third single stranded molecule that displaced the target from the probe restored the initial state of the origami. Operation, dimension and shape were carefully characterized by combining microscopy and fluorescence techniques.
Nano Letters 11/2011; 11(12):5449-54. · 13.20 Impact Factor
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ABSTRACT: Embryonic stem (ES) cell differentiation in specific cell lineages is a major issue in cell biology particularly in regenerative medicine. Differentiation is usually achieved by using biochemical factors and it is not clear whether mechanical properties of the substrate over which cells are grown can affect proliferation and differentiation. Therefore, we produced patterns in polydimethylsiloxane (PDMS) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. We analyzed the effect of different nanostructures on differentiation of ES-derived neuronal precursors into neuronal lineage without adding biochemical factors. Neuronal precursors adhered on PDMS more effectively than on glass coverslips. We demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. On higher pillar neuronal differentiation reaches ∼80% 96 h after plating and the largest differentiation enhancement of pillars over flat PDMS was observed during the first 6 h of culture. We conclude that PDMS nanopillars accelerate and increase neuronal differentiation.
Biotechnology and Bioengineering 06/2011; 108(11):2736-46. · 3.95 Impact Factor
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ABSTRACT: During early development of the central nervous system, there is an excessive outgrowth of neuronal projections, which later need to be refined to achieve precise connectivity. Axon pruning and degeneration are strategies used to remove exuberant neurites and connections in the immature nervous system to ensure the proper formation of functional circuitry. To observe morphological changes and physical mechanisms underlying this process, early differentiating embryonic stem cell-derived neurons were used combining video imaging of live growth cones (GCs) with confocal laser scanning microscopy and atomic force microscopy, both on fixed and living neurons. Using this method, we could highlight the presence of submicrometric fragments in still and in some of the retracting GCs. The observed fragmentation is not an artifact of atomic force microscopy scanning or fixation, or the result of apoptosis. Therefore, the morphology of GCs depends on their overall motility, and fragmentation seems to be the fate of GCs that have not found a correct destination.
Stem cells and development 06/2011; 20(6):1031-41. · 4.15 Impact Factor
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ABSTRACT: Tip-Enhanced Raman spectroscopy (TERS) is a promising microscopy technique which combines, in principle, outstanding spatial resolution with a detailed chemical analysis of the sample. However, as yet, it is not routinely used although an increasing number of research groups are becoming more actively involved in the field. Among the several reasons which can explain the relatively low usage of TERS, the lack of reproducibility of tips as field enhancers is probably the most critical. Here we propose and demonstrate a TERS microscope which uses photonic engineered tips. These tips are based on standard silicon nitride atomic force microscope (AFM) cantilevers. A photonic crystal together with a plasmonic waveguide focuses the Raman excitation laser to the apex of the waveguide, enabling a photon confinement equivalent to the radius of curvature of the nanofabricated tip. These tips were successfully applied here in both AFM imaging and high resolution Raman spectroscopy. The new tips produced AFM imaging performances comparable with the best AFM commercial tips. Moreover, we demonstrate that the photonic crystal combined with the plasmonic waveguide acts effectively as a localized near field emitter.
Micron 09/2010; · 1.53 Impact Factor
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Diane Latawiec,
Fernando Herrera,
Alpan Bek,
Valeria Losasso,
Michela Candotti,
Federico Benetti,
Elvio Carlino,
Agata Kranjc, Marco Lazzarino,
Stefano Gustincich,
Paolo Carloni,
Giuseppe Legname
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ABSTRACT: The action of dopamine on the aggregation of the unstructured alpha-synuclein (alpha-syn) protein may be linked to the pathogenesis of Parkinson's disease. Dopamine and its oxidation derivatives may inhibit alpha-syn aggregation by non-covalent binding. Exploiting this fact, we applied an integrated computational and experimental approach to find alternative ligands that might modulate the fibrillization of alpha-syn. Ligands structurally and electrostatically similar to dopamine were screened from an established library. Five analogs were selected for in vitro experimentation from the similarity ranked list of analogs. Molecular dynamics simulations showed they were, like dopamine, binding non-covalently to alpha-syn and, although much weaker than dopamine, they shared some of its binding properties. In vitro fibrillization assays were performed on these five dopamine analogs. Consistent with our predictions, analyses by atomic force and transmission electron microscopy revealed that all of the selected ligands affected the aggregation process, albeit to a varying and lesser extent than dopamine, used as the control ligand. The in silico/in vitro approach presented here emerges as a possible strategy for identifying ligands interfering with such a complex process as the fibrillization of an unstructured protein.
PLoS ONE 01/2010; 5(2):e9234. · 4.09 Impact Factor
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Microelectronic Engineering 01/2010; 87(5-8):730-733. · 1.56 Impact Factor
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Francesco De Angelis,
Gobind Das,
Patrizio Candeloro,
Maddalena Patrini,
Matteo Galli,
Alpan Bek, Marco Lazzarino,
Ivan Maksymov,
Carlo Liberale,
Lucio Claudio Andreani,
Enzo Di Fabrizio
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ABSTRACT: The fields of plasmonics, Raman spectroscopy and atomic force microscopy have recently undergone considerable development, but independently of one another. By combining these techniques, a range of complementary information could be simultaneously obtained at a single molecule level. Here, we report the design, fabrication and application of a photonic-plasmonic device that is fully compatible with atomic force microscopy and Raman spectroscopy. Our approach relies on the generation and localization of surface plasmon polaritons by means of adiabatic compression through a metallic tapered waveguide to create strongly enhanced Raman excitation in a region just a few nanometres across. The tapered waveguide can also be used as an atomic force microscope tip. Using the device, topographic, chemical and structural information about silicon nanocrystals may be obtained with a spatial resolution of 7 nm.
Nature Nanotechnology 11/2009; 5(1):67-72. · 27.27 Impact Factor
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ABSTRACT: The conversion of the cellular form of the prion protein (PrP(C)) to an abnormal, alternatively folded isoform (PrP(Sc)) is the central event in prion diseases or transmissible spongiform encephalopathies. Recent studies have demonstrated de novo generation of murine prions from recombinant prion protein (recPrP) after inoculation into transgenic and wild-type mice. These so-called synthetic prions lead to novel prion diseases with unique neuropathological and biochemical features. Moreover, the use of recPrP in an amyloid seeding assay can specifically detect and amplify various strains of prions. We employed this assay in our experiments and analyzed in detail the morphology of aggregate structures produced under defined chemical constraints. Our results suggest that changes in the concentration of guanidine hydrochloride can lead to different kinetic traces in a typical thioflavin T(ThT) assay. Morphological and structural analysis of these aggregates by atomic force microscopy indicates a variation in the structure of the PrP molecular assemblies. In particular, ThT positive PrP aggregates produced from rec mouse PrP residues 89 to 230 lead to mostly oligomeric structures at low concentrations of guanidine hydrochloride, while more amyloidal structures were observed at higher concentrations of the denaturant. These findings highlight the presence of numerous and complex pathways in deciphering prion constraints for infectivity and toxicity.
Journal of Molecular Biology 09/2009; 393(5):1033-42. · 4.00 Impact Factor
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ABSTRACT: We have analyzed the morphology of growth cones of differentiating neurons from rat dorsal root ganglia (DRG) with conventional Laser Scanning Confocal Microscopy (LSCM) and Atomic Force Microscopy (AFM). Images of immunofluorescent DRG growth cones colabeled for actin and tubulin were superimposed to images obtained with AFM at different scanning forces. In order to reduce changes of the image surface caused by the pressure of the AFM tip, we have developed a procedure to obtain 0pN AFM images. Further analysis of these images revealed topographical structures with nanoscale dimensions, referred to as "invaginations" or "holes". These holes had an area varying from 0.01 to 3.5 microm(2) with a depth varying from 2 to 178 nm. Comparative analysis with LSCM images showed that these holes correspond to regions where staining of both actin and tubulin was negligible. Filopodia height varied from 40 to 270 nm and their diameter from 113 to 887 nm. These results show that the combination of LSCM and AFM reveal structural details with a nanoscale dimension of DRG growth cones, difficult to resolve with conventional microscopy.
Journal of Structural Biology 09/2009; 168(3):366-77. · 3.41 Impact Factor
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ABSTRACT: Atomic force microscopy (AFM) provides the possibility to map the 3D structure of viewed objects with a nanometric resolution, which cannot be achieved with other imaging methods such as conventional video imaging and confocal fluorescent microscopy. Video imaging with CCD cameras can provide an analysis of biological events with a temporal and spatial resolution not possible with AFM, while confocal imaging allows the simultaneous acquisition of immunofluorescence images. In this communication we present a simple method to combine AFM and confocal images to study differentiating embryonic stem (ES) cells-derived and dorsal root ganglia (DRG) neurons in culture. Neurons were grown on coverslips with micrometric markers that allow finding and imaging the same neuron with different microscopes. AFM and confocal images were registered using conventional methods used in Computer Science. The combination of these two techniques allows relating functional properties to morphological features of imaged neurons.
Journal of Neuroscience Methods 11/2008; 177(1):94-107. · 1.98 Impact Factor
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Faustino Martelli,
Silvia Rubini,
Matteo Piccin,
Giorgio Bais,
Fauzia Jabeen,
Silvano De Franceschi,
Vincenzo Grillo,
Elvio Carlino,
Francesco D'Acapito,
Federico Boscherini,
Stefano Cabrini, Marco Lazzarino,
Luca Businaro,
Filippo Romanato,
Alfonso Franciosi
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ABSTRACT: GaAs nanowires have been grown on SiO2 and GaAs by molecular beam epitaxy using manganese as growth catalyst. Transmission electron microscopy shows that the wires have a wurtzite-type lattice and that alpha-Mn particles are found at the free end of the wires. X-ray absorption fine structure measurements reveal the presence of a significant fraction of Mn-As bonds, suggesting Mn diffusion and incorporation during wire growth. Transport measurements indicate that the wires are p-type, as expected from doping of GaAs with Mn.
Nano Letters 10/2006; 6(9):2130-4. · 13.20 Impact Factor
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ABSTRACT: Planar light-emitting diodes (LEDs) fabricated within a single high-mobility quantum well are demonstrated. Our approach leads to a dramatic reduction of radiative lifetime and junction area with respect to conventional vertical LEDs, promising very high-frequency device operation. Devices were fabricated by UV lithography and wet chemical etching starting from p-type modulation-doped AlGaAs/GaAs heterostructures grown by molecular beam epitaxy. Electrical and optical measurements from room temperature down to 1.8 K show high spectral purity and high external efficiency. Time-resolved measurements yielded extremely short recombination times of the order of 50 ps, demonstrating the relevance of the present scheme for high-frequency device applications in the GHz range. Comment: 5 pages, 3 figures
07/2002;
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ABSTRACT: Resonant transport is demonstrated in a hybrid superconductor-semiconductor heterostructure junction grown by molecular beam epitaxy on GaAs. This heterostructure realizes the model system introduced by de Gennes and Saint-James in 1963 [P. G. de Gennes and D. Saint-James, Phys. Lett. {\bf 4}, 151 (1963)]. At low temperatures a single marked resonance peak is shown superimposed to the characteristic Andreev-dominated subgap conductance. The observed magnetotransport properties are successfully analyzed within the random matrix theory of quantum transport, and ballistic effects are included by directly solving the Bogoliubov-de Gennes equations.
11/2001;
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ABSTRACT: Reflectionless-tunneling was observed in Nb/GaAs superconductor/semiconductor junctions fabricated through a two-step procedure. First, periodic $\delta$-doped layers were grown by molecular beam epitaxy near the GaAs surface, followed by an As cap layer to protect the surface during {\it ex-situ} transfer. Second, Nb was deposited by dc-magnetron sputtering onto the GaAs(001) 2 $\times$ 4 surface {\it in-situ} after thermal desorption of the cap layer. The magnetotransport behavior of the resulting hybrid junctions was successfully analyzed within the random matrix theory of phase-coherent Andreev transport. The impact of junction morphology on reflectionless tunneling and the applicability of the fabrication technique to the realization of complex superconductor/semiconductor mesoscopic systems are discussed. Comment: 10 pages, 3 figures, to be published in Appl. Phys. Lett
02/2001;
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ABSTRACT: The impact of junction transparency in driving phase-coherent charge transfer across diffusive semiconductor-superconductor junctions is demonstrated. We present conductivity data for a set of Nb-InAs junctions differing only in interface transparency. Our experimental findings are analyzed within the quasi-classical Green-function approach and unambiguously show the physical processes giving rise to the observed excess zero-bias conductivity. Comment: 10 pages (RevTex), 4 figures (PostScript), accepted for pubblication in Physical Review B
07/2000;
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Philosophical Magazine B. 04/2000; 80(4):817-823.
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ABSTRACT: A determination of the InAs/GaAs band-offset energy is presented. Electronic-transport analysis, based on capacitance-voltage and deep-level transient spectroscopy techniques, demonstrates high crystalline quality of our sample and yields a band-offset estimate of 0.69 eV, corresponding to a band-offset ratio of 7030. The present results agree well with reported theoretical values and allow the accurate modeling of electronic states in GaAs/AlGaAs heterostructures containing InAs ultrathin layers. © 2000 American Institute of Physics.
Applied Physics Letters 02/2000; 76(9):1146-1148. · 3.84 Impact Factor
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ABSTRACT: Electron transport at high electric fields is investigated in a periodic semiconductor heterostructure. We present the analysis of the magnetic-field dependence of resonant Zener tunneling from a valence-band subband to a conduction-band subband in a multiple-quantum-well heterostructure. It is shown that Zener tunneling can have a different physical origin in such structures with respect to bulk systems. The dominant role of holes trapped in the valence-band quantum wells in driving this process is demonstrated. © 1998 American Institute of Physics.
Applied Physics Letters 12/1998; 73(24):3553-3555. · 3.84 Impact Factor
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ABSTRACT: Andreev-reflection dominated transport is demonstrated in Al/n-In0.38Ga0.62As superconductor-semiconductor junctions grown by molecular beam epitaxy on GaAs(001). High junction transparency was achieved in low-doped devices by exploiting Si interface bilayers to suppress the native Schottky barrier. It is argued that this technique is ideally suited for the fabrication of ballistic transport hybrid microstructures. Comment: 9 REVTEX pages + 3 postscript figures, to be published in APL 73, (28dec98)
11/1998;
