The use of scanning electron microscopy and energy dispersive X-ray spectroscopy in a case of occupational death.

Scanning electron microscopy is a technique that provides high-resolution images at the micro- and nano-scale. The combination of scanning electron microscopy and energy dispersive X-ray spectroscopy analysis is developing fast for application in forensic science. In this work, we report a case of work-related traumatic death of a 50-year-old man. The autopsy showed cranial fractures with cerebral haemorrhage. It was more difficult to understand the accident dynamics because the body had been shifted from the accident site to mask what had really taken place. Scanning electron microscopy/energy dispersive X-ray spectroscopy was used to identify the material of the impacting tool and to establish the possible legal responsibility of the employer. In this study, we demonstrate that scanning electron microscopy/energy dispersive X-ray spectroscopy is a useful forensic tool for the analysis of biological samples. Further, for studying the lacerations on the corpse from doubtful blunt tools, scanning electron microscopy/energy dispersive X-ray spectroscopy can assist in demonstrating that the scene has been falsified, as it was in this case.

Relating the rate of growth of metal nanoparticles to cluster size distribution in electroless deposition.

Electroless deposition on patterned silicon substrates enables the formation of metal nanomaterials with tight control over their size and shape. In the technique, metal ions are transported by diffusion from a solution to the active sites of an autocatalytic substrate where they are reduced as metals upon contact. Here, using diffusion limited aggregation models and numerical simulations, we derived relationships that correlate the cluster size distribution to the total mass of deposited particles. We found that the ratio ξ between the rates of growth of two different metals depends on the ratio γ between the rates of growth of clusters formed by those metals through the linearity law ξ = 14(γ - 1). We then validated the model using experiments. Different from other methods, the model derives k using as input the geometry of metal nanoparticle clusters, decoded by SEM or AFM images of samples, and a known reference.

Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour.

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual's diagnosis and/or help clarify risk in healthy populations.

Silica diatom shells tailored with Au nanoparticles enable sensitive analysis of molecules for biological, safety and environment applications.

Diatom shells are a natural, theoretically unlimited material composed of silicon dioxide, with regular patterns of pores penetrating through their surface. For their characteristics, diatom shells show promise to be used as low cost, highly efficient drug carriers, sensor devices or other micro-devices. Here, we demonstrate diatom shells functionalized with gold nanoparticles for the harvesting and detection of biological analytes (bovine serum albumin-BSA) and chemical pollutants (mineral oil) in low abundance ranges, for applications in bioengineering, medicine, safety, and pollution monitoring.

An Overview of Lipid Droplets in Cancer and Cancer Stem Cells.

For decades, lipid droplets have been considered as the main cellular organelles involved in the fat storage, because of their lipid composition. However, in recent years, some new and totally unexpected roles have been discovered for them: (i) they are active sites for synthesis and storage of inflammatory mediators, and (ii) they are key players in cancer cells and tissues, especially in cancer stem cells. In this review, we summarize the main concepts related to the lipid droplet structure and function and their involvement in inflammatory and cancer processes.

Nano-topography Enhances Communication in Neural Cells Networks.

Neural cells are the smallest building blocks of the central and peripheral nervous systems. Information in neural networks and cell-substrate interactions have been heretofore studied separately. Understanding whether surface nano-topography can direct nerve cells assembly into computational efficient networks may provide new tools and criteria for tissue engineering and regenerative medicine. In this work, we used information theory approaches and functional multi calcium imaging (fMCI) techniques to examine how information flows in neural networks cultured on surfaces with controlled topography. We found that substrate roughness S a affects networks topology. In the low nano-meter range, S a = 0-30 nm, information increases with S a . Moreover, we found that energy density of a network of cells correlates to the topology of that network. This reinforces the view that information, energy and surface nano-topography are tightly inter-connected and should not be neglected when studying cell-cell interaction in neural tissue repair and regeneration.

Imaging and structural studies of DNA-protein complexes and membrane ion channels.

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

Nanoscale reduction of graphene oxide thin films and its characterization.

In this paper, we report on a method to reduce thin films of graphene oxide (GO) to a spatial resolution better than 100 nm over several tens of micrometers by means of an electrochemical scanning probe based lithography. In situ tip-current measurements show that an edged drop in electrical resistance characterizes the reduced areas, and that the reduction process is, to a good approximation, proportional to the applied bias between the onset voltage and the saturation thresholds. An atomic force microscope (AFM) quantifies the drop of the surface height for the reduced profile due to the loss of oxygen. Complementarily, lateral force microscopy reveals a homogeneous friction coefficient of the reduced regions that is remarkably lower than that of native graphene oxide, confirming a chemical change in the patterned region. Micro Raman spectroscopy, which provides access to insights into the chemical process, allows one to quantify the restoration and de-oxidation of the graphitic network driven by the electrochemical reduction and to determine characteristic length scales. It also confirms the homogeneity of the process over wide areas. The results shown were obtained from accurate analysis of the shift, intensity and width of Raman peaks for the main vibrational bands of GO and reduced graphene oxide (rGO) mapped over large areas. Concerning multilayered GO thin films obtained by drop-casting we have demonstrated an unprecedented lateral resolution in ambient conditions as well as an improved control, characterization and understanding of the reduction process occurring in GO randomly folded multilayers, useful for large-scale processing of graphene-based material.

Behaviour of dental pulp stem cells on different types of innovative mesoporous and nanoporous silicon scaffolds with different functionalizations of the surfaces.

Dental pulp stem cells (DPSCs) are stem cells found in the dental pulp. The ability of DPSCs to differentiate towards odontoblastic and osteoblastic phenotype was reported first in the literature, then in the following years, numerous studies on odontogenesis were carried out, starting from mesenchymal stem cells isolated from tissues of dental and oral origin. The aim of this research was to evaluate the behaviour of DPSCs grown on silicon nanoporous and mesoporous matrices and differentiated towards the osteogenic phenotype, but also to investigate the use of DPSCs in pilot studies focused on the biological compatibility of innovative dental biomaterials. Twenty-eight silicon samples were created with standardized procedures. These scaffolds were divided into samples made of silicon bulk, nanoporous silicon, mesoporous silicon, nanoporous silicon functionalized with (3-Aminopropyl) Trimethoxysilane (APTMS) and methanol (MeOH), nanoporous silicon functionalized with (3-Aminopropyl) Trimethoxysilane (APTMS)/toluene, mesoporous silicon functionalized with (3-Aminopropyl) Trimethoxysilane (APTMS) and methanol (MeOH) andmesoporous silicon functionalized with (3-Aminopropyl) Trimethoxysilane (APTMS)/toluene. DPSC proliferation on the tested silicon scaffolds was analyzed at 3 and 5 days. The assay showed that DPSCs proliferated better on mesoporous scaffolds functionalized with APTMS/toluene compared to a silicon one. These results show that the functionalization of silicon scaffold with APTMS/toluene supports the growth of DPSCs and could be used for future applications in tissue engineering.

Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application.

AIM: Consistent expansion of primary human endothelial cells in vitro is critical in the development of engineered tissue. A variety of complex culture media and techniques developed from different basal media have been reported with alternate success. Incongruous results are further confounded by donor-to-donor variability and cellular source of derivation. Our results demonstrate how to overcome these limitations using soluble CD54 (sCD54) as additive to conventional culture medium. METHODS AND RESULTS: Isolated primary fragment of different vessel types was expanded in Ham's F12 DMEM, enriched with growth factors, Fetal Calf Serum and conditioned medium of Human Umbilical Vein Endothelial Cells (HUVEC) collected at different passages. Cytokine content of culture media was analyzed in order to identify the soluble factors correlating with better proliferation profile. sCD54 was found to induce the in vitro expansion of human endothelial cells (HECs) independently from the vessels source and even in the absence of HUVEC-conditioned medium. The HECs cultivated in the presence of sCD54 (50 ng/ml), resulted positive for the expression of CD146 and negative for CD45, and lower fibroblast contamination. Cells were capable to proliferate with an S phase of 25%, to produce vascular endothelial growth factor, VEGF, (10 ng/ml) and to give origin to vessel-like tubule in vitro. CONCLUSION: Our results demonstrate that sCD54 is an essential factor for the in-vitro expansion of HECs without donor and vessel-source variability. Resulting primary cultures can be useful, for tissue engineering in regenerative medicine (e.g. artificial micro tissue generation, coating artificial heart valve etc.) and bio-nanotechnology applications.

An optimized table-top small-angle X-ray scattering set-up for the nanoscale structural analysis of soft matter.

The paper shows how a table top superbright microfocus laboratory X-ray source and an innovative restoring-data algorithm, used in combination, allow to analyze the super molecular structure of soft matter by means of Small Angle X-ray Scattering ex-situ experiments. The proposed theoretical approach is aimed to restore diffraction features from SAXS profiles collected from low scattering biomaterials or soft tissues, and therefore to deal with extremely noisy diffraction SAXS profiles/maps. As biological test cases we inspected: i) residues of exosomes' drops from healthy epithelial colon cell line and colorectal cancer cells; ii) collagen/human elastin artificial scaffolds developed for vascular tissue engineering applications; iii) apoferritin protein in solution. Our results show how this combination can provide morphological/structural nanoscale information to characterize new artificial biomaterials and/or to get insight into the transition between healthy and pathological tissues during the progression of a disease, or to morphologically characterize nanoscale proteins, based on SAXS data collected in a room-sized laboratory.

In vitro expansion of tumour cells derived from blood and tumour tissue is useful to redefine personalized treatment in non-small cell lung cancer patients.

The clinical development of locally and advanced non-small cell lung cancer (NSCLC) suffers from a lack of biomarkers as a guide in the selection of optimal prognostic prediction. Circulating Tumour Cells (CTCs) are correlated to prognosis and show efficacy in cancer monitoring in patients. However, their enumeration alone might be inadequate; it might also be critical to understand the viability, the apoptotic state and the kinetics of these cells. Here, we report what we believe to be a new and selective approach to visually detect tumour specific CTCs. Firstly, using labelled human lung cancer cells, we detected a specific density interval in which NSCL-CTCs were concentrated. Secondly, to better characterize CTCs in respect to their heterogeneous composition and tumour reference, blood and tumour biopsy were performed on specimens taken from the same patient. The approach consisted in comparing phenotype profile of CTCs, and their progenitor Tumour Stem Cells, (TSCs). Moreover, NSCL-CTCs were cultivated in short-time human cultures to provide response to drug sensitivity. Our bimodal approach allowed to reveal two items. Firstly, that one part of a tumour, proximal to the bronchial structure, displays a predominance of CD133+. Secondly, specific NSCL-CTCs Epithelial Cell Adhesion Molecule (EpCAM)+CD29+ can be used as a negative prognostic factor as well the high expression of CTCs EpCAM+. These data were confirmed by drug-sensitivity tests, in vitro, and by the survival curves, in vivo.

Focusing and imaging with increased numerical apertures through multimode fibers with micro-fabricated optics.

The use of individual multimode optical fibers in endoscopy applications has the potential to provide highly miniaturized and noninvasive probes for microscopy and optical micromanipulation. A few different strategies have been proposed recently, but they all suffer from intrinsically low resolution related to the low numerical aperture of multimode fibers. Here, we show that two-photon polymerization allows for direct fabrication of micro-optics components on the fiber end, resulting in an increase of the numerical aperture to a value that is close to 1. Coupling light into the fiber through a spatial light modulator, we were able to optically scan a submicrometer spot (300 nm FWHM) over an extended region, facing the opposite fiber end. Fluorescence imaging with improved resolution is also demonstrated.

Hot-electron nanoscopy using adiabatic compression of surface plasmons.

Surface plasmon polaritons are a central concept in nanoplasmonics and have been exploited to develop ultrasensitive chemical detection platforms, as well as imaging and spectroscopic techniques at the nanoscale. Surface plasmons can decay to form highly energetic (or hot) electrons in a process that is usually thought to be parasitic for applications, because it limits the lifetime and propagation length of surface plasmons and therefore has an adverse influence on the functionality of nanoplasmonic devices. Recently, however, it has been shown that hot electrons produced by surface plasmon decay can be harnessed to produce useful work in photodetection, catalysis and solar energy conversion. Nevertheless, the surface-plasmon-to-hot-electron conversion efficiency has been below 1% in all cases. Here we show that adiabatic focusing of surface plasmons on a Schottky diode-terminated tapered tip of nanoscale dimensions allows for a plasmon-to-hot-electron conversion efficiency of ∼30%. We further demonstrate that, with such high efficiency, hot electrons can be used for a new nanoscopy technique based on an atomic force microscopy set-up. We show that this hot-electron nanoscopy preserves the chemical sensitivity of the scanned surface and has a spatial resolution below 50 nm, with margins for improvement.

Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay.

Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of ß-galactosides and galectin-3--a protein that is correlated to the progress of tumor and metastasis--is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction's strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine.

Integrated microfluidic device for single-cell trapping and spectroscopy.

Optofluidic microsystems are key components towards lab-on-a-chip devices for manipulation and analysis of biological specimens. In particular, the integration of optical tweezers (OT) in these devices allows stable sample trapping, while making available mechanical, chemical and spectroscopic analyses.

Laser synthesis of ligand-free bimetallic nanoparticles for plasmonic applications.

A picosecond laser ablation approach has been developed for the synthesis of ligand-free AuAg bimetallic NPs where the relative amount of Ag is controlled in situ through a laser shielding effect. Various measurements, such as optical spectroscopy, transmission electron microscopy combined with energy dispersive X-ray spectroscopy and inductively coupled plasma optical emission spectrometry, revealed the generation of homogenous 15 nm average size bimetallic NPs with different compositions and tunable localized surface plasmon resonance. Furthermore, ligand-free metallic nanoparticles with respect to chemically synthesized nanoparticles display outstanding properties, i.e. featureless Raman background spectrum, which is a basic requirement in many plasmonic applications such as Surface Enhanced Raman Spectroscopy. Various molecules were chemisorbed on the nanoparticle and SERS investigations were carried out, by varying the laser wavelength. The SERS enhancement factor for AuAg bimetallic NPs shows an enhancement factor of about 5.7 × 10(5) with respect to the flat AuAg surface.

Electron-beam-driven collective-mode metamaterial light source.

We demonstrate experimentally that the energy from a highly localized free-electron-beam excitation can be converted via a planar plasmonic metamaterial to a low-divergence free-space light beam. This emission, which emanates from a collectively oscillating coupled metamolecule nanoantenna ensemble much larger in size than the initial excitation, is distinctly different from cathodoluminescence and bears some similarity with laser light. It offers a novel, flexible paradigm for the development of scalable, threshold-free light sources.

AFM characterization of biomolecules in physiological environment by an advanced nanofabricated probe.

Many relevant questions in biology and medicine require both topography and chemical information with high spatial resolution. Several biological events that occur at the nanometer scale level need to be investigated in physiological conditions. In this regard Atomic Force Microscopy (AFM) is one of the most powerful tools for label-free nanoscale characterization of biological samples in liquid environment. Recently, the coupling of Raman spectroscopy to scanning probe microscopies has opened new perspectives on this subject; however, the coupling of quality AFM spectroscopy with Raman spectroscopy in the same probe is not trivial. In this work we report about the AFM capabilities of an advanced high-resolution probe that has been previously nanofabricated by our group for coupling with Raman spectroscopy applications. We investigate its use for liquid AFM measurements on biological model samples like lipid bilayers, amyloid fibrils, and titin proteins. We demonstrate topography resolution down to nanometer level, force measurement and stable imaging capability. We also discuss about its potential as nanoscale chemical probe in liquid phase.

Multi-scheme approach for efficient surface plasmon polariton generation in metallic conical tips on AFM-based cantilevers.

We report on the possibility of realizing adiabatic surface plasmon polaritons compression on metallic conical tips built-in on AFM cantilevers by means of different approaches. The problem is faced considering the role of the source, when linear and radial polarizations are assumed, associated to different fabrication schemes. Nano-patterned devices properly combined with metallic conical tips can affect the adiabatic characteristic of the surface electric field. The results are analyzed in terms of tradeoff between fabrication difficulties and device performances. Suggestions on the best possible scheme are provided.