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Lipid Droplets (LDs) are emerging as crucial players in colon cancer development and maintenance. Their expression has been associated with high tumorigenicity in Cancer Stem Cells (CSCs), so that they have been proposed as a new functional marker in Colorectal Cancer Stem Cells (CR-CSCs). They are also indirectly involved in the modulation of the tumor microenvironment through the production of pro-inflammatory molecules. There is growing evidence that a possible connection between metabolic alterations and malignant transformation exists, although the effects of nutrients, primarily glucose, on the CSC behavior are still mostly unexplored. Glucose is an essential fuel for cancer cells, and the connections with LDs in the healthy and CSC populations merit to be more deeply investigated. Here, we showed that a high glucose concentration activated the PI3K/AKT pathway and increased the expression of CD133 and CD44v6 CSC markers. Additionally, glucose was responsible for the increased amount of Reactive Oxygen Species (ROS) and LDs in both healthy and CR-CSC samples. We also investigated the gene modulations following the HG treatment and found out that the healthy cell gene profile was the most affected. Lastly, Atorvastatin, a lipid-lowering drug, induced the highest mortality on CR-CSCs without affecting the healthy counterpart.

Imaging neuronal activity with high and homogeneous spatial resolution across the field-of-view (FOV) and limited invasiveness in deep brain regions is fundamental for the progress of neuroscience, yet is a major technical challenge. We achieved this goal by correcting optical aberrations in gradient index lens-based ultrathin (≤ 500 µm) microendoscopes using aspheric microlenses generated through 3D-microprinting. Corrected microendoscopes had extended FOV (eFOV) with homogeneous spatial resolution for two-photon fluorescence imaging and required no modification of the optical set-up. Synthetic calcium imaging data showed that, compared to uncorrected endoscopes, eFOV-microendoscopes led to improved signal-to-noise ratio and more precise evaluation of correlated neuronal activity. We experimentally validated these predictions in awake head-fixed mice. Moreover, using eFOV-microendoscopes we demonstrated cell-specific encoding of behavioral state-dependent information in distributed functional subnetworks in a primary somatosensory thalamic nucleus. eFOV-microendoscopes are, therefore, small-cross-section ready-to-use tools for deep two-photon functional imaging with unprecedentedly high and homogeneous spatial resolution.

We report the development and application of a new approach to correct aberrations in GRIN microendoscopes using 3D printed polymer microlenses. Corrected microendoscopes are validated performing functional imaging experiments in the mouse brain in vivo.

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.

Gold cuboid nanostructures with edge size in the range of 40-65 nm and inter-particle separation of 20 nm were fabricated by electron beam lithography. The aim of the present work is to investigate the effect of cuboid size on surface enhanced Raman scattering intensity and, thereby, to optimize the size of nanostructures in order to maximize signal enhancement. The electric field distribution of 4 × 4 array of Au cuboid nanostructures was numerically simulated by commercial software. A monolayer of Rhodamine-6G is deposited on the device using chemisorption technique, finding an enhancement factor ∼10 4, which candidates the cuboid nanostructures as a promising enhanced Raman substrate.

Graphical abstractDisplay Omitted Highlights? Fabrication and characterization of a polarization-selective Raman biosensor. ? Surface Plasmon Resonances in nanoantenna structures enhance the Raman signal. ? Optical transmittance and SERS signal confirm the polarization-dependent response. We report on the fabrication and spectroscopic characterization of a polarization-selective Raman sensor composed of an ordered array of planar nanoantennas. The device shows two distinct plasmon resonances associated with the main axes of the elongated nanostructures and is able to significantly enhance the Raman signal when the excitation wavelength matches one of these resonances.

The detection of a few molecules in a highly diluted solution is of paramount interest in fields including biomedicine, safety and eco-pollution in relation to rare and dangerous chemicals. Nanosensors based on plasmonics are promising devices in this regard, in that they combine the features of high sensitivity, label-free detection and miniaturization. However, plasmonic-based nanosensors, in common with general sensors with sensitive areas on the scale of nanometres, cannot be used directly to detect molecules dissolved in femto- or attomolar solutions. In other words, they are diffusion-limited and their detection times become impractical at such concentrations. In this Article, we demonstrate, by combining super-hydrophobic artificial surfaces and nanoplasmonic structures, that few molecules can be localized and detected even at attomolar (10−18 mol l−1) concentration. Moreover, the detection can be combined with fluorescence and Raman spectroscopy, such that the chemical signature of the molecules can be clearly determined.

Antonella Pujia1, Francesco De Angelis1,2, Domenica Scumaci3, Marco Gaspari3, Carlo Liberale1,2, Patrizio Candeloro1, Giovanni Cuda3, Enzo Di Fabrizio1,21BIONEM Laboratory, Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia”, Germaneto (CZ), Italy; 2IIT, Italian Institute of Technology, Genova, Italy; 3Proteomics and Mass Spectrometry Laboratory, Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Graecia”, Germaneto (CZ), ItalyBackground: Human serum has the potential to become the most informative source of novel biomarkers, but its study is very difficult due to the incredible complexity of its molecular composition. We describe a novel tool based on biodegradable nanoporous nanoparticles (NPNPs) that allows the harvesting of low-molecular-weight fractions of crude human serum or other biofluids. NPNPs with a diameter of 200 nm and pore size of a few nm were obtained by ultrasonication of nanoporous silicon. When incubated with a solution, the NPNPs harvest only the molecules small enough to be absorbed into the nanopores. Then they can be recovered by centrifugation and dissolved in water, making the harvested molecules available for further analyses.Results: Fluorescence microscopy, gel electrophoresis, and mass spectrometry were used to show the enrichment of low-molecular-weight fraction of serum under physiological conditions, with a cut-off of 13 kDa and an enrichment factor >50.Conclusion: From these findings, we conclude that ability to tune pore size, combined with the availability of hundreds of biomolecule cross-linkers, opens up new perspectives on complex biofluid analysis, discovery of biomarkers, and in situ drug delivery.Keywords: nanoporous silicon, nanoparticle, biomarker discovery, human serum proteomics, harvesting

In the last couple of decades we have been witnessing an enormous technological advancement in the field of micro-technology to the extent that nowadays we talk about nanotechnology. Faster computers, LCD based mobiles, nanoparticles for UV absorption in suntan lotions are just few of many examples where nanotechnology plays a fundamental role. The merit of this is mainly in the advance of the fabrication methods. Present techniques such as Focused Ion Beam (FIB) lithography guarantee a resolution of less than 10 nanometers which is about five times more precise than ten years before. Also Photonic Crystals (PhCs), among the others, take advantage from this extremely high resolution level allowing a downscale that permits the realization of structures which in principle can work at vey high energy. Historically PhCs were known as Bragg mirrors and only in 1987 (Yablonovitch, 1987; Sajeev, 1987) with the works of Yablonovitch and Sajeev the term Photonic Crystals was introduced. Nowadays, besides their natural application as filters in particular under full band gap conditions, PhCs see a number of applications: optical fibers (Birks et al., 1997; Zhao et al., 2010), vertical cavity surface emitting lasers (Yokouchi et al., 2003), high reflection coatings, temperature sensors (Song et al., 2006), high efficiency solar cells (Bermel et al., 2007), electric field detectors (Song & Proietti Zaccaria, 2007), non-linear analysis (Malvezzi et al., 2002; Malvezzi et al., 2003), just to name a few. Many are the techniques for the fabrication of PhCs, for example by means of focused-ion beam (Cabrini et al., 2005), two-photon fabrication (Deubel et al., 2004), laser-interference (Proietti Zaccaria et al., 2008a) or waver-fusion techniques (Takahashi et al., 2006). Here we shall focus on the role that PhCs can play for another exciting discipline known as Plasmonics. It refers to the capability of some devices of sustaining an optical surface mode, namely an electromagnetic wave travelling at the interface between two different materials such as a dielectric and a metal. Such a wave originates from the coupling of incident photons on the interface with

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., 5 pages, 3 figures

The fabrication of complex 3D plasmonic nanostructures opens new scenarios towards the realization of high electric field confinement and enhancement. We exploit the unique properties of these nanostructures for performing Raman spectroscopy in the single/few molecules detection limit.

We report on the possibility of realizing adiabatic compression of polaritonic wave on a metallic conical nano-structure through an oscillating electric potential (quasi dynamic regime). By comparing this result with an electromagnetic wave excitation, we were able to relate the classical lighting-rod effect to adiabatic compression. Furthermore, we show that while the magnetic contribution plays a marginal role in the formation of adiabatic compression, it provides a blue shift in the spectral region. In particular, magnetic permeability can be used as a free parameter for tuning the polaritonic resonances. The peculiar form of adiabatic compression is instead dictated by both the source and the metal permittivity. The analysis is performed by starting from a simple electrostatic system to end with the complete electromagnetic one through intermediate situations such as the quasi-electrostatic and quasi-dynamic regimes. Each configuration is defined by a particular set of equations which allows to clearly determine the individual role played by the electric and magnetic contribution in the generation of adiabatic compression. We notice that these findings can be applied for the realization of a THz nano-metric generator.

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.

In this work the design, fabrication and optical characterization of a polarization-sensitive L-shape nanoantenna device are reported. Such configuration supports plasmon-polariton modes that are combinations of in-phase and out-of-phase single antenna long-axis surface plasma oscillations. In the former case charges distributions induce in the gap region an intense hot spot while in the latter one a "zero-field spot"occurs in a plasmonic mode which can be referred to a non-zero dipolar momentum.

The aim of this chapter is to expound on the theoretical analysis and experimental assessment of NanoParticles (NPs) for imaging, early detection and therapeutic applications. NPs are extremely small particulates with dimensions ranging from few micrometers down to few tens of nanometers. Their characteristics, including size, shape, physical and chemical properties, can be tailored during the fabrication/synthesis process and, on account of these, they would feature certain aspects that may be exploited for applications ranging from drug delivery to the enhancement of the local electric field, and thus the detection of few molecules. In particular, intravascularly injectable NPs (that are sometimes called nanovectors or nanocarriers) are probably the major class of nanotechnological devices of interest for use in cancer or, in general, for the treatment of diseases. On the other hand, aggregates of metallic NPs, either of silver or gold, represent extremely efficient SERS (Surface Enhanced Raman Scattering) substrates. In the following, after a brief description of NPs as a whole, a number of different applications will be discussed.

Modification and functionalization of substrates can lead to the presence at materials surfaces of molecular nanostructures. Their 2D packing and chemical functionalities may impart to surfaces particular properties that can be very different from the pristine ones. Modification is a very general term concerning every kind of treatment leading to change the physical morphology or surface chemistry of a given material in order to obtain tailored properties with dependence on the material application field. Functionalization is mainly related to the introduction at a surface of chemical groups with a variable surface density which are requested to subsequently react with other species. When a process or a group of processes are applied to the surface of an inorganic or organicmaterial, affecting both topography/morphology and surface chemistry at the micro- and/or nanoscale level, the material surface results physically and chemically patterned. In the following the main modification, functionalization, and patterning surface techniques will be described according to process methods, substrate chemistry, and nanotechnological application fields

Porous silicon (PSi) is a promising material in several biomedical applications because of its biocompatibility and biodegradability. Despite the plethora of studies focusing on the interaction of cells with micrometer and submicro geometrical features, limited information is available on the response of cells to substrates with a quasi-regular distribution of nanoscopic pores. Here, the behavior of four different cell types is analyzed on two mesoporous (MeP) silicon substrates, with an average pore size of ∼5 (MeP1) and ∼20 nm (MeP2), respectively. On both MeP substrates, cells are observed to spread and adhere in a larger number as compared to flat silicon wafers. At all considered time points, the surface density of the adhering cells nd is larger on the PSi substrate with the smaller average pore size (MeP1). At 60 h, nd is from ∼1.5 to 5 times larger on MeP1 than on MeP2 substrates, depending on the cell type. The higher rates of proliferation are observed for the two neuronal cell types, the mouse neuroblastoma cells (N2A) and the immortalized human cortical neuronal cells (HCN1A). It is speculated that the higher adhesion on MeP1 could be attributed to a preferential matching of the substrate topography with the recently observed multiscale molecular architecture of focal adhesions. These results have implications in the rational development of PSi substrates for supporting cell adhesion and controlling drug release in implants and scaffolds for tissue engineering applications.

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.

Surface-enhanced Raman scattering (SERS) substrates were fabricated using electro-plating and e-beam lithography techniques. Nano-structures were obtained comprising regular arrays of gold nanoaggregates with a diameter of 80 nm and a mutual distance between the aggregates (gap) ranging from 10 to 30 nm. The nanopatterned SERS substrate enabled to have better control and reproducibility on the generation of plasmon polaritons (PPs). SERS measurements were performed for various proteins, namely bovine serum albumin (BSA), myoglobin, ferritin, lysozyme, RNase-B, α-casein, α-lactalbumin and trypsin. Principal component analysis (PCA) was used to organize and classify the proteins on the basis of their secondary structure. Cluster analysis proved that the error committed in the classification was of about 14%. In the paper, it was clearly shown that the combined use of SERS measurements and PCA analysis is effective in categorizing the proteins on the basis of secondary structure.

Doxorubicin (DOXO) is a powerful anthracycline antibiotic used to treat many human neoplasms, including acute leukemias, lymphomas, stomach, breast and ovarian cancer, and bone tumors, yet causing cardiotoxicity at the same time. For this reason, there is a great interest in medical field to gain deep insight and knowledge of this molecule. Raman, Fourier Transform Infrared (FT-IR) absorption spectroscopy, and Resonance Raman scattering were performed for the vibrational characterization of DOXO molecule. Density function theorem (DFT) modeling of Raman and FT-IR spectra were used for the assignment of the vibrational frequencies. The optimized molecular structured was obtained, first, on the basis of potential energy distribution. The simulation for vibrational bands is based on the calculations for internal force constants and potential energy distribution matrices. The calculated DOXO vibrational bands show qualitative agreement with the experimental observations (FT-IR absorption and Raman scattering).

Micro and nano-patterned devices were fabricated which disclose the possibility of concentrating, localizing, detecting and thus analyzing with unprecedented accuracy few molecules (at the limit a single molecule) of biological and medical interest. Important applications are envisioned where proteins poorly concentrated in blood are detected thus (early) revealing the appearance of diseases. The major advance of the paper is that it combines micro/nano-fabrication and spectroscopical nano-optics based techniques to study and individualize the biomolecules at study. Super hydrophobic surface would concentrate the diluted solution into a small area whereas spectroscopical nano-optics techniques would gather the molecular/chemical information of the residual solute. Fluorescence and SERS techniques were used to analyze Rhodamine molecules initially highly diluted (10^-^1^8M).

We describe the fabrication of different micro-optical structures on top of optical fibers using two-photon polymerization. We show the convenience of this approach to quickly create generic three-dimensional shapes using a single setup by comparison to previous shape-dependent methods. A set of different structures, designed for different optical functions, are fabricated and characterized to demonstrate the versatility of this approach and their high optical quality.

The aim of this paper is to demonstrate that efficient fiber-waveguide optical coupling can be achieved using a multilevel phase diffractive element (PDE) fabricated directly on the top of the fiber by means of e-beam lithography. The diffractive phase element is calculated to focus and reshape the gaussian symmetric beam exiting a single-mode fiber into a desired asymmetric intensity distribution at the waveguide input plane. Phase modulation is obtained by multilevel profiling a polymeric material coated on the top of the fiber by means of a specific fabrication process including e-beam lithography and chemical etching. Experimental results obtained for fiber-waveguide coupling with a 20-µm diameter diffractive element are also presented.

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.