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Integration of plasmonic nanostructures with fiber-optics-based neural probes enables label-free detection of molecular fingerprints via surface-enhanced Raman spectroscopy (SERS), and it represents a fascinating technological horizon to investigate brain function. However, developing neuroplasmonic probes that can interface with deep brain regions with minimal invasiveness while providing the sensitivity to detect biomolecular signatures in a physiological environment is challenging, in particular because the same waveguide must be employed for both delivering excitation light and collecting the resulting scattered photons. Here, a SERS-active neural probe based on a tapered optical fiber (TF) decorated with gold nanoislands (NIs) that can detect neurotransmitters down to the micromolar range is presented. To do this, a novel, nonplanar repeated dewetting technique to fabricate gold NIs with sub-10 nm gaps, uniformly distributed on the wide (square millimeter scale in surface area), highly curved surface of TF is developed. It is experimentally and numerically shown that the amplified broadband near-field enhancement of the high-density NIs layer allows for achieving a limit of detection in aqueous solution of 10−7 m for rhodamine 6G and 10−5 m for serotonin and dopamine through SERS at near-infrared wavelengths. The NIs-TF technology is envisioned as a first step toward the unexplored frontier of in vivo label-free plasmonic neural interfaces., M.D.V. and Fe.P. contributed equally to this work and are co-last-authors. D.Z., Li.C., R.M.-B., A.B., C.C., F.T., F.D.A., L.M.P., M.V., M.D.V., and Fe.P. acknowledge funding from the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 828972. Fi.P., A.B., B.S., and Fe.P. acknowledge funding from the European Research Council under the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 677683. Lu.C. acknowledges financial support by the Italian Ministry of Economic Development through the Project "GENESI"-Development of innovative radiopharmaceuticals and biomarkers for the diagnosis of tumors of the male and female reproductive apparatus (cod. F/180003/01-03/X43, Call MISE "Intelligent Factory, Agri food and Life Sciences"). Fi.P., M.D.V., and Fe.P. acknowledge that this project has received funding from the European Union's Horizon 2020 Research and Innovation Program under Grant Agreement No. 101016787. M.P., Fe.P., and M.D.V. were funded by the U.S. National Institutes of Health (Grant No. 1UF1NS108177-01).

Deciphering the neural patterns underlying brain functions is essential to understanding how neurons are organized into networks. This deciphering has been greatly facilitated by optogenetics and its combination with optoelectronic devices to control neural activity with millisecond temporal resolution and cell type specificity. However, targeting small brain volumes causes photoelectric artefacts, in particular when light emission and recording sites are close to each other. We take advantage of the photonic properties of tapered fibres to develop integrated ‘fibertrodes’ able to optically activate small brain volumes with abated photoelectric noise. Electrodes are positioned very close to light emitting points by non-planar microfabrication, with angled light emission allowing the simultaneous optogenetic manipulation and electrical read-out of one to three neurons, with no photoelectric artefacts, in vivo. The unconventional implementation of two-photon polymerization on the curved taper edge enables the fabrication of recoding sites all around the implant, making fibertrodes a promising complement to planar microimplants., B.S., A.B., M.B., F. Pisano and F. Pisanello acknowledge funding from the European Research Council under the European Union's Horizon 2020 research and innovation programme (no. 677683); M.P. and M.D.V. acknowledge funding from the European Research Council under the European Union's Horizon 2020 research and innovation programme (no. 692943). M.B., M.D.V. and F.Pisanello acknowledge funding from the European Research Council under the European Union's Horizon 2020 research and innovation programme (no. 966674). F. Pisano, M.D.V. and F. Pisanello acknowledge funding from the European Union's Horizon 2020 research and innovation programme (no. 101016787). L.S., M.D.V. and B.L.S. are funded by the US National Institutes of Health (U01NS094190). M.P., L.S., F. Pisanello, M.D.V. and B.L.S. are funded by the US National Institutes of Health (1UF1NS108177-01). A.B., F. Pisanello and M.D.V. also acknowledge funding from the European Union's Horizon 2020 research and innovation programme (no. 828972). We also acknowledge J. Lee for help setting up the optrode fibre launch system.

Two-photon (2P) lithography shows great potential for the fabrication of three dimensional micro- and nano- mechanical elements, for applications ranging from microelectromechanical systems to tissue engineering, by virtue of its high resolution (, The authors thank Marco Pisanello, Roberto Fiammengo and Alfonso Maffezzoli for useful discussion on data analysis.

Fiber photometry is widely used in neuroscience labs for in vivo detection of functional fluorescence from optical indicators of neuronal activity with a simple optical fiber. The fiber is commonly placed next to the region of interest to both excite and collect the fluorescence signal. However, the path of both excitation and fluorescence photons is altered by the uneven optical properties of the brain, due to local variation of the refractive index, different cellular types, densities and shapes. Nonetheless, the effect of the local anatomy on the actual shape and extent of the volume of tissue that interfaces with the fiber has received little attention so far. To fill this gap, we measured the size and shape of fiber photometry efficiency field in the primary motor and somatosensory cortex, in the hippocampus and in the striatum of the mouse brain, highlighting how their substructures determine the detected signal and the depth at which photons can be mined. Importantly, we show that the information on the spatial expression of the fluorescent probes alone is not sufficient to account for the contribution of local subregions to the overall collected signal, and it must be combined with the optical properties of the tissue adjacent to the fiber tip.

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In the present study we describe the in vitro pharmacological characterization of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) ligand Ac-RYYRWK-NH2 and the synthesis and biological evaluation of 13 Trp5 substituted Ac-RYYRWK-NH2 analogs. Results indicate that Ac-RYYRWK-NH2 behaves as a highly potent and selective partial agonist at the NOP receptors and that the whole indole moiety of the Trp5 side chain is not required, being a phenyl-ethyl side chain already sufficient for maintaining high potency.

Optogenetics promises spatiotemporal precise control of neural processes using light. However, the spatial extent of illumination within the brain is difficult to control and cannot be adjusted using standard fiber optics. We demonstrate that optical fibers with tapered tips can be used to illuminate either spatially restricted or large brain volumes. Remotely adjusting the light input angle to the fiber varies the light-emitting portion of the taper over several millimeters without movement of the implant. We use this mode to activate dorsal versus ventral striatum of individual mice and reveal different effects of each manipulation on motor behavior. Conversely, injecting light over the full numerical aperture of the fiber results in light emission from the entire taper surface, achieving broader and more efficient optogenetic activation of neurons when compared to the standard flat-faced fiber stimulation. Thus, tapered fibers permit focal or broad illumination that can be precisely and dynamically matched to experimental needs.

Physical and mechanical properties of extracellular matrix (ECM) have been proved to be crucial in the metastatic process. However, currently available studies on the interplay between ECM stiffness and cancer cell invasive behaviour are performed on planar assays, while the in vivo interaction takes place in three-dimensions. To take into consideration the ECM structural and mechanical complexity in the cell/structure interactions, we fabricated 3D microscaffolds through two-photon lithography (2PL) and tested how they are invaded by human colorectal adenocarcinoma (LS-174T) tumor cells, showing that it is possible to detect significant differences in cells/structure interaction when structural parameters are modified. In particular, both scaffold geometry and 2PL fabrication parameters were optimized to obtain 3D polymeric cylindrical structures with controlled Young's modulus and with linear stiffness gradients. The ability of LS-174T to migrate in the scaffolds was tested in different experimental conditions, including scaffolds functionalization and under β-catenin downregulation. It was observed that high Young's modulus scaffolds are always less invaded than softer ones, confirming the role of the 3D micro-environmental stiffness in mediating cells migration, including when specific functionalization or pharmacological treatments are performed.

Sperm cells progressive motility is the most important parameter involved in the fertilization process. Sperm middle piece contains mitochondria, which play a critical role in energy production and whose proper operation ensures the reproductive success. Notably, sperm progressive motility is strictly related to mitochondrial membrane potential (MMP) and consequently to mitochondrial functionality. Although previous studies presented an evaluation of mitochondrial function through MMP assessment in entire sperm cells samples, a quantitative approach at single-cell level could provide more insights in the analysis of semen quality. Here we combine laser scanning confocal microscopy and functional fluorescent staining of mitochondrial membrane to assess MMP distribution among isolated spermatozoa. We found that the sperm fluorescence value increases as a function of growing progressive motility and that such fluorescence is influenced by MMP disruptors, potentially allowing for the discrimination of different quality classes of sperm cells in heterogeneous populations.

Cells are highly dynamic elements, continuously interacting with the extracellular environment. Mechanical forces sensed and applied by cells are responsible for cellular adhesion, motility, and deformation, and are heavily involved in determining cancer spreading and metastasis formation. Cell/extracellular matrix interactions are commonly analyzed with the use of hydrogels and 3D microfabricated scaffolds. However, currently available techniques have a limited control over the stiffness of microscaffolds and do not allow for separating environmental properties from biological processes in driving cell mechanical behavior, including nuclear deformability and cell invasiveness. Herein, a new approach is presented to study tumor cell invasiveness by exploiting an innovative class of polymeric scaffolds based on two-photon lithography to control the stiffness of deterministic microenvironments in 3D. This is obtained by fine-tuning of the laser power during the lithography, thus locally modifying both structural and mechanical properties in the same fabrication process. Cage-like structures and cylindric stent-like microscaffolds are fabricated with different Young's modulus and stiffness gradients, allowing obtaining new insights on the mechanical interplay between tumor cells and the surrounding environments. In particular, cell invasion is mostly driven by softer architectures, and the introduction of 3D stiffness “weak spots” is shown to boost the rate at which cancer cells invade the scaffolds. The possibility to modulate structural compliance also allowed estimating the force distribution exerted by a single cell on the scaffold, revealing that both pushing and pulling forces are involved in the cell–structure interaction. Overall, exploiting this method to obtain a wide range of 3D architectures with locally engineered stiffness can pave the way for unique applications to study tumor cell dynamics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cancer cell motility is one of the major events involved in metastatic process. Tumor cells that disseminate from a primary tumor can migrate into the vascular system and, being carried by the bloodstream, transmigrate across the endothelium, giving rise to a new tumor site. However, during the invasive process, tumor cells must pass through the extracellular matrix, whose structural and mechanical properties define the parameters of the migration process. Here, we propose 3D-complex cage-like microstructures, realized by two-photon (TP) direct laser writing (DLW), to analyze cell migration through pores significantly smaller than the cell nucleus. We found that the ability to traverse differently sized pores depends on the metastatic potential and on the invasiveness of the cell lines, allowing to establish a pore-area threshold value able to discriminate between non-tumorigenic and tumorigenic human breast cells.

Two-photon (2P) lithography shows great potential for the fabrication of three-dimensional (3-D) micro- and nanomechanical elements, for applications ranging from microelectromechanical systems to tissue engineering, by virtue of its high resolution (