Tapered fibertrodes for optoelectrical neural interfacing in small brain volumes with reduced artefacts.

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.

Holographic Manipulation of Nanostructured Fiber Optics Enables Spatially-Resolved, Reconfigurable Optical Control of Plasmonic Local Field Enhancement and SERS.

Integration of plasmonic structures on step-index optical fibers is attracting interest for both applications and fundamental studies. However, the possibility to dynamically control the coupling between the guided light fields and the plasmonic resonances is hindered by the turbidity of light propagation in multimode fibers (MMFs). This pivotal point strongly limits the range of studies that can benefit from nanostructured fiber optics. Fortunately, harnessing the interaction between plasmonic modes on the fiber tip and the full set of guided modes can bring this technology to a next generation progress. Here, the intrinsic wealth of information of guided modes is exploited to spatiotemporally control the plasmonic resonances of the coupled system. This concept is shown by employing dynamic phase modulation to structure both the response of plasmonic MMFs on the plasmonic facet and their response in the corresponding Fourier plane, achieving spatial selective field enhancement and direct control of the probe's work point in the dispersion diagram. Such a conceptual leap would transform the biomedical applications of holographic endoscopic imaging by integrating new sensing and manipulation capabilities.

Depth-resolved fiber photometry with a single tapered optical fiber implant.

Fiber photometry is increasingly utilized to monitor fluorescent sensors of neural activity in the brain. However, most implementations are based on flat-cleaved optical fibers that can only interface with shallow tissue volumes adjacent to the fiber. We exploit modal properties of tapered optical fibers (TFs) to enable light collection over an extent of up to 2 mm of tissue and multisite photometry along the taper. Using a single TF, we simultaneously observed distinct dopamine transients in dorsal and ventral striatum in freely moving mice performing a simple, operant conditioning task. Collection volumes from TFs can also be engineered in both shape and size by microstructuring the nonplanar surface of the taper, to optically target multiple sites not only in the deep brain but, in general, in any biological system or organ in which light collection is beneficial but challenging because of light scattering and absorption.

Numerical Calculation of the Light Propagation in Tapered Optical Fibers for Optical Neural Interfaces.

As implantable optical systems recently enabled new approaches to study the brain with optical radiations, tapered optical fibers emerged as promising implantable waveguides to deliver and collect light from sub-cortical structures of the mouse brain. They rely on a specific feature of multimodal fiber optics: as the waveguide narrows, the number of guided modes decreases and the radiation can gradually couple with the environment. This happens along a taper segment whose length can be tailored to match with the depth of functional structures of the mouse brain, and can extend for a few millimeters. This anatomical requirement results in optical systems which have an active area that is very long compared to the wavelength of the light they guide and their behavior is typically estimated by ray tracing simulations, because finite element methods are too computationally demanding. Here we present a computational technique that exploits the beam-envelope method and the cylindrical symmetry of the fibers to provide an efficient and exact calculation of the electric field along the fibers, which may enable the design of neural interfaces optimized to meet different goals.

Two-photon fluorescence-assisted laser ablation of non-planar metal surfaces: fabrication of optical apertures on tapered fibers for optical neural interfaces.

We propose a feedback-assisted direct laser writing method to perform laser ablation of fiber optic devices in which their light-collection signal is used to optimize their properties. A femtosecond-pulsed laser beam is used to ablate a metal coating deposited around a tapered optical fiber, employed to show the suitability of the approach to pattern devices with a small radius of curvature. During processing, the same pulses generate two-photon fluorescence in the surrounding environment and the signal is monitored to identify different patterning regimes over time through spectral analysis. The employed fs beam mostly interacts with the metal coating, leaving almost intact the underlying silica and enabling fluorescence to couple with a specific subset of guided modes, as verified by far-field analysis. Although the method is described here for tapered optical fibers used to obtain efficient light collection in the field of optical neural interfaces, it can be easily extended to other waveguide-based devices and represents a general approach to support the implementation of a closed-loop laser ablation system of fiber optics.

Ray tracing models for estimating light collection properties of microstructured tapered optical fibers for optical neural interfaces.

Tapered optical fibers (TFs) were recently employed for depth-resolved monitoring of functional fluorescence in subcortical brain structures, enabling light collection from groups of a few cells through small optical windows located on the taper edge [Pisano et al., Nat. Methods16, 1185 (2019)1548-709110.1038/s41592-019-0581-x]. Here we present a numerical model to estimate light collection properties of microstructured TFs implanted in scattering brain tissue. Ray tracing coupled with the Henyey-Greenstein scattering model enables the estimation of both light collection and fluorescence excitation fields in three dimensions, whose combination is employed to retrieve the volume of tissue probed by the device.

High transmission from 2D periodic plasmonic finite arrays with sub-20 nm gaps realized with Ga focused ion beam milling.

Fabricating plasmonic nanostructures with good optical performances often requires lengthy and challenging patterning processes that can hardly be transferred to unconventional substrates, such as optical fiber tips or curved surfaces. Here we investigate the use of a single Ga focused ion beam process to fabricate 2D arrays of gold nanoplatelets for nanophotonic applications. While observing that focused ion beam milling of crossing tapered grooves inherently produces gaps below 20 nm, we provide experimental and theoretical evidence for the spectral features of grooves terminating with a sharp air gap. We show that transmission near 10% can be obtained via two-dimensional nano-focusing in a finite subset of 2D arrays of gold nanoplatelets. This enables the application of our nanostructure to detect variations in the refractive index of thin films using either reflected or transmitted light when a small number of elements are engaged.

Focused ion beam nanomachining of tapered optical fibers for patterned light delivery.

With the advent of optogenetic techniques, a major need for precise and versatile light-delivery techniques has arisen from the neuroscience community. Driven by this demand, research on innovative illuminating devices has opened previously inaccessible experimental paths. However, tailoring light delivery to functionally and anatomically diverse brain structures still remains a challenging task. We progressed in this endeavor by micro-structuring metal-coated tapered optical fibers and exploiting the resulting mode-division multiplexing/demultiplexing properties. To do this, a non-conventional Focused Ion Beam (FIB) milling method was developed in order to pattern the non-planar surface of the taper around the full 360°, by equipping the FIB chamber with a micromanipulation system. This led us to develop three novel typologies of micro-structured illuminating tools: (a) a tapered fiber that emits light from a narrow slot of adjustable length; (b) a tapered fiber that emits light from four independently addressable optical windows; (c) a tapered fiber that emits light from an annular aperture with 360° symmetry. The result is a versatile technology enabling reconfigurable light-delivery that can be tailored to specific experimental needs.

Toward Plasmonic Neural Probes: SERS Detection of Neurotransmitters through Gold-Nanoislands-Decorated Tapered Optical Fibers with Sub-10 nm Gaps.

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.

An open source three-mirror laser scanning holographic two-photon lithography system.

Two-photon polymerization is a widely adopted technique for direct fabrication of 3D and 2D structures with sub-diffraction-limit features. Here we present an open-hardware, open-software custom design for a holographic multibeam two-photon polymerization system based on a phase-only spatial light modulator and a three-mirror scanhead. The use of three reflective surfaces, two of which scanning the phase-modulated image along the same axis, allows to overcome the loss of virtual conjugation within the large galvanometric mirrors pair needed to accommodate the holographic projection. This extends the writing field of view among which the hologram can be employed for multi-beam two-photon polymerization by a factor of ~2 on one axis (i.e. from ~200µm to ~400µm), with a voxel size of ~250nm × ~1050nm (lateral × axial size), and writing speed of three simultaneous beams of 2000 voxels/s, making our system a powerful and reliable tool for advanced micro and nano-fabrications on large area.

Orthogonalization of far-field detection in tapered optical fibers for depth-selective fiber photometry in brain tissue.

The field of implantable optical neural interfaces has recently enabled the interrogation of neural circuitry with both cell-type specificity and spatial resolution in sub-cortical structures of the mouse brain. This generated the need to integrate multiple optical channels within the same implantable device, motivating the requirement of multiplexing and demultiplexing techniques. In this article, we present an orthogonalization method of the far-field space to introduce mode-division demultiplexing for collecting fluorescence from the implantable tapered optical fibers. This is achieved by exploiting the correlation between the transversal wavevector k t of the guided light and the position of the fluorescent sources along the implant, an intrinsic property of the taper waveguide. On these bases, we define a basis of orthogonal vectors in the Fourier space, each of which is associated with a depth along the taper, to simultaneously detect and demultiplex the collected signal when the probe is implanted in fixed mouse brain tissue. Our approach complements the existing multiplexing techniques used in silicon-based photonics probes with the advantage of a significant simplification of the probe itself.

Comparative study of autofluorescence in flat and tapered optical fibers towards application in depth-resolved fluorescence lifetime photometry in brain tissue.

As the scientific community seeks efficient optical neural interfaces with sub-cortical structures of the mouse brain, a wide set of technologies and methods is being developed to monitor cellular events through fluorescence signals generated by genetically encoded molecules. Among these technologies, tapered optical fibers (TFs) take advantage of the modal properties of narrowing waveguides to enable both depth-resolved and wide-volume light collection from scattering tissue, with minimized invasiveness with respect to standard flat fiber stubs (FFs). However, light guided in patch cords as well as in FFs and TFs can result in autofluorescence (AF) signal, which can act as a source of time-variable noise and limit their application to probe fluorescence lifetime in vivo. In this work, we compare the AF signal of FFs and TFs, highlighting the influence of the cladding composition on AF generation. We show that the autofluorescence signal generated in TFs has a peculiar coupling pattern with guided modes, and that far-field detection can be exploited to separate functional fluorescence from AF. On these bases, we provide evidence that TFs can be employed to implement depth-resolved fluorescence lifetime photometry, potentially enabling the extraction of a new set of information from deep brain regions, as time-correlating single photon counting starts to be applied in freely-moving animals to monitor the intracellular biochemical state of neurons.

Influence of the anatomical features of different brain regions on the spatial localization of fiber photometry signals.

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.

Single-cell micro- and nano-photonic technologies.

Since the advent of optogenetics, the technology development has focused on new methods to optically interact with single nerve cells. This gave rise to the field of photonic neural interfaces, intended as the set of technologies that can modify light radiation in either a linear or non-linear fashion to control and/or monitor cellular functions. This set includes the use of plasmonic effects, up-conversion, electron transfer and integrated light steering, with some of them already implemented in vivo. This article will review available approaches in this framework, with a particular emphasis on methods operating at the single-unit level or having the potential to reach single-cell resolution.

The Three-Dimensional Signal Collection Field for Fiber Photometry in Brain Tissue.

Fiber photometry is used to monitor signals from fluorescent indicators in genetically-defined neural populations in behaving animals. Recently, fiber photometry has rapidly expanded and it now provides researchers with increasingly powerful means to record neural dynamics and neuromodulatory action. However, it is not clear how to select the optimal fiber optic given the constraints and goals of a particular experiment. Here, using combined confocal/2-photon microscope, we quantitatively characterize the fluorescence collection properties of various optical fibers in brain tissue. We show that the fiber size plays a major role in defining the volume of the optically sampled brain region, whereas numerical aperture impacts the total amount of collected signal and, marginally, the shape and size of the collection volume. We show that ~80% of the effective signal arises from 105 to 106 µm3 volume extending ~200 µm from the fiber facet for 200 µm core optical fibers. Together with analytical and ray tracing collection maps, our results reveal the light collection properties of different optical fibers in brain tissue, allowing for an accurate selection of the fibers for photometry and helping for a more precise interpretation of measurements in terms of sampled volume.

A Wireless Head-mountable Device with Tapered Optical Fiber-coupled Laser Diode for Light Delivery in Deep Brain Regions.

Optogenetics sets new experimental paradigms that can reveal cell type-specific contributions on the neural basis of behavior. Since most of the available systems for this purpose are based on approaches that tether animals to a set of cables, recent research activities have been focused on minimizing external factors that can alter animal movements. Current wireless optogenetic systems are based on waveguide-coupled LED and implanted LEDs. However, each configuration separately suffers from significant limitations, such as low coupling efficiency, penetration depth and invasiveness of waveguide-coupled LED, and local heat generated by implanted µLEDs. This work presents a novel wireless head-mountable stimulating system for a wide-volume light delivery. The device couples the output of a semiconductor laser diode (LD) to a tapered optical fiber (TF) on a wireless platform. The LD-TF coupling was engineered by setting up far-field analysis, which allows the full exploitation of the mode division demultiplexing properties of TFs. The output delivered light along the tapered segment is capable of stimulating structures of depths up to ~2mm. TFs are tapered to a gradual taper angle (2° to 10°) that ends with a sharp tip (~500 nm) for smooth insertion and less invasiveness. Thus, the proposed system extends the capabilities of wireless optogenetic by offering a novel solution for wide volume light delivery in deep brain regions.

Tailoring light delivery for optogenetics by modal demultiplexing in tapered optical fibers.

Optogenetic control of neural activity in deep brain regions ideally requires precise and flexible light delivery with non-invasive devices. To this end, Tapered Optical Fibers (TFs) represent a versatile tool that can deliver light over either large brain volumes or spatially confined sub-regions, while being sensibly smaller than flat-cleaved optical fibers. In this work, we report on the possibility of further extending light emission length along the taper in the range 0.4 mm-3.0 mm by increasing the numerical aperture of the TFs to NA = 0.66. We investigated the dependence between the input angle of light (θin) and the output position along the taper, finding that for θin > 10° this relationship is linear. This mode-division demultiplexing property of the taper was confirmed with a ray tracing model and characterized for 473 nm and 561 nm light in quasi-transparent solution and in brain slices, with the two wavelengths used to illuminate simultaneously two different regions of the brain using only one waveguide. The results presented in this manuscript can guide neuroscientists to design their optogenetic experiments on the base of this mode-division demultiplexing approach, providing a tool that potentially allow for dynamic targeting of regions with diverse extension, from the mouse VTA up to the macaque visual cortex.

Tapered Fibers Combined With a Multi-Electrode Array for Optogenetics in Mouse Medial Prefrontal Cortex.

Optogenetics offers many advantages in terms of cell-type specificity, allowing to investigate functional connectivity between different brain areas at high spatial and neural population selectivity. In order to obtain simultaneous optical control and electrical readout of neural activity, devices called "optrodes" are employed. They are typically composed of a linear array of microelectrodes integrated on a slender probe shafts combined with flat-cleaved optical fibers (FF) placed above the recording sites. However, due to tissue absorption and scattering, light delivered by the FF unevenly illuminates the region of interest. This issue is of particular relevance when cellular populations are disposed along the dorso-ventral axis, such as in medial prefrontal cortex (mPFC) where cortical layers are aligned vertically. The study presented here aims at using tapered optical fibers (TFs) in combination with a 16-electrode neural probe to better access neural populations distributed along the dorso-ventral axis in the mPFC of newborn mice, restricting light delivery over a specific portion of the cortical layer of interest. Half of the TF surface is coated with a reflecting metal blocking the light to enable light delivery from one side of the probe's shaft only, with the probe base being designed to host the fiber without interfering with the wire-bonds that connect the recording sites to a printed circuit board. Monte-Carlo simulations have been implemented to define the relative TF-probe position and to identify the light intensity distribution above the recording sites. In vivo recordings indicate that simultaneous optical stimulation and electrical readout of neural activity in the mPFC benefit from the use of the engineered TF-based optrode in terms of a more uniform light distribution along the dorso-ventral axis and the possibility of restricting light delivery to a subset of electrical recording sites of interest.

Microenvironmental Stiffness of 3D Polymeric Structures to Study Invasive Rates of Cancer Cells.

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.

Single-cell-based evaluation of sperm progressive motility via fluorescent assessment of mitochondria membrane potential.

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.