Implantable photonic nano-modulators open perspectives for advanced optical interfaces with deep brain areas.

An emerging trend at the forefront of optical neural interfaces leverages the optical properties of photonic nanostructures to modulate light delivery and collection patterns in deep brain regions. This perspective article surveys the early works that have spearheaded this promising strategy, and discusses its promise towards the establishment of a class of augmented nano-neurophotonic probes.

Exploiting holographically encoded variance to transmit labelled images through a multimode optical fiber.

Artificial intelligence has emerged as promising tool to decode an image transmitted through a multimode fiber (MMF) by applying deep learning techniques. By transmitting thousands of images through the MMF, deep neural networks (DNNs) are able to decipher the seemingly random output speckle patterns and unveil the intrinsic input-output relationship. High fidelity reconstruction is obtained for datasets with a large degree of homogeneity, which underutilizes the capacity of the combined MMF-DNN system. Here, we show that holographic modulation can encode an additional layer of variance on the output speckle pattern, improving the overall transmissive capabilities of the system. Operatively, we have implemented this by adding a holographic label to the original dataset and injecting the resulting phase image into the fiber facet through a Fourier transform lens. The resulting speckle pattern dataset can be clustered primarily by holographic label, and can be reconstructed without loss of fidelity. As an application, we describe how color images may be segmented into RGB components and each color component may then be labelled by distinct hologram. A ResUNet architecture was then used to decode each class of speckle patterns and reconstruct the color image without the need for temporal synchronization between sender and receiver.

Optimizing the internal phase reference to shape the output of a multimode optical fiber.

Pre-shaping light to achieve desired amplitude distributions at the tip of a multimode fiber (MMF) has emerged as a powerful method allowing a wide range of imaging techniques to be implemented at the distal facet. Such techniques rely on measuring the transmission matrix of the optically turbid waveguide which scrambles the coherent input light into an effectively random speckle pattern. Typically, this is done by measuring the interferogram between the output speckle and a reference beam. In recent years, an optical setup where the reference beam passes through the MMF has become an attractive configuration because of the high interferometric stability of the common optical path. However, the merits and drawbacks of an internal reference beam remain controversial. The measurement of the transmission matrix is known to depend on the choice of internal reference and has been reported to result in "blind spots" due to phase singularities of the reference beam. Here, we describe how the focussing efficiency of the calibration can be increased by several percent by optimising the choice of internal reference beam.

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.

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.

Neurophotonic tools for microscopic measurements and manipulation: status report.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

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.

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.

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.

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.

Disrupting cortico-cerebellar communication impairs dexterity.

To control reaching, the nervous system must generate large changes in muscle activation to drive the limb toward the target, and must also make smaller adjustments for precise and accurate behavior. Motor cortex controls the arm through projections to diverse targets across the central nervous system, but it has been challenging to identify the roles of cortical projections to specific targets. Here, we selectively disrupt cortico-cerebellar communication in the mouse by optogenetically stimulating the pontine nuclei in a cued reaching task. This perturbation did not typically block movement initiation, but degraded the precision, accuracy, duration, or success rate of the movement. Correspondingly, cerebellar and cortical activity during movement were largely preserved, but differences in hand velocity between control and stimulation conditions predicted from neural activity were correlated with observed velocity differences. These results suggest that while the total output of motor cortex drives reaching, the cortico-cerebellar loop makes small adjustments that contribute to the successful execution of this dexterous movement.

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.

Multimode Optical Fibers for Optical Neural Interfaces.

Although multiphoton microscopy enables optical control and monitoring of neural activity with single cells resolution over a depth of several hundreds of micrometers, the scattering nature of the brain tissue requires implantable optical neural interfaces to access subcortical structures. If micro light-emitting devices (µLEDs) and solid-state waveguides represent important technological advancements for the field, multimodal optical fibers (MMFs) are still the most diffused tool in neuroscience labs to interface with deep regions of the brain. At a first glance, MMFs can be seen as very limited systems. However, new studies and discoveries in optics, photonics, and technological solutions for their application to neuroscience research have enabled applications of MMF where competing technologies fail. In this framework, the chapter starts with a description of optical neural interfaces based on MMF, with specific reference on recent works analyzing the performances of this approach to deliver and collect light from scattering tissue. The discussion then focuses on how peculiar features of MMFs can be exploited to obtain unconventional applications, including brain imaging through a single multimode fiber, multifunctional neural interfaces, and depth-resolved light delivery and functional fluorescence collection.

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.

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.

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.

3D-microenvironments initiate TCF4 expression rescuing nuclear ß-catenin activity in MCF-7 breast cancer cells.

Mechanical cues sensed by tumor cells in their microenvironment can influence important mechanisms including adhesion, invasion and proliferation. However, a common mechanosensitive protein and/or pathway can be regulated in different ways among diverse types of tumors. Of particular interest are human breast epithelial cancers, which markedly exhibit a heterogeneous pattern of nuclear ß-catenin localization, a protein known to be involved in both mechanotransduction and tumorigenesis. ß-catenin can be aberrantly accumulated in the nucleus wherein it binds to and activates lymphoid enhancer factor/T cell factor (LEF/TCF) transcription factors. At present, little is known about how mechanical cues are integrated into breast cancer cells harboring impaired mechanisms of ß-catenin's nuclear uptake and/or retention. This prompted us to investigate the influence of mechanical cues on MCF-7 human breast cancer cells which are known to fail in relocating ß-catenin into the nucleus due to very low baseline levels of LEF/TCFs. Exploiting three-dimensional (3D) microscaffolds realized by two-photon lithography, we show that surrounding MCF-7 cells have not only a nuclear pool of ß-catenin, but also rescue from their defective expression of TCF4 and boost invasiveness. Together with heightened amounts of vimentin, a ß-catenin/TCF-target gene regulator of proliferation and invasiveness, such 3D-elicited changes indicate an epithelial-to-mesenchymal phenotypic switch of MCF-7 cells. This is also consistent with an increased in situ MCF-7 cell proliferation that can be abrogated by blocking ß-catenin/TCF-transcription activity. Collectively, these data suggest that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of ß-catenin nuclear activity in MCF-7 cells. The employed methodology could, therefore, provide a mechanism-based rationale to dissect further aspects of mechanotranscription in breast cancerogenesis, somewhat independent of ß-catenin's nuclear accumulation. More importantly, by considering the heterogeneity of ß-catenin signaling pathway in breast cancer patients, these data may open alternative avenues for personalized disease management and prevention. STATEMENT OF SIGNIFICANCE: Mechanical cues play a critical role in cancer pathogenesis. Little is known about their influence in breast cancer cells harboring impaired mechanisms of ß-catenin's nuclear uptake and/or retention, involved in both mechanotransduction and tumorigenesis. We engineered 3D scaffold, by two-photon lithography, to study the influence of mechanical cues on MCF-7 cells which are known to fail in relocating ß-catenin into the nucleus. We found that 3D microenvironments are per se sufficient to prime a TCF4-dependent rescuing of ß-catenin nuclear activity that boost cell proliferation and invasiveness. Thus, let us suggest that our system could provide a mechanism-based rationale to further dissect key aspects of mechanotranscription in breast cancerogenesis and progression, somewhat independent of ß-catenin's nuclear accumulation.

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.

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.