Marco Pisanello

Multipoint-Emitting Optical Fibers for Spatially Addressable In Vivo Optogenetics

Optical stimulation and silencing of neural activity is a powerful technique for elucidating the structure and function of neural circuitry. In most in vivo optogenetic experiments, light is delivered into the brain through a single optical fiber. However, this approach limits illumination to a fixed volume of the brain. Here a focused ion beam is used to pattern multiple light windows on a tapered optical fiber. We show that such fibers allow selective and dynamic illumination of different brain regions along the taper. Site selection is achieved by a simple coupling strategy at the fiber input, and the use of a single tapered waveguide minimizes the implant invasiveness. We demonstrate the effectiveness of this approach for multipoint optical stimulation in the mammalian brain in vivo by coupling the fiber to a microelectrode array and performing simultaneous extracellular recording and stimulation at multiple sites in the mouse striatum and cerebral cortex.

Dynamic illumination of spatially restricted or large brain volumes via a single tapered optical fiber

Optogenetics promises precise spatiotemporal 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, compared to standard flat-faced fiber stimulation. Thus, tapered fibers permit focal or broad illumination that can be precisely and dynamically matched to experimental needs.

Modal demultiplexing properties of tapered and nanostructured optical fibers for in vivo optogenetic control of neural activity

Optogenetic approaches to manipulate neural activity have revolutionized the ability of neuroscientists to uncover the functional connectivity underlying brain function. At the same time, the increasing complexity of in vivo optogenetic experiments has increased the demand for new techniques to precisely deliver light into the brain, in particular to illuminate selected portions of the neural tissue. Tapered and nanopatterned gold-coated optical fibers were recently proposed as minimally invasive multipoint light delivery devices, allowing for site-selective optogenetic stimulation in the mammalian brain [Pisanello , Neuron82, 1245 (2014)]. Here we demonstrate that the working principle behind these devices is based on the mode-selective photonic properties of the fiber taper. Using analytical and ray tracing models we model the finite conductance of the metal coating, and show that single or multiple optical windows located at specific taper sections can outcouple only specific subsets of guided modes injected into the fiber.

Photonic technologies for optogenetics

Light-induced stimulation and inhibition of neuronal activity has been recently made possible by optogenetics. In optogenetics, specific light-sensitive proteins, called opsins, are genetically targeted into specific neuronal cell types in an animal model. When light of the appropriate wavelength is delivered into the brain, the light-activated proteins respond by stimulating or inhibiting the firing of action potentials in neurons. It is therefore possible to switch on and off different types of neurons merely by turning on a light. Delivering of light in vivo to specific neurons at specific locations in the brain is still a challenge, however. Here we review recent advances on micro and nanotechnologies for the fabrication of optogenetic devices to deliver light in vivo at specific, controlled sites in mammalian brains, while simultaneously monitoring their electrical activity.

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.

Optical fiber technologies for in-vivo light delivery and optogenetics

In optogenetics, light-sensitive proteins are genetically targeted into specific classes of neurons in living animal models (typically mice), making possible to control their neural activity by means of visible light delivered into the brain tissue. In this paper, recent advances on techniques for in-vivo optical stimulation and inhibition of neuronal activity in optogenetic experiments are reported, with particular emphasis on new a new generation of fiber-optic technologies.

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.

Influence of laser beam quality on modal selection in tapered optical fibers for multipoint optogenetic control of neural activity

Spatial-resolved light delivery in the living mammalian brain is of utmost importance in optogenetics experiments, since it allows for combining cell-type specificity with spatial selective optical control of neural activity. This can be achieved at sub-cellular resolution in first cortical layers with two-photon microscopy, while sub-cortical regions can be accessed by using waveguide-based devices. Tapered and nanostructured optical fibers have been recently proposed as viable devices to dynamically switch light stimuli between different points of deep-brain structures, by exploiting both mode division demultiplexing operated by the fiber taper and excitation of well-defined subsets of guided modes into the fiber [Pisanello et al., Neuron, vol. 82, p.1245 (2014); Pisanello et al., Biomed. Opt. Express, vol. 6, p. 4014 (2015)]. This latter is achieved by selecting the injection angle of a high-quality Gaussian beam, therefore exciting guided modes with defined transversal propagation constants. In this contribution we analyze the effectiveness of this technique as a function of the laser beam profile used to guide light into the fiber, complementing experimental results with ray tracing simulations. Two different commercial laser sources, with sensibly different beam qualities, were used to determine the extent of the injected modal subsets in a 0.22 N.A. optical fiber. This was obtained by measuring the transversal component kt of the wavevector through far-field imaging. We show that even poor quality beams might return satisfactory selection accuracy, provided that care is taken in the design of the coupling strategy.

Exploiting modal demultiplexing properties of tapered optical fibers for tailored optogenetic stimulation

Optogenetic control of neural activity in deep brain regions requires precise and flexible light delivery with non-invasive devices. To this end, Tapered Optical Fibers (TFs) represent a minimally-invasive tool that can deliver light over either large brain volumes or spatially confined subregions. This work links the emission properties of TFs with the modal content injected into the fiber, finding that the maximum transversal propagation constant (kt) and the total number of guided modes sustained by the waveguide are key parameters for engineering the mode demultiplexing properties of TFs. Intrinsic features of the optical fiber (numerical aperture and core/cladding diameter) define the optically active segment of the taper (up to ∼3mm), along which a linear relation between the propagating set of kt values and the emission position exists. These site-selective light-delivery properties are preserved at multiple wavelengths, further extending the range of applications expected for tapered fibers for optical control of neural activity.

Multipoint and large volume fiber photometry with a single tapered optical fiber implant

Techniques to monitor functional fluorescence signal from the brain are increasingly popular in the neuroscience community. However, most implementations are based on flat cleaved optical fibers (FFs) that can only interface with shallow tissue volumes adjacent to the fiber opening. To circumvent this limitation, we exploit modal properties of tapered optical fibers (TFs) to structure light collection over the wide optically active area of the fiber taper, providing an approach to efficiently and selectively collect light from the region(s) of interest. While being less invasive than FFs, TF probes can uniformly collect light over up to 2 mm of tissue and allow for multisite photometry along the taper. Furthermore, by micro-structuring the non-planar surface of the fiber taper, collection volumes from TFs can also be engineered arbitrarily in both shape and size. Owing to the abilities offered by these probes, we envision that TFs can set a novel, powerful paradigm in optically targeting not only the deep brain, but, more in general, any biological system or organ where light collection from the deep tissues is beneficial but challenging because of tissue scattering and absorption.