Leonardo Sileo

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.

Three-dimensional cage-like microscaffolds for cell invasion studies

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.

Micro- and Nanotechnologies for Optical Neural Interfaces

In last decade, the possibility to optically interface with the mammalian brain in vivo has allowed unprecedented investigation of functional connectivity of neural circuitry. Together with new genetic and molecular techniques to optically trigger and monitor neural activity, a new generation of optical neural interfaces is being developed, mainly thanks to the exploitation of both bottom-up and top-down nanofabrication approaches. This review highlights the role of nanotechnologies for optical neural interfaces, with particular emphasis on new devices and methodologies for optogenetic control of neural activity and unconventional methods for detection and triggering of action potentials using optically-active colloidal nanoparticles.

Fabrication of multipoint light emitting optical fibers for optogenetics

Multipoint Light Emitting Optical Fibers (MPF) has been recently demonstrated as a versatile tool for spatially addressable optogenetics experiments. Their fabrication has been possible thanks to a number of key microfabrication technologies, in particular the unique nanofabrication capabilities of a Focused Ion Beam. This work provides the complete description of MPF fabrication, detailing the optimization process for each fabrication step.

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.

A fully integrated GaAs-based three-axis Hall magnetic sensor exploiting self-positioned strain released structures

In this work, we demonstrate a fully integrated three-axis Hall magnetic sensor by exploiting microfabrication technologies applied to a GaAs-based heterostructure. This allows us to obtain, by the same process, three mutually orthogonal sensors: an in-plane Hall sensor and two out-of-plane Hall sensors. The micromachined devices consist of a two-dimensional electron gas AlGaAs/InGaAs/GaAs multilayer which represents the sensing structure, grown on the top of an InGaAs/GaAs strained bilayer. After the release from the substrate, the strained bilayer acts as a hinge for the multilayered structure allowing the out-of-plane self-positioning of devices. Both the in-plane and out-of-plane Hall sensors show a linear response versus the magnetic field with a sensitivity for current-biased devices higher than 1000 V A−1 T−1, corresponding to an absolute sensitivity more than 0.05 V T−1 at 50 µA. Moreover, Hall voltage measurements, as a function of the mechanical angle for both in-plane and out-of-plane sensors, demonstrate the potential of such a device for measurements of the three vector components of a magnetic field.

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.