Patterned photostimulation via visible-wavelength photonic probes for deep brain optogenetics.

Optogenetic methods developed over the past decade enable unprecedented optical activation and silencing of specific neuronal cell types. However, light scattering in neural tissue precludes illuminating areas deep within the brain via free-space optics; this has impeded employing optogenetics universally. Here, we report an approach surmounting this significant limitation. We realize implantable, ultranarrow, silicon-based photonic probes enabling the delivery of complex illumination patterns deep within brain tissue. Our approach combines methods from integrated nanophotonics and microelectromechanical systems, to yield photonic probes that are robust, scalable, and readily producible en masse. Their minute cross sections minimize tissue displacement upon probe implantation. We functionally validate one probe design in vivo with mice expressing channelrhodopsin-2. Highly local optogenetic neural activation is demonstrated by recording the induced response-both by extracellular electrical recordings in the hippocampus and by two-photon functional imaging in the cortex of mice coexpressing GCaMP6.

Introduction

An overarching technological goal in the field of optogenetics is the development of new methods for stimulating neural circuits with very high spatiotemporal precision. Ongoing efforts seek to address large functional ensembles of neurons, i.e., “brain circuits,” through realization of tools providing fine enough resolution to interrogate and control each constituent neuron individually and independently. Significant advances in the development of excitatory and inhibitory opsins have been made over the past decade that now permit direct optical control of cellular processes. To realize the full potential of these technologies, complementary methods for delivering light with cellular precision in vivo are now essential., Existing, state-of-the-art approaches involve the use of spatially patterned light, projected via free-space optics, to stimulate small and transparent organisms or excite neurons within superficial layers of the cortex., However, light scattering and absorption in neural tissue, characterized by the optical attenuation length, cause ballistic light penetration to be extremely short. This makes it impossible to employ free-space optical methods to probe brain regions deeper than about . This statement holds true even if we take into account methods for two-photon and three-photon excitation, and recent efforts made to develop opsins that operate in the red or near infrared. With these limitations in mind, we advance here an alternative approach, involving implantable photonic devices, as the most promising paradigm for delivering and projecting high-resolution patterned light at “arbitrary depths” and with minimal perturbation in the brain.

We identify five critical requirements for realizing widely useful, implantable photonic devices, which we term “visible-wavelength photonic probes”: (i) the probes should provide a multiplicity of microscale illumination sources (hereafter “emitter pixels,” or “E-pixels”), each individually controllable and capable of delivering fine illumination, with cellular-scale cross-section dimensions. Ideally, emission from these microscale E-pixels should have minimal spatial overlap, while collectively covering the entire brain volume of interest. (ii) This patterned illumination must be delivered with sufficient intensity to activate optogenetic effectors (actuators/silencers) within the interrogated region. (iii) Associated thermal perturbations of neural tissue at, or adjacent to, the implanted devices must minimally affect neural circuits. Recent studies show that temperature elevation of as small as 1°C can change the neural firing rate and behavior of mice. (iv) The cross-sectional dimensions of the probes must be made as small as possible—to reduce displacement of brain tissue upon implantation, to minimize tissue damage, and to suppress potential immunological response. (v) Finally, photonic nanoprobe fabrication should be compatible with, and ultimately transferrable to, foundry (factory)-based methods for mass production. This will permit wide deployment of this new technology in the near-term to the neuroscience community. Here, we present a class of photonic probes satisfying these requirements; they are based on integrated, silicon-based nanophotonic components adapted to operate at visible wavelengths and embedded onto implantable silicon probes patterned by microelectromechanical systems (MEMS) processes.

Implantable Photonic Neural Probes

Various architectures for implantable optical probes have recently been proposed. For example, one approach relies upon multiple optical fibers to excite individually addressed illumination points, each driven by a dedicated laser source., Given the complexity of coupling many fibers to a probe, this approach is capable of providing only a few illumination points. To surmount this issue, coupling a fiber-bundle to on-chip photonic waveguides has been proposed, but neither in vitro nor in vivo validation of this particular approach has been reported. Another approach implements modal multiplexing to address several illumination points along an implantable multimode optical fiber. However, this approach necessitates a rather large distance between illumination points () to avoid overlap between adjacent illumination beams. Neither approach is readily upscalable to many emission points, nor easily produced en masse.

An alternative approach involves the integration of microscale light emitting diodes () directly onto the probe shanks. A variation on this theme integrates laser diodes upon the probe head, with their light output routed by on-chip integrated photonic waveguides to emission points located along the shanks. In both cases, however, the power dissipated by these active devices must be strictly limited, given that neuronal activity thresholds are highly sensitive to minute temperature variations., Minimizing the total heat delivered to brain tissue by the probe, which is dominated by the heat generated by the in these architectures, can significantly restrict the number of active illumination sources that can be integrated. Unless the efficiency of or laser diode sources is dramatically increased, it will not be feasible to include more than a limited number of active light emitters on implantable photonic probes.

Here, we present a new paradigm for photonic probes that employs wavelength division multiplexing (WDM). It provides the potential for massive upscaling of the number of E-pixels that can be incorporated and individually addressed within implantable, ultra-compact neural probes. The technique of WDM employs a multiplicity of independent data streams, each imprinted on individual carrier wavelengths (spectral channels), that are combined (i.e., multiplexed) and transmitted via a single optical fiber. At the receiving end, these multispectral signals are subsequently demultiplexed and delivered to their intended destinations. In our application of WDM, each temporally modulated carrier wavelength is delivered to an independent E-pixel at a specific, spatial location located along an implantable photonic probe shank. Spectral separation is achieved by photonic circuitry for WDM integrated within the probe head. Our technique is exceptionally well suited for optogenetic effectors, because currently employed opsins respond to a relatively broad spectrum of light, typically spanning ., This permits accommodating many spectral channels within the opsin absorption band. Additionally, this unique assignment of different wavelengths to specifically located E-pixels can be accomplished solely using “passive” components, which neither requires power to operate, nor generate additional heat.

The photonic neural probes described herein provide a first proof-of-concept of our paradigm. The prototype devices we report here comprise implantable shanks, initially with nine E-pixels, which are spectrally addressed through “one” single-mode optical fiber. We implement the E-pixels themselves using large diffractive grating couplers that produce beams with low divergence angles, as small as 1.7 deg. This low-light divergence offers beam cross-section dimensions that are comparable to the size of neural cell bodies—even after traversing several hundreds of micrometers. Other recent implementations of implantable probes based on photonic technology do not provide a route toward the goal of generating complex illumination patterns with narrow illumination beams at arbitrary locations within the brain.

Probe Architecture and Fabrication

The overall structure of our prototype photonic probes is patterned using standard nanophotonic and MEMS fabrication processes (Appendix A, Fig. 6). The implantable, needle-like probe shanks have widths of near the probe head decreasing to only near the tip, with a uniform thickness of throughout. The shank tips are wedge-shaped [Fig. 1(b)], with tips having a radius of curvature; this ensures smooth penetration of brain tissue with minimal dimpling. Our approach yields implantable probes with overall cross sections representing the state-of-the-art for optical probes. They are far smaller than the optical fibers or endoscopes currently implanted for optogenetic experiments (Appendix A, Fig. 9). The shanks of the prototype probes reported here have a pitch of and lengths of either 3 or 5 mm, yet they remain straight after fabrication through our careful engineering of the ubiquitous internal stresses present within thin-film multilayers (Appendix A).

Fig. 6

Schematic of the fabrication process flow. (a) Schematic of the SOI wafer after deposition of the photonic layer. (b) Photonic circuitry patterning using electron-beam lithography and pseudo-Bosch etch processes. (c) Encapsulation of the photonic layer by deposition of PECVD . (d) Photonic probe front side patterning using photolithography and ICP etch processes. Front (e) and side (f) views of the photonic probe after the backside etch but before the HF etch step that removes the thin BOX remaining between the shanks.

Fig. 1

Prototype photonic probe architecture. (a) Optical micrograph showing photonic probes operating at visible wavelengths. This specific design contains three 3-mm-long, -thick shanks that taper in width from , at the head, down to , near the tip. The photonic elements comprise three AWG demultiplexers; one per shank. Each is driven by a single-input waveguide (left) and subsequently drives a multiplicity of output waveguides (right) that traverse the shanks, carrying light to their ultimate destinations on the shank tips. At their termini, the photonic waveguides drive grating couplers (termed E-pixels), which couple light off-shank into brain tissue. All the on-chip photonic elements are patterned from a 200-nm thick silicon nitride layer, which is deposited on top of the oxidized silicon structural layer used to form the probe body. (b) Photograph showing the top view of two grating couplers that constitute the E-pixels near the tip of a shank. The tapered waveguides transform the small optical cross section of the submicron waveguides to the larger neuronal-scale spot size delivered by the E-pixels. (c) Side view of a photonic probe. Although the shank thickness is , the thicker probe head (on left) facilitates handling and mounting of the device.

Fig. 9

Probe dimensions. (a) Photograph showing the relative size of a probe having 5-mm long shanks compared to a U.S. penny. Optical micrographs showing (b) top and (c) side views of the tip of the shank compared to a diameter optical fiber.

The probe head [Fig. 1(a)] contains integrated nanophotonic circuitry required to couple multispectral light delivered from a single external optical fiber onto the probe chip and, subsequently, to route the individual spectral components (channels) to specific emitters on the shank(s). E-pixels arrays [Fig. 1(b)] can be placed at any location along the implantable shanks; in the first prototypes reported here, we include nine E-pixels, spaced on a pitch. It is straightforward to achieve spacing between adjacent E-pixels without changing our fabrication protocols (Appendix A).

Nanophotonic Circuitry

The visible-wavelength photonic circuitry on the probe is fabricated from an optical multilayer comprising a 200-nm thick silicon nitride () layer encapsulated between two layers of silicon dioxide (), to yield a total thickness of . This multilayer is grown upon a commercially available silicon-on-insulator (SOI) substrate, itself comprising a -thick Si (structural) layer atop, a buried oxide (BOX) layer, above a -thick Si wafer. The photonic circuit is comprised of grating couplers, photonic waveguides, and arrayed waveguide gratings (AWGs). In Appendix A, we describe a microprism coupling method (hereafter, μ-prism) bridging the external input fiber’s terminus to the on-chip grating coupler. This efficiently couples the light to the photonic waveguides on-chip. Once on-chip, this multispectral light is routed by a single waveguide to an AWG located on the probe head [Fig. 1(a)]. The AWGs function as passive optical demultiplexers that spectrally separate the incoming wavelength-multiplexed signal to the nine output waveguides. These separated post-AWG signals are subsequently routed by separate photonic waveguides onto the shank, and then to their termini at individual E-pixels. These E-pixels (described in Appendix B) comprise small-footprint diffractive grating couplers patterned on the surface of the shanks [Fig. 2(a) inset]; light routed to each is emitted off-shank, almost perpendicularly, into adjacent neural tissue. Our prototype devices require a single optical fiber per AWG or shank. Future designs will incorporate a hierarchical on-probe photonic circuit, in which a master AWG drives subsequent AWGs, to reduce the total number of required optical fibers to one.

Fig. 2

Characterization of E-pixel illumination. (a) Optical micrograph showing the side view of a shank immersed in a fluorescein–water solution to visualize the E-pixel illumination profiles. In this image, blue light (473 nm) is emitted from an E-pixel located away from the tip of the shank. This light stimulates the green photoluminescence (PL) visible in the image. (Inset, top) Optical micrograph showing another E-pixel (here, from the shank tip) emitting blue light. Scale-bar corresponds to . (Inset, bottom) Normalized iso-intensity contours for light measured at the surface of an E-pixel. (b) and (c) Measured green PL intensity pattern, covering a distance of , generated by the blue (473 nm) illumination beam emitted by the photonic E-pixel. Image (b) was obtained in a fluorescein–water solution at ; image (c) was obtained from a fluorescein-stained mouse brain slice. The dashed line delineates the beam trajectory. (d) Simulated E-pixel illumination intensity profile in water. Results were obtained using the nominal probe design parameters. (e) PL beam profile analysis. (Top) Continuous lines correspond to measured and simulated normalized PL intensity, calculated along the beam trajectory as a function of distance from the E-pixel. Normalization of the experimental and simulation result is done relative to the maximum beam intensity (measured at a distance of 70 to ) and the simulated intensity at a distance of , respectively. Dashed lines show fit results of the far-field intensity to an exponential decaying function. (Bottom) Analysis of the beam width (FWHM, i.e., full width at half maximum) as a function of the distance from the E-pixel.

The critical integrated photonic elements on the probes—the AWGs and the grating couplers—require spatiotemporally coherent light for their operation. We drive them with multispectral light generated and modulated off-probe, and then delivered to the probe head by a single external optical fiber. The ratio of the incident power delivered by the fiber, to the total power emitted by the E-pixels, defines the probe insertion loss (IL). The total IL of these first unoptimized prototypes is about . Roughly, of this arises from coupling loss into and out of the probe, dominated by nonideal coupling between the fiber and the on-chip photonic circuitry. The various losses present are fully delineated in Appendix B, Fig. 11. In future device generations, these ILs can be reduced significantly through advanced engineering design and, especially, by use of the highly optimized fabrication processes available at commercial photonic foundries. We emphasize that the majority of these losses, 18 dB in our current prototypes occurs within the probe base, rather than at the point of emission, as is the case for .

Fig. 11

(a) Normalized waveguide transmittance as a function of the waveguide length. Error bars represent the standard deviation of the measured transmittance within each group of six waveguides. (b) and (d) Grating coupler images and simulated spectra. (b) and (c) Scanning electron microscope (SEM) images of an input grating coupler. The grating couplers have dimensions of . Image (c) was obtained at an angle of 52 deg to better visualize the partial etch of the photonic layer between the grating teeth. (d) Finite-difference time-domain (FDTD) simulation results showing the transmission spectra of an improved input grating coupler using an in-line angle-polished fiber for coupling, and the designed E-pixel (output grating coupler) used in the probes demonstrated in this work. The input grating coupler spectrum is the transmission between the fiber mode and the fundamental transverse-electric (TE) mode of the waveguide with a coupling angle of 5 deg; the E-pixel spectrum is the percentage of optical power radiated toward brain tissue with a fundamental TE mode input to the waveguide.

Validation of the capability of our photonic probes to stimulate neural activity, as described in the next section, has been achieved with E-pixel emission power that ranged between 5 to . With IL of 20 dB, incident laser power of 1 mW per E-pixel is required. Such power is readily available over the relevant wavelength range with supercontinuum lasers.

Characterization of Single E-Pixel Illumination

Our measurement and simulations results demonstrate that E-pixels emit beams with a propagation direction angle of 2 to 30 deg from the normal to the probe surface [Fig. 2(a)]. The exact angle of each individual E-pixel can be engineered during the probe design phase by setting the period of the grating couplers. Once probes are fabricated, this angle is fixed. The low divergence of the beams minimizes overlap between adjacent beams, while preserving light intensity over significant propagation distances from the E-pixel. We have capitalized on the highly collimated photonic probe beamshape to enable local optogenetic activation of neurons.

The beam profile at the surface of the E-pixel is (FWHM) along both transverse axes of the beam [Fig. 2(a) inset]. We characterize the beam profile versus distance from probe shank by: (i) imaging in a fluorescein solution [Figs. 2(b) and 13], (ii) imaging in, thick, adult mouse brain slices soaked overnight in a fluorescein solution [Fig. 2(c)], and (iii) comparison with numerical simulations [Figs. 2(d), 14, and 15]. We find Fresnel diffraction determines the beam intensity profile up to a distance of from the probe; beyond that, it is characterized by far-field Fraunhofer diffraction. (Appendix B; Fig. 15). The minimal beam width, observed at the transition between the Fresnel and far-field regions at a distance of [Fig. 2(d)], is in the fluorescein solution, at a distance of in the brain slice, and at distances smaller than in our simulations. Light scattering results in a slightly larger beam divergence in tissue [Fig. 2(c)] than in the fluorescein solution [Fig. 2(b)]. However, all beam widths measured up to a distance of are less than, or of order, the size of an individual neuronal cell body. This property of E-pixel illumination permits reducing the E-pixel pitch to , while still maintaining negligible overlap between adjacent beams.

Fig. 13

Fluorescein imaging setup. The photonic nanoprobe is inserted sideways into a small reservoir containing a fluorescein–water droplet. The probe is positioned vertically to be as close as possible to the top coverslip; this minimizes scattering along the imaging axis. (Inset) Photograph showing insertion of the probe into the fluorescein–water solution.

Fig. 14

Far-field beam illumination pattern. (a) Simulation showing the emission beam intensity pattern spanning a distance of 1 mm from an E-pixel, which is implemented as an output grating coupler. The intensity was truncated at a value of 0.5 to improve image contrast. The inset shows a photograph of the beam shape in fluorescein. Image was taken using the setup depicted in Fig. 13, inset. Beam extends over a distance of almost 10 mm. (b) Analysis of the simulated beam in panel (a). Blue and red curves show the peak intensity and the FWHM transverse beam width, respectively, as a function of the distance away from the grating coupler.

Fig. 15

Fresnel diffraction pattern. (a) Optical image showing the green PL intensity pattern of the E-pixel emission. This image magnifies an area of close to the E-pixel, where the pattern is dominated by Fresnel diffraction. The weak illumination beams emitted at large angles are generated by the diffraction of light that is reflected back from the bottom silicon substrate (panel d). (b) Color map and (c) line-plot showing FDTD simulation results of the corresponding E-pixel normalized intensity emission pattern as a function of the lateral and perpendicular distances from the origin of the grating couplers. Lines in panel (c) are spaced by half a unit for clarity. (d) Color map showing the refractive indexes of the structure, calculated numerically by FTDT. The plotted white line shows the near-field emission intensity pattern calculated above the grating structure.

Multibeam Illumination

Our use of WDM makes it possible to independently address on-shank E-pixels by separate temporally modulated multispectral components of the light delivered to the probe. Figure 3(a) shows an illustration of our WDM approach. Coherent light from a broadband (multispectral) source is split into discrete spectral bins; each is employed as an independently controllable transmission channel. Temporal modulation providing the unique, arbitrarily complex illumination pattern required for simultaneous excitation of specific locations within the brain is imposed on each of these spectral channels. Figure 16 depicts a possible approach for temporal modulation, using of the shelf components.

Fig. 3

Multibeam illumination. (a) Schematic elucidating the concept of WDM applied to photonic neural probes. The inset in the demultiplexer block shows an electron micrograph of one of our blue-wavelength AWGs (the scale bar represents ; Fig. 12). (b) Transmission measurements of the various output channels of the AWG versus input light wavelength. Measurements are obtained by delivering a broadband light input to the AWG and measuring the output spectrum emitted from each output channel with a spectrometer. (c) Schematic showing the optical setup used to address individual channels of the AWG. We employ a Ti:Sapphire pulsed laser (946 nm) to pump a BBO crystal, which generates blue (473 nm) light by exploiting a second harmonic generation process. Unconverted infrared light is redirected into a beam blocker by a dichroic mirror; the converted blue light is coupled to the optical fiber. The 13-nm bandwidth of the infrared pump signal reduces, after conversion, to blue light with a bandwidth of . This is subsequently narrowed spectrally with a manually tuned bandpass filter (Alluxa, FWHM 1 nm). Rotation of the filter tunes the central wavelength of this filter, thereby enabling dynamic tuning of the passband. A shutter or an acousto-optic modulator can be added at any point along the beam to temporally modulate the light. (d) Optical micrograph showing a shank with nine simultaneously driven E-pixels. The center-to-center pitch between E-pixels is in these prototypes. The annotations denote the mapping between the spectral channels plotted in panel (b) and the spatial location of the corresponding E-pixels to which they are coupled. The scale bar here represents . (e) and (f) Measured fluorescein PL patterns generated by simultaneous illumination from several E-pixels. The spectrum (bandwidth and central wavelength) was set to 5.5 nm at 470 nm, and 1.8 nm at 473 nm, in panels (e) and (f), respectively. The inset in panel (f) superimposes this spectrum with the spectral response of the AWG. Here, scale bars represent .

Fig. 16

WDM setup. Schematic of a WDM setup based on an AOTF.

Fig. 12

AWG measurement setup. (a) SEM image of an AWG test chip. The image shows an AWG having one input channel on the left and nine output channels on the right. Above and below the AWG are two waveguides used to de-embed the fiber-to-chip coupling losses from the AWG measurement. (b) Optical microscope image of the AWG, obtained while the AWG is excited with broadband multicolor light, thus all its output channels emit simultaneously. (c) Schematic of the optical setup used to measure the AWG. (d) and (e) SEM images of one of the AWGs designed to route blue light. This array is composed of waveguides, each having a width of 240 nm.

Our embodiment of the on-chip optical demultiplexer is realized with a visible wavelength AWG [Fig. 3(a), inset; Fig. 12]. AWGs are now perfected and commercially available for use with infrared light in telecommunications technology, however, given their need for much tighter dimensional tolerances and smoother structures (to suppress diffuse sidewall scattering), there are only a couple of reports of AWGs configured for the visible spectrum to date. The blue-wavelength AWGs we have developed for this work have a compact footprint of ; accordingly, they are ideally suited for integration within the heads of our miniature photonic probes [Fig. 1(b)]. By appropriately synthesizing the multispectral light input [Figs. 3(b) and 3(c)], either individual E-pixels or a multiplicity of them can be independently and simultaneously addressed [Figs. 3(e) and 3(f)]. Figure 16 presents a schematic of one possible setup for addressing E-pixels, which provides high temporal bandwidth using a tunable acousto-optic filter.

The maximum number of E-pixels addressable by a single AWG is determined by the ratio of the absorption bandwidth of the optogenetic effector to the bandwidth of the individual spectral channels. For example, a typical optogenetic effector such as ChR2 has an absorption bandwidth of centered near a wavelength of 460 nm, whereas the spectral channel width, an engineerable parameter, can be much narrower (Appendix C). In the designs here, we set the latter to [Fig. 3(b)], thus permitting each AWGs to address up to E-pixels with only a single external fiber input to the chip. Upscaling this number to even more E-pixels outputs per fiber input is readily achievable with precise foundry-based fabrication methods, which can permit definition of spectral channels with a roughly narrower bandwidth. However, one must keep in mind that upscaling spectral channel density must be accompanied by a proportional increase in applied laser power per unit bandwidth, as the light will be distributed over a larger number of E-pixels within the effector’s absorption band.

Functional Validation

We validate the functional capabilities of our prototype photonic probes in vivo, in two separate experimental implementations. In the first, we optogenetically activate neurons in the hippocampus of Thy1:18-ChR2-EYFP transgenic mice, while simultaneously recording induced extracellular electrical activity close to the point of light stimulation. To achieve this, an electrically insulated tungsten wire was glued directly atop the photonic probe [Figs. 4(a) and 4(c)]. The uninsulated distal electrode tip is positioned above the E-pixel. This composite probe was then advanced into the CA3 region of the hippocampus of an anesthetized, head-fixed mouse [Fig. 4(c)]. In this brain region, pyramidal neurons express high levels of ChR2-EYFP. Shortly after implantation, the illumination beam was directed rostrally and electrical measurements were recorded in response to optical stimulation pulses. Several temporal patterns of illumination were tested. The corresponding extracellular electrical recording [Figs. 4(d) and 4(e)] reveals repeated and intense multiunit spiking activity in direct response to light pulses delivered by the photonic probe. No significant degradation in the rate or amplitude is observed for these multiunit bursts, or even for pulses as long as 10 s. In these experiments, we estimate the optical power emitted by the E-pixel to be .

Fig. 4

In vivo photoactivation of hippocampal CA3 pyramidal neurons in a mouse with concomitant electrophysiological recording. (a) Schematic depicting the relative configuration of a recording electrode tip, the photonic probe, and light that is emitted from one E-pixel. (b) The combined probe setup that is carefully implanted into the mouse brain within the CA3 region of the hippocampus. Optical emission from the E-pixel is directed toward the anterior region of the brain. (c) Photograph of the electrical probe (A-M Systems, tungsten electrode, , ), which is affixed immediately above the photonic probe. The probe shank length is 3 mm. (d) Recordings obtained with the electrical probe, showing the response evoked from a 400-ms long optical excitation pulse (blue bar). Black lines above the recording denote spikes. Photoelectric transients, which occur at the location of the red lines, are generated when the optical excitation is switched on and off; these artifacts have been removed from the data for clarity. (e) Raster plot showing the spiking response evoked during repeated illumination trials, demonstrating the activation of ChR2 by blue light from the E-pixel. Several illumination patterns were tested, including 100-, 200-, and 400-ms long pulses (blue bars) with repetition rates of 4, 2, 1 Hz, respectively, as well as 2- and 10-s long pulses.

In a second experimental implementation, the functionality of the photonic probes was assessed via simultaneous free-space, two-photon functional imaging of cortical neurons in a mouse coexpressing both ChR2 and GCaMP6s (Fig. 5). The photonic probe was inserted at an angle of into cortical layer . Although minor dimpling was observed during photonic probe insertion at this angle, the probe was sufficiently sharp to penetrate the dura with only moderate pressure. Probe illumination was directed upward from the surface of the probe into the brain tissue and a local population of neurons was imaged above the probe tip [dashed white circles in Figs. 5(c) and 5(g)]. The approximate FWHM beam width at the imaging plane was .

Fig. 5

Cortical neural stimulation with concomitant two-photon optical functional imaging. (a) Schematic of the experimental setup. Measurements are carried out in an anesthetized, head-fixed mouse, placed under a custom two-photon microscope. The photonic probe is implanted at an angle of 35 deg relative to the surface of the optical dissection, providing access to the brain through the narrow gap between the microscope objective and the surgical opening. A fiber-coupled 473-nm diode laser drives the E-pixel at the tip of the photonic probe. To prevent saturation of the PMTs during application of optical stimuli, mechanical shutters are used to block light emitted by the probe. The inset shows a photograph of the experimental configuration. The narrow profile of the probe enables it to fit under the microscope objective. (b) Illustration of the light excitation sequence. Each sequence includes a single-stimulation event and a subsequent control event, during which only the mechanical shutter is activated while no light is emitted by the probe. This precaution permits identifying the level of response evoked by auditory stimuli; no response is observed. (c) Visualization of the expression levels of optogenetic actuators and reporters of the imaging site in mouse cortical layer (920 nm excitation, Nikon -NA objective; scale bar represents ). By overlaying two-photon photometry images, it is possible to identify neurons coexpressing both GCaMP6s and ChR2-mCherry (insets; scale bars represent ). This imaging site is located above the tip of the probe, whose lateral position is marked by the dashed black line. The dashed blue circle marks the approximate probe beam position and width at the imaging plane of the microscope. The four dashed white circles, labeled N1 to N4, delineate four coexpressing neurons located in close proximity to the illumination site. (d)–(f) Results from neural excitation. (d)  transients, measured for neuron #1, showing evoked neural response during sequential excitation and control events, as marked by blue and gray lines, respectively. Traces are normalized by the base fluorescence level, integrated over the first 30 ms prior to the stimuli event. (e) Peristimulus time histogram of neurons 1 to 4 calculated over 19 stimulation cycles. (f) Peristimulus time histogram of neurons 1 to 4 calculated over 15 pulses of wide-field blue (473 nm) illumination delivered through the microscope objective. (g) An overlaid image showing the peristimulus activation calculated at single pixel resolution, across the entire imaging site. Baseline fluorescence in green is calculated over a period of 40 ms prior to the stimuli. The black–red–yellow layer shows the peak peristimulus fluorescence, calculated as an average over a 30 ms interval following the stimulus event. The dashed circle marks are duplicated from panel b.

For this second set of experiments, 200-ms light pulses with an estimated optical output power of were delivered by the photonic probe with a repetition rate of 0.2 Hz. The fluorescence response of the ChR2/GCaMP6s-expressing cells was simultaneously recorded by two-photon imaging. To prevent saturation of the photomultiplier tubes (PMTs) during the optical stimulation pulses from the photonic probe, a mechanical shutter (Uniblitz TS6B) was used as a blanker during E-pixel illumination pulses. To rule out the potentially confounding effect of the audible “click” generated by this shutter (and heard by the mouse), stimulation pulses were interleaved with control events, in which only the shutter was activated without concomitant light emission [Fig. 5(b)].

In this second proof-of-concept experiment, an individual neuron was reliably activated by light pulses delivered from an implanted photonic probe. Figure 5(d) shows the absence of any response for control events. Analysis of the optical beam trajectory in this experiment indicates that the light emitted by the probe did not impinge upon the cell body of neuron #1 directly, but instead activated a basal dendrite projecting into the center of the beam illumination profile [Fig. 5(g)]. Given the highly collimated nature of light emitted from the probe, simultaneous activation of neighboring neurons—labeled #2, #3, and #4—was not induced. To confirm that these untriggered neurons were indeed activatable, wide-field pulses of blue light illumination were subsequently delivered through the microscope objective. This resulted in widespread, simultaneous activation of the neurons within the illumination field [Fig. 5(f)].

Discussion

To exploit the full potential of optogenetics, it is essential to deliver light with high temporal and spatial resolution at arbitrary locations within the brain. Here, we demonstrate photonic probes operating at visible wavelengths that permit realization of this goal. Our photonic probes leverage technological developments achieved over the past two decades in the field of optical communications at infrared wavelengths, realizing them within the visible range of relevance for present-day optogenetic reporters and effectors operating at visible wavelengths. Specifically, we employ wavelength division demultiplexing using AWG’s, integrated photonic waveguides, and E-pixels realized as diffractive grating couplers. The technology of visible photonics is rapidly advancing, and this makes it feasible to create a spectrum of components for assembling future complex photonic neural probe architectures. Exceptionally promising candidate technologies will enable fast switching and lensless beam focusing. An important attribute of our photonic probe paradigm is their mass producibility via existing photonics foundry protocols. With our achievement of the proof-of-concept reported here, significantly upscaling of E-pixel density and multiplexing is now underway, enabled both with robust and precise foundry-based fabrication protocols and with recent improvements in laser source technology. This will permit their widespread deployment in the near term to the neuroscience and neuromedical research communities.

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