Nongenetic optical neuromodulation with silicon-based materials

Abstract

Optically controlled nongenetic neuromodulation represents a promising approach for the fundamental study of neural circuits and the clinical treatment of neurological disorders. Among the existing material candidates that can transduce light energy into biologically relevant cues, silicon (Si) is particularly advantageous due to its highly tunable electrical and optical properties, ease of fabrication into multiple forms, ability to absorb a broad spectrum of light, and biocompatibility. This protocol describes a rational design principle for Si-based structures, general procedures for material synthesis and device fabrication, a universal method for evaluating material photoresponses, detailed illustrations of all instrumentation used, and demonstrations of optically controlled nongenetic modulation of cellular calcium dynamics, neuronal excitability, neurotransmitter release from mouse brain slices, and brain activity in the mouse brain in vivo using the aforementioned Si materials. The entire procedure takes ~4-8 d in the hands of an experienced graduate student, depending on the specific biological targets. We anticipate that our approach can also be adapted in the future to study other systems, such as cardiovascular tissues and microbial communities.

Figure 1. A rational design principle of Si structures for optically-controlled neuromodulation.

a, A schematic diagram illustrating the biology-guided neural interface design. The biological targets place criteria on the material structure (Selection I) and function (Selection II) to establish functional neural with efficient signal transductions across the tight junctions upon light illumination. b, Selected Si structures in this protocol for optically-controlled neuromodulation. They are nanowires (Steps 83–90) for organelle level interfaces, nanowires (Steps 61–75), particles (Steps 61–75), and membranes (Steps 76–82 and Steps 91–105) for cell and tissue level interfaces, and meshes (Steps 106–122) for organ level interfaces. a and b are adapted with permission from Ref., Nature Publishing Group.

Figure 2. Schematic overview of the entire protocol.

Four major blocks of procedures are illustrated on the left side, with details of different combinations between Si materials and neural systems connected on the right side.

Figure 3. Experimental setup for the CVD growth of Si materials.

a, A schematic diagram of the home-build CVD system. Three subsystems for gas delivery, reactor, and pumping are highlighted. b, A schematic diagram of the gas flow valve manifolds, including the pneumatic valves, mass flow controllers, and manual valves. c, A photograph of the CVD control modules for the vacuum level, gas flow rates, and valves of the growth chamber. d, A photograph of the growth chamber (i.e., a quartz tube), the tube furnace for temperature control, gas flow valve manifolds, vacuum and pressure transducer, and the vacuum sensor. The orange box marks the region with detailed schematics in b. e, Photographs showing the growth substrates for Si materials. Mesoporous SiO₂ template inside an inner quartz test tube can be used for porous particle growths. A Si wafer placed at the center of the quartz tube can be used for both nanowire or membrane growths. a is adapted with permission from Ref., Nature Publishing Group.

Figure 4. Structural characterizations of Si material building blocks.

a, Scanning electron microscope (SEM) image (left), transmission electron microscope (TEM) image (middle), and atom probe tomography (APT) reconstruction (right) of mesoporous Si particles produced in Steps 1–12. The particles are consisting of aligned Si nanowire bundles with a hexagonal mesoscale ordering. Scale bars, 100 nm (left); 100 nm (middle); 20 nm (right). b, SEM (left), side-view TEM (middle), cross-sectional TEM (upper right), and selected area electron diffraction (SAED) pattern (lower right) of coaxial Si nanowires produced in Steps 13–18. These nanowires have a single crystalline core and a nanocrystalline shell. Notably, both the dopant type and concentration can be controlled during the CVD growth to yield uniformly doped or diode junction structures. Scale bars, 1 µm (left); 100 nm (middle); 50 nm (upper right). c, TEM, SEM, and SAED of a p-i-n Si diode junction produced in Steps 19–22. The nanocrystalline intrinsic and the n-type layers were grown epitaxially from a single crystalline p-type SOI wafer as evidenced by the SAED patterns taken at different locations of the diode junction. Scale bars, 200 nm (upper left); 200 nm (upper right). a is adapted with permission from Ref., Nature Publishing Group. b and c are adapted with permission from Ref., Nature Publishing Group.

Figure 5. The fabrication process of the PDMS-supported Si mesh (Steps 25–43).

Fabrication of the distributed Si mesh through photolithography (Steps 25–29), RIE (Steps 30–31), and wet etching (Steps 32–33) processes. Fabrication of the holey PDMS membrane using soft lithography (Steps 34–39). Transfer of the Si mesh onto the PDMS membrane (Steps 40–43) and corresponding photos and SEM image of the final device. Photomask designs for the Si mesh and the SU-8 pillar array are included. Black denotes chromium; White denotes the exposed area on the soda lime glass mask. Scale bars in 7/VII, 500 µm (left); 2 mm (upper right); 200 µm (lower right). Adapted with permission from Ref., Nature Publishing Group.

Figure 6. Experimental setup of the photo-response measurement (Steps 44–50).

a, Schematic diagrams illustrating the measurement configuration. Light pulses (530 nm LED or 532 nm laser) are delivered through a water-immersion objective to the Si material immersed in a PBS solution. A glass micropipette is placed near the material surface with a ~ 2 µm distance. Light-induced currents are recorded at different pipette command potentials (V_p) under a voltage-clamp mode. R_feedback is the resistance of a feedback resistor for the amplifier. b, Front view of an upright microscope showing the light path for the laser stimulation. c, Side view of the microscope showing the light path for the LED stimulation. d, A photograph of a micro-manipulator controller (to move the micropipette), an amplifier (to record ionic currents), and a digitizer (to send TTL signals for light pulse controls and to interface the amplifier and the computer). a is adapted with permission from Ref., Nature Publishing Group.

Figure 7. Experimental setups for the in vitro photostimulation of cultured neurons (Steps 51–90).

a, Schematic diagrams illustrating the connections between individual pieces of equipment of the patch clamp setup. Si particles and Si nanowires can be applied onto a cultured DRG neuron for the photostimulation with laser pulses. A1 and A3 are high gain amplifiers whereas A2 has gain of one and it just works as a differential amplifier. b, Photographs of the associated amplifier, AD/DA converter, waveform generator, and the AOM controller (left) and the inverted microscope equipped with a laser setup for the photostimulation experiment (right). c, Photographs highlighting important components of the setup, including the 532 nm laser and the AOM (left), the ND filters (middle), and the electrodes (right). d, Photographs of an inverted laser scanning confocal microscope that can monitor calcium dynamics during the photostimulation experiment (top). A 592 nm stimulated emission depletion (STED) laser is used as the stimulation light source, and the laser scanner allows arbitrary aiming of the laser spot onto the point of interest for the stimulation (bottom). a is adapted with permission from Refs.^,, Nature Publishing Group.

Figure 8. Experimental setup for the photostimulation of acute brain slices (Steps 91–105).

a, A schematic diagram of the photostimulation setup and optical micrographs showing the Si/slice interface and the patched neuron. Scale bars, 250 µm (upper right); 10 µm (lower right). b, A photograph of an upright microscope equipped with an 850 nm IR LED for imaging. c, A photograph of external devices to control the stimulation light sources (473 nm laser or multi-wavelength LEDs), the Pockels cell for laser modulation, the stage heater, and the micro-manipulator for the photostimulation experiment. d, Side views of the microscope showing the light paths for LED (top) and laser (bottom) stimulation. The laser scanning system allows the photostimulation of multiple sites of the Si mesh for functional circuit mapping. a is adapted with permission from Ref., Nature Publishing Group.

Figure 9. Experimental setup for the in vivo photostimulation experiment (Steps 106–122).

a, A block diagram of the entire setup combining the laser scanning photostimulation (LSPS) system, the linear array system with five probes for electrophysiology, the video system, the input/output (I/O) interface, and the graphical user interface (GUI). b, A schematic diagram highlighting the layout of the photostimulation experiment. The mouse brain covered by the Si mesh is stimulated by the laser from the scanning assembly and a linear array recording electrode is inserted into the nearby cortex. c, Photographs of the operating platform overview (left), highlighting the laser scanning assembly (middle), and the electrophysiological recording geometry (right). d, Photographs of the I/O interface (left) and the GUI (right) for controls of manipulators, laser scanning, electrophysiology, camera, and data analysis. b is adapted with permission from Ref., Nature Publishing Group.

Figure 10. Expected results of Si-based optically-controlled neuromodulation.

a, Local solution temperature (top) and bilayer capacitance (bottom) dynamics following laser pulses onto a sheet of mesoporous Si particles supported by a suspended lipid bilayer with different input energies (laser power, 22.4 mW; black, 0.5 ms; red, 1.0 ms; blue, 1.5 ms; olive, 2.0 ms). b, Porous Si particle-enabled modulation of cultured neurons. Representative membrane potential recordings of a DRG neuron with trains of laser pulses (5.32 μJ) delivered to a membrane supported particle at different frequencies, with corresponding FFTs (right). f and f₀ are output and input frequencies, respectively. Green bars indicate when 532 nm laser pulses were delivered. c, p-i-n coaxial Si nanowire-enabled modulation of cultured neurons. Patch-clamp electrophysiology current-clamp traces of membrane voltage in DRG neurons illuminated by 532 nm laser pulses at the neuron/nanowire interface with different durations and powers. In general, longer or stronger laser pulses can elicit action potentials while shorter or weaker ones can only depolarize the membrane. d, Glial cell-internalized intrinsic coaxial Si nanowire-enabled remote modulation of cultured neurons. Calcium imaging time series (green, calcium; blue, Si nanowires) show that the initial calcium surge of the glial cell due to photostimulation of an internalized nanowire can propagate to both adjacent glial cells and neurons. Scale bars, 20 µm. e, p-i-n Si diode junctions enable high spatiotemporal-resolution extracellular stimulation of calcium dynamics. Laser illumination (592 nm, ~ 14.4 mW) of cells in a DRG culture (green: calcium) on a p-i-n Si diode junction, induced localized and fast calcium elevation near the stimulation site and subsequent calcium wave propagation both intra- and inter-cellularly. Laser stimulation was 1-ms long before the time point of 2.722 s. The white arrow marks the laser stimulation site. Scale bars, 20 µm. f, p-i-n Si mesh-enabled modulation of acute brain slices. Data traces from voltage-clamp recordings of the patched neuron with 1-ms-long laser stimulations (left, cyan bar). The grey dashed box marks the time frame for zoomed-in views on the right. EPSCs are marked by stars following the illuminations of the Si mesh (right). The cyan shaded area marks the illumination period in each trial. # denotes the photocapacitive artifact from the Si. g, Au-decorated p-i-n Si mesh-enabled modulation of brain activities. Data traces of neural responses from four adjacent channels in one stimulation. The light blue band marks the illumination period (left). A mean neuron-firing waveform (orange) superposed on individual waveforms (grey) of both spontaneous and stimulation-evoked activities (right). The maroon shaded area denotes standard deviations. h, Au-decorated p-i-n Si mesh-enabled modulation of animal motions. The left limb (green dot) of the mouse moves up and down following the laser illumination of a Si mesh attached to the right side of the forelimb primary motor cortex. Time-dependent mean limb movements show a preferred motion of the left forelimb after the stimulation. The 0 ms time point represents the start of the light pulse. Shaded areas denote s.e.m. of the data. Scale bars, 1 cm. All rat procedures described here were approved by the Institutional Animal Care and Use Committee at the University of Chicago, and all mouse procedures were approved by the Northwestern University Animal Care and Use Committee. a and b are adapted with permission from Ref., Nature Publishing Group. c is adapted with permission from Ref., Nature Publishing Group. d-h are adapted with permission from Ref., Nature Publishing Group.