Non-genetic photoacoustic stimulation of single neurons by a tapered fiber optoacoustic emitter

Abstract

Neuromodulation at high spatial resolution poses great significance in advancing fundamental knowledge in the field of neuroscience and offering novel clinical treatments. Here, we developed a tapered fiber optoacoustic emitter (TFOE) generating an ultrasound field with a high spatial precision of 39.6 µm, enabling optoacoustic activation of single neurons or subcellular structures, such as axons and dendrites. Temporally, a single acoustic pulse of sub-microsecond converted by the TFOE from a single laser pulse of 3 ns is shown as the shortest acoustic stimuli so far for successful neuron activation. The precise ultrasound generated by the TFOE enabled the integration of the optoacoustic stimulation with highly stable patch-clamp recording on single neurons. Direct measurements of the electrical response of single neurons to acoustic stimulation, which is difficult for conventional ultrasound stimulation, have been demonstrated. By coupling TFOE with ex vivo brain slice electrophysiology, we unveil cell-type-specific responses of excitatory and inhibitory neurons to acoustic stimulation. These results demonstrate that TFOE is a non-genetic single-cell and sub-cellular modulation technology, which could shed new insights into the mechanism of ultrasound neurostimulation.

Fig. 1. TFOE for high-precision optoacoustic stimulation.

TFOE for high-precision optoacoustic stimulation.a Schematic of TFOE enabling single-neuron stimulation. b Multiwall CNT/PDMS mixture as coating material casted on a metal mesh followed by a punch-through method to coat the tapered fiber. Bottom left: optical image of TFOE, Bottom right: zoom-in showing the tip. c Acoustic signal waveforms detected by the hydrophone at the distances ranging from 0 to 282 µm from the TFOE. The curves shown from the top to the bottom were obtained at the distances of 0, 2, 6, 10, 17, 29, 49, 82, 145, 185 and 282 µm, respectively. d Detected pressure plotted as a function of the distance. e Frequency spectra of acoustic signals acquired at 0, 49 and 145 µm, respectively. All measurements in (c–e) were done with single laser pulse with a pulse energy of 6.7 µJ. f Surface temperature of TFOE tip during laser excitation of 50 ms (7.8 mW, Red) and 1 ms (11.4 mW, Blue), respectively. Shaded area in (f): standard deviation taken from three measurements from the same TFOE. Black arrow: laser onset.

Fig. 2. Fluorescence images of GCaMP6f-expressing neurons in response to TFOE stimulation.

a, b Fluorescence images and calcium traces in sparse population stimulated by TFOE with a laser duration of 50 ms (a) and 1 ms (b). The fluorescence images with the peak intensity after stimulation are shown as “after”. Blue arrows: laser onset. c Max ∆F/F images of one neuron undergone repeated TFOE stimulation three times. Right: calcium signal of the neuron. Blue arrows: laser onset. d Sequential stimulation of three neurons. Max ∆F/F labeled in red, yellow, green respectively superimposed with the fluorescence image. Scale bars: 50 µm. e Merged fluorescence image and transmission image of neuron culture. Inset: zoom-in of the targeted neuron and the TFOE tip. Scale bar: 10 µm. f Representative fluorescence signals evoked at the targeted neuron by TFOE stimulation. Average Calcium signals induced by strong neuron stimulation when the TFOE was placed at $<5$ µm (Red), subtle stimulation when the TFOE was placed at 5–10 µm (Yellow) and no activation when the distance was $>10$ µm (Black). Shaded areas: the standard deviation from 10 neurons. g Calcium response as a function of distance at targeted neurons upon TFOE stimulation. 10 neurons were tested individually. For each neuron, four different distances were tested. Calcium responses were characterized by color bars with the red bars corresponding to the strong stimulation similar to the red curve in (f), the yellow bars corresponding to the subtle stimulation and the black bars for no activation. h Average distances to evoke strong and subtle activations. Laser condition for (e–h): 11.4 mW, 1 ms, 1.7 kHz.

Fig. 3. Pulse energy dependence of TFOE stimulation.

a–c Fluorescence images of GCaMP6f expressing neurons before and after TFOE stimulation with a single pulse. d Calcium trace of the targeted neuron undergone single-pulse stimulation. Blue vertical line: onset of optoacoustic stimulation with zoom-in showing a representative optoacoustic waveform. e Pulse energy threshold for successful neuron stimulation as a function of pulse number (N = 5–7).

Fig. 4. TFOE evoked sub-cellular stimulation on neurites.

a–b TFOE evoked neurites activation with calcium wave propagating along the neuron network. Colored arrows: targeted area (purple), neuron 1 (cyan) and neuron 2, 3 (red). c ΔF/F of calcium signal at 4 s after laser onset. d Calcium traces of targeted area (purple), neuron 1 (cyan), and neuron 2 and 3 (red), as labeled in (a). Inset: Zoom-in of calcium signals immediately after the laser onset. Blue arrow: laser onset. e A multipolar neuron stimulated with a TFOE selectively targeting the axon (red arrow) and dendrites (yellow and green arrows). f–h Maximum ΔF of calcium signal upon stimulation of different areas. i Calcium traces measured at the targeted neurites as labeled in (e) by red, yellow and green arrows, respectively. j Calcium traces measured at the soma upon stimulation of different neurites. Blue arrows in (i–j): laser onset. Scale bars in (a–c), (e–h): 50 µm

Fig. 5. Single neuron patch clamp with TFOE stimulation.

a–b Two-photon imaging of patch clamp integrated with TFOE in a mouse brain slice targeting GAD2-tdTomato negative pyramidal neurons and GAD2-tdTomato positive inhibitory interneurons. The patch pipette is visualized using the cyan-green fluorescent dye Alexa Fluor 488 in the intracellular electrode solution. c, d Membrane voltage response in an excitatory pyramidal cell upon TFOE stimulation (5 ms) at a distance of ~5 µm (c) and ~10 µm (d). e, f Voltage response in an inhibitory interneuron upon TFOE stimulation at ~5 µm at the membrane voltages of −75 mV (e) and −40 mV (f). Laser: 11.4 mW, 1.7 kHz, 5 ms duration.