•Non-genetic neural stimulation using photoacoustic nanoparticles is reported•Nanotransducers generate photoacoustic signals upon 1,030-nm nanosecond laser excitation•Nanotransducer stimulation targets a mechanosensitive channel on neuron membrane•Single-cell stimulation through nanotransducers is reported Precise neuromodulation is critical to understanding how the brain functions under healthy and diseased conditions. In this work, we introduce the photoacoustic nanoparticles (PANs) that generate acoustic waves locally on the neuronal membrane. Non-genetic neural stimulation was achieved both in vitro and in vivo with nanosecond laser excitation in the near-infrared second widow (NIR-II). Specificity of the stimulation was further improved by targeting of mechanosensitive TRPV4 channels on the neuronal membrane. With its unique absorption in the NIR-II and the absence of genetic modification, PANs open up the potential for non-invasive neuromodulation with high spatial resolution in deep tissue for rodents as well as primates and humans. Neuromodulation is an invaluable approach for the study of neural circuits and clinical treatment of neurological diseases. Here, we report semiconducting polymer nanoparticles based photoacoustic nanotransducers (PANs) for neural stimulation in vitro and in vivo. Our PANs strongly absorb the nanosecond pulsed laser in the near-infrared second window (NIR-II) and generate localized acoustic waves. PANs are shown to be surface modified and selectively bind onto neurons. PAN-mediated activation of primary neurons in vitro is achieved with ten 3-ns laser pulses at 1,030 nm over a 3-ms duration. In vivo neural modulation of mouse brain activities and motor activities is demonstrated by PANs directly injected into brain cortex. With submillimeter spatial resolution and negligible heat deposition, PAN stimulation is a new non-genetic method for precise control of neuronal activities, opening up potentials in non-invasive brain modulation. Neuromodulation is an invaluable approach for the study of neural circuits and clinical treatment of neurological diseases. Here, we report semiconducting polymer nanoparticles based photoacoustic nanotransducers (PANs) for neural stimulation in vitro and in vivo. Our PANs strongly absorb the nanosecond pulsed laser in the near-infrared second window (NIR-II) and generate localized acoustic waves. PANs are shown to be surface modified and selectively bind onto neurons. PAN-mediated activation of primary neurons in vitro is achieved with ten 3-ns laser pulses at 1,030 nm over a 3-ms duration. In vivo neural modulation of mouse brain activities and motor activities is demonstrated by PANs directly injected into brain cortex. With submillimeter spatial resolution and negligible heat deposition, PAN stimulation is a new non-genetic method for precise control of neuronal activities, opening up potentials in non-invasive brain modulation. Neural stimulation is an important tool enabling our understanding of how brains function and treatments of neurological disorders. Electrical stimulation is the basis of current implantable devices and has already been used in the clinical treatment of depression, Parkinson's disease, and Alzheimer's disease. These devices, often made of metal electrodes, are limited by their invasive nature,1Perlmutter J.S. Mink J.W. Deep brain stimulation.Annu. Rev. Neurosci. 2006; 29: 229-257Crossref PubMed Scopus (568) Google Scholar inability to target precisely due to current spread, and magnetic resonance imaging incompatibility. Non-invasive clinical or preclinical methods, such as transcranial magnetic stimulation2Hallett M. Transcranial magnetic stimulation and the human brain.Nature. 2000; 406: 147-150Crossref PubMed Scopus (1059) Google Scholar and transcranial direct current stimulation,3Brunoni A.R. Nitsche M.A. Bolognini N. Bikson M. Wagner T. Merabet L. Edwards D.J. Valero-Cabre A. Rotenberg A. Pascual-Leone A. et al.Clinical research with transcranial direct current stimulation (tDCS): challenges and future directions.Brain Stimul. 2012; 5: 175-195Abstract Full Text Full Text PDF PubMed Scopus (817) Google Scholar do not require a surgical procedure but offer a spatial resolution on the order of several millimeters. Optogenetics has been shown to be a powerful method for modulating population neural activities in rodents more precisely and with cell specificity.4Zhang F. Gradinaru V. Adamantidis A.R. Durand R. Airan R.D. de Lecea L. Deisseroth K. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures.Nat. Protoc. 2010; 5: 439-456Crossref PubMed Scopus (516) Google Scholar,5Yizhar O. Fenno L.E. Davidson T.J. Mogri M. Deisseroth K. Optogenetics in neural systems.Neuron. 2011; 71: 9-34Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar Optogenetics requires genetic modification through viral infection, which makes it challenging to be applied to humans.6Gilbert F. Harris A.R. Kapsa R.M.I. Controlling brain cells with light: ethical considerations for optogenetic clinical trials.AJOB Neurosci. 2014; 5: 3-11Crossref Scopus (37) Google Scholar Ultrasound neuromodulation, an emerging non-invasive neuromodulation method, has been demonstrated to evoke action potentials in vitro and behavioral responses in vivo in rodents,7Tufail Y. Matyushov A. Baldwin N. Tauchmann M.L. Georges J. Yoshihiro A. Tillery S.I.H. Tyler W.J. Transcranial pulsed ultrasound stimulates intact brain circuits.Neuron. 2010; 66: 681-694Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar,8Tufail Y. Yoshihiro A. Pati S. Li M.M. Tyler W.J. Ultrasonic neuromodulation by brain stimulation with transcranial ultrasound.Nat. Protoc. 2011; 6: 1453-1470Crossref PubMed Scopus (236) Google Scholar non-human primates,9Deffieux T. Younan Y. Wattiez N. Tanter M. Pouget P. Aubry J.-F. Low-intensity focused ultrasound modulates monkey visuomotor behavior.Curr. Biol. 2013; 23: 2430-2433Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar and even in human subjects.10Legon W. Sato T.F. Opitz A. Mueller J. Barbour A. Williams A. Tyler W.J. Transcranial focused ultrasound modulates the activity of primary somatosensory cortex in humans.Nat. Neurosci. 2014; 17: 322Crossref PubMed Scopus (410) Google Scholar, 11Legon W. Bansal P. Tyshynsky R. Ai L. Mueller J.K. Transcranial focused ultrasound neuromodulation of the human primary motor cortex.Sci.Rep. 2018; 8: 10007Crossref PubMed Scopus (83) Google Scholar, 12Legon W. Rowlands A. Opitz A. Sato T.F. Tyler W.J. Pulsed ultrasound differentially stimulates somatosensory circuits in humans as indicated by EEG and FMRI.PLoS One. 2012; 7: e51177Crossref PubMed Scopus (57) Google Scholar, 13Mueller J. Legon W. Opitz A. Sato T.F. Tyler W.J. Transcranial focused ultrasound modulates intrinsic and evoked EEG dynamics.Brain Stimul. 2014; 7: 900-908Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar However, the spatial resolution for conventional ultrasound neuromodulation is still limited to several millimeters. More recently, a fiber-based optoacoustic converter has been proposed and demonstrated to achieve neuromodulation with submillimeter spatial resolution utilizing the optoacoustic effect,14Jiang Y. Lee H.J. Lan L. Tseng H.-a. Yang C. Man H.-Y. Han X. Cheng J.-X. Optoacoustic brain stimulation at submillimeter spatial precision.Nat. Commun. 2020; 11https://doi.org/10.1038/s41467-020-14706-1Crossref Scopus (17) Google Scholar although it requires surgical implantation for in vivo applications. Nanostructures target neuron membrane locally and convert and amplify the external excitation to local stimuli, offering new interfaces as promising alternative neural stimulation approaches. Gold nanoparticles and nanorods have been studied for photothermal neural stimulation in vitro.15Carvalho-de-Souza J.L. Treger J.S. Dang B. Kent S.B. Pepperberg D.R. Bezanilla F. Photosensitivity of neurons enabled by cell-targeted gold nanoparticles.Neuron. 2015; 86: 207-217Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 16Yong J. Needham K. Brown W.G.A. Nayagam B.A. McArthur S.L. Yu A. Stoddart P.R. Gold-nanorod-assisted near-infrared stimulation of primary auditory neurons.Adv. Healthc. Mater. 2014; 3: 1862-1868Crossref PubMed Scopus (87) Google Scholar, 17Eom K. Im C. Hwang S. Eom S. Kim T.S. Jeong H.S. Kim K.H. Byun K.M. Jun S.B. Kim S.J. Synergistic combination of near-infrared irradiation and targeted gold nanoheaters for enhanced photothermal neural stimulation.Biomed. Opt. Express. 2016; 7: 1614-1625Crossref PubMed Scopus (19) Google Scholar, 18Carvalho-de-Souza J.L. Nag O.K. Oh E. Huston A.L. Vurgaftman I. Pepperberg D.R. Bezanilla F. Delehanty J.B. Cholesterol functionalization of gold nanoparticles enhances photoactivation of neural activity.ACS Chem. Neurosci. 2019; 10: 1478-1487Crossref PubMed Scopus (13) Google Scholar Gold nanoparticles and carbon nanotubes have also been used for photothermal-driven optocapacitive stimulation in vitro.19Carvalho-de-Souza J.L. Pinto B.I. Pepperberg D.R. Bezanilla F. Optocapacitive generation of action potentials by microsecond laser pulses of nanojoule energy.Biophys. J. 2018; 114: 283-288Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 20Farah N. Zoubi A. Matar S. Golan L. Marom A. Butson C.R. Brosh I. Shoham S. Holographically patterned activation using photo-absorber induced neural-thermal stimulation.J. Neural Eng. 2013; 10https://doi.org/10.1088/1741-2560/10/5/056004Crossref PubMed Scopus (42) Google Scholar, 21Weissler Y. Farah N. Shoham S. Simulation of morphologically structured photo-thermal neural stimulation.J. Neural Eng. 2017; 14https://doi.org/10.1088/1741-2552/aa7805Crossref PubMed Scopus (5) Google Scholar The Tian and Bezanilla groups reported photoelectrical stimulations with silicon nanostructures.22Parameswaran R. Carvalho-de-Souza J.L. Jiang Y.W. Burke M.J. Zimmerman J.F. Koehler K. Phillips A.W. Yi J. Adams E.J. Bezanilla F. et al.Photoelectrochemical modulation of neuronal activity with free-standing coaxial silicon nanowires.Nat. Nanotechnol. 2018; 13: 260-266Crossref PubMed Scopus (96) Google Scholar In these light-driven stimulations, the wavelengths used were mostly in the range of 520–808 nm, which has limited penetration through skulls and brain tissue. In pursuit of deeper penetration, thermal stimulation triggered by nanoparticles absorbing longer-wavelength light or magnetic field has also been investigated. The Pu group demonstrated photothermal neural stimulation in vitro using bioconjugated polymer nanoparticles absorbing 808 nm and binding to transient receptor potential cation channel subfamily V member 1 (TRPV1).23Lyu Y. Xie C. Chechetka S.A. Miyako E. Pu K. Semiconducting polymer nanobioconjugates for targeted photothermal activation of neurons.J. Am. Chem. Soc. 2016; 138: 9049-9052Crossref PubMed Scopus (282) Google Scholar,24Jiang Y. Upputuri P.K. Xie C. Zeng Z. Sharma A. Zhen X. Li J. Huang J. Pramanik M. Pu K. Metabolizable semiconducting polymer nanoparticles for second near-infrared photoacoustic imaging.Adv. Mater. 2019; 31: 1808166Crossref Scopus (190) Google Scholar The Anikeeva group used gene transfection to overexpress the thermally sensitive ion channels in TRPV1 and then utilized the magnetothermal effect of the paramagnetic nanoparticles to activate these channels.25Chen R. Romero G. Christiansen M.G. Mohr A. Anikeeva P. Wireless magnetothermal deep brain stimulation.Science. 2015; 347: 1477-1480Crossref PubMed Scopus (319) Google Scholar In these studies, significant local temperature rise, exceeding the thermal threshold of the ion channels, e.g., 43°C in the case of TRPV 1, for a period longer than several seconds was observed, thus raising concerns over the safety of thermally activated neural stimulation. The Khizroev group used the magnetoelectric nanoparticles under an applied magnetic field to perturb the voltage-sensitive ion channels for neuron modulation.26Yue K. Guduru R. Hong J.M. Liang P. Nair M. Khizroev S. Magneto-electric nano-particles for non-invasive brain stimulation.PLoS One. 2012; 7https://doi.org/10.1371/journal.pone.0044040Crossref Scopus (65) Google Scholar Notably, these magnetic stimuli-based techniques deliver a spatial precision relying on the confinement of the magnetic field, which is on the millimeter-to-centimeter scale. New technologies and concepts are still sought to achieve non-invasive, genetic free and precise neural stimulation. Here, we report the development and application of photoacoustic nanotransducers (PANs) to enable non-genetic neural stimulation in cultured primary neurons and in mouse brain in vivo (Figure 1A). Our PANs, based on synthesized semiconducting polymer nanoparticles, efficiently generate localized ultrasound by a photoacoustic process upon absorption of nanosecond pulsed light in the near-infrared second window (NIR-II; 1,000–1,700 nm) (Figure 1B). NIR-II light has the capability of centimeter-deep tissue penetration,27Henderson T.A. Morries L.D. Near-infrared photonic energy penetration: can infrared phototherapy effectively reach the human brain?.Neuropsychiatr. Dis. Treat. 2015; 11: 2191-2208Crossref PubMed Scopus (185) Google Scholar,28Hamblin M.R. Shining light on the head: photobiomodulation for brain disorders.BBA Clin. 2016; 6: 113-124Crossref PubMed Scopus (192) Google Scholar which is beyond the reach of visible light currently used in optogenetics. We modified the PAN surface for non-specific binding to neuronal membrane and specific targeting of mechanosensitive ion channels, respectively. We showed that upon excitation at 1,030 nm, PANs on the neuronal membrane successfully activated rat cortical neurons, confirmed by real-time fluorescence imaging of the fluorescent calcium indicator GCaMP6f. The spatial resolution of the PAN stimulation was shown to be completely determined by the illumination area of the light, and single-neuron stimulation was demonstrated under excitation of NIR-II light delivered by a tapered fiber. We then demonstrated in vivo motor cortex activation and evoked subsequent motor responses through PANs directly injected into a mouse living brain. Importantly, the heat generated by the nanosecond laser pulses is confined inside the PAN, resulting in a transient temperature rise during the photoacoustic process, evidenced by finite element modeling simulations. Collectively, our findings propose PANs as a new platform for modulating neuronal activities. Triggered by NIR-II light and showing negligible temperature increase, PANs open up opportunities for deep-penetrating-light controlled neural activation with high precision. We first synthesized NIR-II absorbing semiconducting polymer bis-isoindigo-based polymer (BTII).29Luo X. Tran D.T. Sun H. Mi T. Kadlubowski N.M. Zhao Y. Zhao K. Mei J. Bis-isoindigos: new electron-deficient building blocks for constructing conjugated polymers with extended electron delocalization.Asian J. Org. Chem. 2018; 7: 2248-2253Crossref Scopus (12) Google Scholar To obtain nanoparticles and modified the polymer with polystyrene-block-poly(acryl acid) (PS-b-PAA) via a nanoprecipitation method (Figure 1C). The PS-b-PAA was chosen due to the amphiphilic nature of its chemical structure. The hydrophobic polystyrene portion forms a π-π stacking with the polymer, while the hydrophilic poly(acryl acid) (PAA) makes the polymer into water-soluble nanoparticles with carboxyl groups decorated on the surface. The Fourier transform infrared (FTIR) spectrum confirmed the presence of carboxyl groups (Figure S1), indicating the successful modification. The PANs were dispersed in aqueous solution for characterization. The size of nanoparticles prepared was measured to be 58.0 ± 5.2 nm using dynamic light scattering (DLS) (Figure 1D). Transmission electron microscopy (TEM) imaging of PANs (Figure S2) shows an average particle diameter of 52.9 ± 12.2 nm, consistent with the DLS measurement results. The nanoparticles were found to be negatively charged, indicated by a potential of −79.79 ± 4.04 mV through the zeta-potential measurement. To confirm that the surface negative charge is introduced by the surfactant PS-b-PAA, we performed surface modification using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)-2000 (DSPE-PEG), a neutrally charged surfactant, as a comparison. DSPE-PEG-modified PANs were found to be charged with −4.88 ± 3.06 mV (Figure 1E). The planar backbone of the semiconducting polymer chain pushed the absorption to the NIR-II.30Wu J.Y.Z. You L.Y. Lan L. Lee H.J. Chaudhry S.T. Li R. Cheng J.X. Mei J.G. Semiconducting polymer nanoparticles for centimeters-deep photoacoustic imaging in the second near-infrared window.Adv. Mater. 2017; 29https://doi.org/10.1002/adma.201703403Crossref Scopus (84) Google Scholar We confirmed this by UV-visible NIR spectroscopy. The nanoparticles broadly absorb NIR-II light from 800 to 1,300 nm with a peak at 1,100 nm (Figure 1F). Next, we tested whether PANs can generate sufficient optoacoustic waves. In the optoacoustic process, an optoacoustic wave is generated following a transient temperature increase and thermal expansion of the nanoparticle. Importantly, two conditions, stress confinement and thermal confinement, need to be met for efficient photoacoustic generation. The initial pressure p0 generated is related to light absorption by the following expression: p0 = ΓμaF, where μa is the absorption coefficient of the absorber, F is the local light fluence, and Γ is the Grüneisen parameter. The Grüneisen parameter can be expressed as Γ = βvs2/Cp = β/(κρCp), where β is the isobaric volume expansion coefficient, Cp is the heat capacity, vs is the acoustic speed, κ is the isothermal compressibility, and ρ is the mass density.31Wang L.V. Photoacoustic Imaging and Spectroscopy. CRC Press, 2017Crossref Scopus (1) Google Scholar Per the stress confinement, to build up the thermoelastic pressure within a nanoparticle with a diameter of less than 100 nm, considering the speed of sound, a laser pulse of less than 67 ps is required. Yet a mode-locked picosecond pulsed laser usually has several orders of magnitude lower pulse energy than a Q-switched nanosecond pulsed laser. Therefore, nanosecond pulsed lasers are widely used for photoacoustic applications. Regarding thermal confinement, the thermal conduction time must be longer than the laser excitation pulse width to generate photoacoustic waves efficiently. The thermal conduction time can be approximated by τth=L2/4D, where L is the length of diffusion and D is the thermal diffusivity of local environment. In the case of PAN, the local environment is water around the cell body. Water has a thermal diffusivity of 1.4 × 10−3 cm2/s, and the thermal diffusion length is approximated by the nanoparticle size, which is ~60 nm. The thermal diffusion time constant τth is thus approximately 6 ns. Therefore, we utilized a nanosecond laser pulse of 3 ns to achieve efficient photoacoustic generation. Measured with an ultrasound transducer with a central frequency at 5 MHz, 1.0 mg/mL nanoparticle solution exhibits a photoacoustic signal showing a waveform in time domain of approximately 2 μs in width and a peak-to-peak amplitude of 33.95 mV (Figure 1G), under 1,030-nm nanosecond laser with a pulse width of 3 ns, a repetition rate of 3.3 kHz, and an energy density of 21 mJ/cm2. The peak pressure was measured to be 1.36 kPa using a needle hydrophone. Since these nanoparticles generate a strong photoacoustic signal under pulsed NIR-II light, we termed them “photoacoustic nanotransducers” (PANs) and studied their potential for neural binding and stimulation, as detailed below. As recently reported, nanoparticles with negatively charged surface can bind onto neuronal membrane, whereas positive nanostructures showed no interactions with neurons.32Dante S. Petrelli A. Petrini E.M. Marotta R. Maccione A. Alabastri A. Quarta A. De Donato F. Ravasenga T. Sathya A. et al.Selective targeting of neurons with inorganic nanoparticles: revealing the crucial role of nanoparticle surface charge.ACS Nano. 2017; 11: 6630-6640Crossref PubMed Scopus (49) Google Scholar To examine whether negatively charged PANs can bind onto the neuron membrane, we cultured PANs with embryonic cortical neurons collected from Sprague-Dawley rats. The neurons were first cultured for 15–18 days (days in vitro, DIV 15–18). We then added 150 μL of 20 μg/mL PAN solution into the culture, reaching a concentration of 2 μg/mL. The same concentration was used in all experiments in this work unless otherwise noted. Confirming and quantifying the binding of PANs to neurons is critical for successful stimulation. Since the semiconducting polymer shows strong intrinsic transient absorption (TA) signals, we then used label-free TA microscopy to visualize binding of PANs to neurons. In TA microscopy, two synchronized femtosecond laser pulse trains, pump and probe respectively, are focused onto the sample. The electronically resonant pump laser pulse excites the molecule to its excited state, after which the probe laser pulse probes the TA change induced by the pump. Such non-linear absorption signals originate from the signature excited state dynamics of the molecule.33Zhu Y. Cheng J.-X. Transient absorption microscopy: technological innovations and applications in materials science and life science.J. Chem. Phys. 2020; 152: 020901Crossref PubMed Scopus (20) Google Scholar, 34Zhu T. Snaider J.M. Yuan L. Huang L. Ultrafast dynamic microscopy of carrier and exciton transport.Annu. Rev. Phys. Chem. 2019; 70: 219-244Crossref PubMed Scopus (39) Google Scholar, 35Fischer M.C. Wilson J.W. Robles F.E. Warren W.S. Invited review article: pump-probe microscopy.Rev. Sci. Instr. 2016; 87: 031101Crossref PubMed Scopus (130) Google Scholar, 36Beane G. Devkota T. Brown B.S. Hartland G.V. Ultrafast measurements of the dynamics of single nanostructures: a review.Rep. Prog. Phys. 2018; 82: 016401Crossref PubMed Scopus (27) Google Scholar With outstanding chemical specificity, TA microscopy has been applied to visualize molecular content in biological samples37Chen A.J. Yuan X. Li J. Dong P. Hamza I. Cheng J.-X. Label-free imaging of heme dynamics in living organisms by transient absorption microscopy.Anal. Chem. 2018; 90: 3395-3401Crossref PubMed Scopus (19) Google Scholar, 38Dong P.-T. Lin H. Huang K.-C. Cheng J.-X. Label-free quantitation of glycated hemoglobin in single red blood cells by transient absorption microscopy and phasor analysis.Sci. Adv. 2019; 5: eaav0561Crossref PubMed Scopus (10) Google Scholar, 39Fu D. Ye T. Matthews T.E. Grichnik J.M. Hong L. Simon J.D. Warren W.S. Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin.J. Biomed. Opt. 2008; 13: 054036Crossref PubMed Scopus (44) Google Scholar, 40Matthews T.E. Piletic I.R. Selim M.A. Simpson M.J. Warren W.S. Pump-probe imaging differentiates melanoma from melanocytic nevi.Sci. Transl. Med. 2011; 3: 71ra15Crossref PubMed Scopus (125) Google Scholar as well as characterization of nanomaterials41Jung Y. Slipchenko M.N. Liu C.H. Ribbe A.E. Zhong Z. Yang C. Cheng J.X. Fast detection of the metallic state of individual single-walled carbon nanotubes using a transient-absorption optical microscope.Phys. Rev. Lett. 2010; 105: 217401Crossref PubMed Scopus (41) Google Scholar, 42Tong L. Liu Y. Dolash B.D. Jung Y. Slipchenko M.N. Bergstrom D.E. Cheng J.X. Label-free imaging of semiconducting and metallic carbon nanotubes in cells and mice using transient absorption microscopy.Nat. Nanotechnol. 2011; 7: 56-61Crossref PubMed Scopus (80) Google Scholar, 43Huang K.-C. McCall J. Wang P. Liao C.-S. Eakins G. Cheng J.-X. Yang C. High-speed spectroscopic transient absorption imaging of defects in graphene.Nano Lett. 2018; 18: 1489-1497Crossref PubMed Scopus (14) Google Scholar, 44Guo Z. Wan Y. Yang M. Snaider J. Zhu K. Huang L. Long-range hot-carrier transport in hybrid perovskites visualized by ultrafast microscopy.Science. 2017; 356: 59-62Crossref PubMed Scopus (275) Google Scholar, 45Huang L. Hartland G.V. Chu L.-Q. Feenstra R.M. Lian C. Tahy K. Xing H. Ultrafast transient absorption microscopy studies of carrier dynamics in epitaxial graphene.Nano Lett. 2010; 10: 1308-1313Crossref PubMed Scopus (143) Google Scholar, 46Lo S.S. Shi H.Y. Huang L. Hartland G.V. Imaging the extent of plasmon excitation in Au nanowires using pump-probe microscopy.Opt. Lett. 2013; 38: 1265-1267Crossref PubMed Scopus (33) Google Scholar including semiconducting polymer nanoparticles.47Wu J. Lee H.J. You L. Luo X. Hasegawa T. Huang K.C. Lin P. Ratliff T. Ashizawa M. Mei J. Functionalized NIR-II semiconducting polymer nanoparticles for single-cell to whole-organ imaging of PSMA-positive prostate cancer.Small. 2020; 16: 2001215Crossref Scopus (13) Google Scholar,48Wu J. Zhu Y. You L. Dong P.T. Mei J. Cheng J.X.J.A.F.M. Polymer electrochromism driven by metabolic activity facilitates rapid and facile bacterial detection and susceptibility evaluation.Adv. Funct. Mater. 2020; 293: 2005192Crossref Scopus (4) Google Scholar Specifically, we used 200-fs laser pulses at 1,045 nm and 845 nm as the pump and probe beams, respectively, with laser power fixed at 20 mW for both beams for TA imaging. To quantify the effective density of PANs bound to neurons, we first measured the signal-to-noise ratio (SNR) of the TA signals of PAN solutions with concentrations ranging from 2.0 to 55.0 μg/mL to obtain a TA calibration curve (Figure S3). The SNR of TA signals was found to be linear to the PAN concentration with a slope of 14.24 mL/μg. Next, we incubated neurons in culture supplemented with PANs for 15 min, rinsed three times with PBS to remove unbound PANs, and fixed the cells for TA imaging. The PANs were found to bind onto the neurons at an estimated density of 40.2 ± 15.9 PANs per soma (Figure 1H). The number of PANs was calculated on the basis of effective TA concentration estimated according to the measured TA intensity and TA calibration curve, focused spot volume, and estimated molecular weight of PANs. Through depth-resolved TA imaging, the PANs were found to bind mainly on the neuronal membrane instead of entering the neuron through endocytosis (Figure S4). By increasing the culture time to 1 h, a higher binding density was achieved and the number of PANs per neuron on the soma area was found to be 78.1 ± 26.7 (Figures S5 and 1I). In aqueous solution, the PANs prepared show no aggregation. Based on the TA images of PANs co-cultured with neurons, some clusters of PANs were observed when binding to the membranes, possibly due to the complex cellular membrane environment. Different from TA image taken at 15 min co-culture, depth-resolved TA imaging performed at 3 h after PAN addition reveals strong TA signal from PANs located in the cytoplasm, which indicates endocytosis of PANs into the soma (Figure S6). To test the cytotoxicity of PANs, we performed an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on cultured neurons (DIV 15–18) following incubation with PANs for 1 h and 24 h, respectively. Cell viabilities over 80% were observed in all experimental groups with PAN concentrations ranging from 20 to 60 μg/mL (Figure 1J), indicating low toxicity of PANs to neurons. To further test whether laser excitation introduces cellular damage and to determine the damage threshold for in vitro neural stimulation, we also performed a cell viability assay after laser application with SYTOX Green staining of nuclei.49Jones L.J. Singer V.L. Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain.Anal. Biochem. 2001; 293: 8-15Crossref PubMed Scopus (39) Google Scholar Neuron cultures at DIV 15–18 were incubated with 150 μL of 20 μg/mL PAN solution for 15 min. Nanosecond laser at 1,030 nm was delivered to the culture via a 200-μm diameter optical fiber with 0.22 numerical aperture (NA). Conditions of the pulsed laser include a pulse width of 3 ns, a repetition rate of 3.3 kHz, and a laser train of 3 ms (corresponding to 10 laser pulses). As shown in Figure 1K, 1 h after laser excitation, only neurons exposed to 57-μJ laser pulses showed slightly decreased viability, while neurons exposed to laser pulses of 35 and 23 μJ showed similar viability compared with neurons without PAN and laser exposure. Thus, we chose a laser pulse energy of 17 μJ/pulse (pulse energy density of 2.1 mJ/cm2) for future stimulation experiments. The laser energy chosen is well below the damage threshold from the viability assay as well as American National Standards Institute standard for maximum permissible skin exposure (80 mJ/cm2 per pulse). These results collectively show that negatively charged PANs can sufficiently bind onto neuronal membranes via a charge-charge interaction, without obvious cytotoxicity upon desired laser excitation. After showing that PANs bind to neurons, we further investigated their potential for neural stimulation. Calcium imaging was performed on Sprague-Dawley rat primary cortical neurons transfected with GCaMP6f on an in-house built wide-field fluorescence microscope. Imaging was performed on five culture batches for each group. Data from a total of 60 neurons, all of which were within 100 μm proximity to the surface of the fiber, were analyzed. The chosen distance of 100 μm was based on the estimated illumination area of the optical fiber. A representative fluorescence image of the neuron culture is shown in Figure 2A, with the dashed circle showing the position of the fiber. Increase in fluorescence intensity of GCaMP6f at individual neurons was clearly observed immediately after applying pulsed laser, as shown in the real-time video (Vid