German Edition: DOI: 10.1002/ange.201703033Electroanalytical Chemistry Hot PaperInternational Edition: DOI: 10.1002/anie.201703033

Plasmonic-Based Electrochemical Impedance Imaging of ElectricalActivities in Single CellsXian-Wei Liu+, Yunze Yang+, Wei Wang, Shaopeng Wang, Ming Gao, Jie Wu, and Nongjian Tao*

Abstract: Studying electrical activities in cells, such as actionpotential and its propagation in neurons, requires a sensitiveand non-invasive analytical tool that can image local electricalsignals with high spatial and temporal resolutions. Here wereport a plasmonic-based electrochemical impedance imagingtechnique to study transient electrical activities in single cells.The technique is based on the conversion of the electrical signalinto a plasmonic signal, which is imaged optically withoutlabels. We demonstrate imaging of the fast initiation andpropagation of action potential within single neurons, andvalidate the imaging technique with the traditional patch clamptechnique. We anticipate that the plasmonic imaging techniquewill contribute to the study of electrical activities in variouscellular processes.

Electrical activities are ubiquitous in biological processesand functions, including brain information processing,[1]

cardiac functions,[2] wound healing,[3] and organism growthand development.[4] Understanding them at the cellular andsubcellular levels requires a technique that can map localelectrical signals with both high spatial and temporal reso-lutions. Microelectrode-based electrical recording has playeda unique role in the study of neurons, but it has limited spatialresolution and can be invasive.[5] Fluorescence imaging usingcalcium-sensitive[6] and voltage-sensitive labels[1,7] is powerfulin resolving local electrical activities in cells with high spatialresolution.[8] However, developing suitable labels withoutaffecting the intrinsic cellular activities, and applying them todifferent samples can be time consuming.[9] Additionally, one

must also consider phototoxicity and photobleaching duringfluorescence imaging. A label-free imaging technique formapping local electrical activities in cells is thus a long-standing goal in analytical science.

Here we report plasmonic-based electrochemical impe-dance microscopy (P-EIM) for label-free imaging of actionpotential and propagation in single neurons with high spatialand temporal resolution. The measured action potentialresembles the simultaneously recorded action potential witha patch clamp, but the imaging capability allows us to observethe local electrical activities, and resolve the fast initiation andpropagation of action potentials within a single neuron. Thebasic principle of P-EIM is to optically image a plasmonicresponse in a metal film to an electrical field (Figure 1). This ispossible because the surface plasmon resonance frequencydepends on the electron density in a thin metal film, whichchanges with the electric field near the metal film.[10] Whenaction potential is fired in a neuron on the metal film, ionsflow in and out of the neuron via the ion channels, creatinga local and transient electric field, and induces a change insurface charge density on the metal film, which is imaged withP-EIM.

The P-EIM in the present work was built on top of aninverted optical microscope using a high-numerical apertureoil immersion objective (Figure 1, left panel).[11] Placed on theobjective was a glass slide coated with 47 nm-thick gold film,

Figure 1. Setup of plasmonic imaging of action potential in neurons. Ap-polarized light beam is directed onto a gold-coated glass coverslipthrough an oil immersion objective to excite plasmons on the goldsurface, which is imaged optically with a fast camera. Neurons arecultured on the poly-l-lysine-coated gold surface, and a micropipette ispatched on one neuron to trigger action potential that is recorded withboth the patch clamp electronics and plasmonic imaging. When anaction potential spike is triggered, ions move in and out the neuronvia the ion channels, creating a transient charge near the gold surface,which affects surface plasmons in the gold film, which is imagedoptically with P-EIM.

[*] Prof. X.-W. Liu,[+]

CAS Key Laboratory of Urban Pollutant ConversionSchool of Chemistry and Materials ScienceUniversity of Science & Technology of ChinaHefei 230026 (China)

Prof. X.-W. Liu[+] Dr. Y. Yang,[+] Dr. S. Wang, Prof. N. TaoBiodesign Center for Bioelectronics and BiosensorsArizona State UniversityTempe, AZ 85287 (USA)E-mail: njtao@asu.edu

Prof. W. WangState Key Laboratory of Analytical Chemistry for Life ScienceSchool of Chemistry and Chemical Engineering, Nanjing UniversityNanjing 210093 (China)

Dr. M. Gao, Prof. J. WuDivision of Neurology, Barrow Neurological Institute, St. Joseph’sHospital and Medical CenterPhoenix, AZ 85013 (USA)

[++] These authors contributed equally to this work.

Supporting information for this article can be found under:https://doi.org/10.1002/anie.201703033.

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on which a silicone cell was mounted to hold buffer solution.Incident light from a super-luminescent diode was directedonto the gold film from the objective to excite surfaceplasmons, and the reflected light was collected with the sameobjective and then directed to a CMOS camera to createa plasmonic image (Figure 1, top right panel). The temporalresolution was limited by the CMOS camera with a frame rateof 100 000 frames per second (fps), corresponding to 10 mstemporal resolution. To allow neurons to grow and attach, thegold film was coated with a thin layer of poly-l-lysine. Actionpotential in the neurons was triggered with a patch-clamprecording setup in the current-clamp mode, and the corre-sponding membrane voltage was recorded electrically witha current amplifier, and optically with P-EIM, simultaneously(Figure 1, bottom right panel).

Figure 2a and b show both bright field and plasmonicimages of a hippocampal neuron cultured from embryonicday 18 rat. We evoked action potential in the whole-cellcurrent clamp mode by injecting current pulses with ampli-tude of about 500 pA and duration of 4 ms into the neuron,and recorded a stack of plasmonic images during actionpotential firing (see Figure S1 in the Supporting Information).P-EIM measures a change in the plasmonic signal associatedwith electrical stimulation, which was obtained by subtractingthe first frame from the rest of the frames in each stack. Tostudy transient changes in the plasmonic signal associatedwith action potential, we averaged the P-EIM images overmultiple action potential excitations. We then obtained thetransient plasmonic intensity changes of a neuron by selectingthe regions of the neuron and plotting the intensity profile.The transient plasmonic intensity resembles closely theelectrically recorded action potential spike (Figure 2c andd), which demonstrates plasmonic imaging of action potentialin single neurons.

Unlike electrical recording with the traditional patchclamp method, P-EIM imaging provides both temporal andspatial resolutions. A movie of plasmonic imaging of theaction potential is presented in Supporting Information(video S1). Figure 2e,f show several snapshots of the movieat different stages of action potential, as marked in Figure 2d.The video reveals the action potential triggering process atdifferent stages, including initial depolarization, spontaneousaction potential firing, and action potential dissipation.

We introduced tetrodotoxin (TTX), a Na+ ion channelinhibitor, to block action potential initiation.[12] The plas-monic images show little response to current injection(Figure 3a,b), showing that the P-EIM image intensityresponse is caused by action potential. To further validatethe P-EIM response and test the repeatability of plasmonicimaging, we stimulated the neurons at different frequencies,and observed similar transient P-EIM intensity profilesresembling action potential spikes at the frequencies (Fig-ure 3c,d). The P-EIM images at the peaks of repeatedly firedaction potential spikes (Figure 3e–j) reveal local distributionwithin the neuron, and demonstrate repeatability of themeasurement. The capability of tracking the firing of actionpotential at different frequencies allows one to identify thefrequency changes of action potential, which is critical forresolving frequency coding of information in neurons.

P-EIM can resolve local electrical activities in single cells.This capability allowed us to study action potential initiationand propagation within a single neuron. Figure 4a,b showbright field and plasmonic images of two neurons (outlinedwith white dashed lines), 1 and 2, and the location whereaction potential was triggered (red dot in neuron 1). Figure 4cplots the transient plasmonic imaging intensity profilesmeasured at different locations of the two neurons. Well-defined action potential spikes are clearly resolved in differ-ent regions of neuron 1 (regions 1–4), but not in neuron 2

Figure 2. Plasmonic imaging of action potential in single neurons.a) Bright-field and b) plasmonic images of a hippocampal neuron,where the dashed lines mark the edge of a neuron. c) Patch clamprecording of action potential and d) simultaneous P-EIM recording ofaction potential of the whole cell (frame rate of 10000 fps). To clearlyillustrate the data, we normalized the intensity change DI with meanintensity I, and plotted the plasmonic intensity in negative intensitychange for fair comparison. e–h) Snapshot P-EIM images of actionpotential at the moments marked by the gray spots in (d). The P-EIMimages were averaged over 90 cycles of repeated action potential firingat 23 Hz repeat rate to reduce random noise.

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(region 5), indicating that the two neurons were not electri-cally connected. Analysis of the action potential spikes atdifferent locations of neuron 1 reveals time delays betweenthem. For example, Figure 4d shows a 0.5-ms lag between theaction potentials at two locations, one (outlined with greenline) is near the triggering location, and the other one(outlined with purple line) is near the end of the axon (seeFigure 4b). This observation shows that P-EIM imaging candirectly monitor action potential propagation within a singleneuron, which has been a difficult task with traditionalmethods.

To clearly visualize the time delay in the action potentialbetween different regions of a neuron, and propagation ofaction potential within a neuron, we recorded a P-EIM moviewith 10 ms temporal resolution (video S2). The movie showshow the action potential is initiated near the trigger point andpropagates along the putative axon and to the dendriticregions. Figure 4 e< xfigr4 shows several snapshots of theprocess. From the high-speed P-EIM movie, we constructeda map of action potential time delay (Figure 4 f,g). The mapallowed us to estimate the propagation speed of actionpotential in a neuron to be about 0.1 ms@1 inside a single cell,which agrees with the reported values in literature.[13] Themap further shows that the time delay does not simply

increase with the distance to the triggering location. Thisobservation is consistent with the report that the initiationand propagation of action potential are highly dependent onthe density of sodium ion channels.[14] The capability ofmonitoring action potential propagation within neurons isparticularly useful for the study of the heterogeneousdistribution of sodium ion channels and the associatedfunctions in neurons.[15]

P-EIM measures the local plasmonic response to anelectrical field (potential) near the metal film. In the case ofa bare gold film, the electric field induces a surface charge inthe metal film, which changes the surface plasmon resonancefrequency according to the free electron gas model.[10] Themeasured plasmonic signal is resonant angle shift (Dq), whichis proportional to the surface charge change (electric field),given by Dq = Ds/a, where a is a constant that measures howsensitive the plasmonic signal is to the surface charge (electricfield). For bare gold film, a is 28 C·m@2 deg@1, which agreeswith the free electron gas model calculation.[10] In the presentwork, we coated the gold film with a layer of poly-l-lysinelayer (ca. 1 nm in thickness[16]) and grew neurons on its top.Our calibration shows that a is 2 C·m@2 deg@1, much smallerthan that for the bare gold film (Supporting Information),indicating a larger plasmonic signal. The smaller a is due toelectric field-induced conformational change in the poly-l-lysine layer, which leads to signal amplification.[17] From themeasured a, we estimate that action potential induces a spikeamplitude of about 0.2 m Deg@1 in the P-EIM signal, whichagrees with the observation.

Associated with action potential, Na+ flows into theneuron, and then followed by K+ flowing out of the neuron.This re-distribution of ions can, in principle, change the localrefractive index near the metal film, and thus contributes tothe P-EIM. However, this change in the plasmonic signal isabout 30 times smaller the observed values (see section 4c inthe Supporting Information). Another possible contributionto the measured P-EIM signal is structural changes in theneuronal membrane, including the orientations of the lipidmolecules that leads to birefringence,[18] and mechanicaldeformation of the membrane (see section 4b in the Support-ing Information).[19] The membrane deformation modelpredicts that a softer cell membrane will lead to a largerdeformation. To examine the contribution of membranedeformation, we measured action potential in neurons treatedwith cytochalasin D, which is known to soften cells byinhibiting polymerization of actin, a major component ofcytoskeleton.[20] We found that the action potential peak inneurons treated by cytochalasin D is slightly greater than thatin neurons without the treatment (Figure S7), suggestingpossible contribution of the structural change to the plas-monic action potential peaks. Although we cannot rule outthe contribution from the membrane structural change,potential-induced change in the surface charge and poly-l-lysine layers alone explains the observed plasmonic signal,and the present work demonstrates a label-free imaging ofaction potential and its propagation in single neurons.

In summary, we have demonstrated P-EIM imaging ofaction potential and ion channel activities in single mamma-lian neurons. The imaging technique resolves the individual

Figure 3. TTX inhibited Na+ ion channel (blue) and control experiment(red) measured with the patch clamp (a) and P-EIM recordings (b).Spike train imaging. Micropipette electrode recording action potentialat different frequency (c). P-EIM recording of action potential ofa whole neuron area at a frame rate of 10000 fps (d). Opticaltransmission images of hippocampal neuron (e). Pixel-by-pixel mapsof action potential (f–j) at five peaks (d).

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action potential spikes with width and shape closely resemblethose simultaneously recorded with the traditional patchclamp technique. The imaging technique is label-free, whichsimplifies the sample preparation as required by fluorescenceimaging techniques, and removes the possible effects of thelabeling on the native functions of the neurons. This label-freeimaging capability allows us to image the fast initiation andpropagation of action potential in single neurons at subcel-lular level. The optics of the technique is compatible with thetraditional patch clamp recording and fluorescence micros-copy, allowing the study of the same sample with multipletechniques. Although the present work is focused on culturedneurons, we anticipate the plasmonic imaging technique canbe further improved and applied to study various cellularelectrical activities (e.g., neuronal signaling processes, woundhealing and tissue development,) and contribute to theunderstanding of the related biological functions and pro-cesses by combining it with other methods, such as theoptotrode techniques.

Acknowledgements

We thank NIH (R21DA033839), Gordon, Betty MooreFoundation and National Natural Science Foundation ofChina (NSFC, grant number 21327008, and 21676260) forsupport.

Conflict of interest

The authors declare no conflict of interest.

Keywords: electrical activity · electroanalytical chemistry ·electrochemical impedance microscopy · single cells ·surface plasmon resonances

How to cite: Angew. Chem. Int. Ed. 2017, 56, 8855–8859Angew. Chem. 2017, 129, 8981–8985

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Figure 4. Plasmonic imaging of action potential propagation within a single neuron. a) Optical transmission and b) plasmonic images of twohippocampal neurons, where the dash lines mark the edges of the neurons, and the red dot marks the location of micropipette. c) Time course ofaction potential at locations outlined by solid lines with the corresponding colors in (b). d) Comparison of action potential spikes at two locations(Regions 1 and 3) showing time decay due to finite propagation speed of action potential. The green and purple solid lines are Lorentzian fittingto the corresponding P-EIM data. e) Snapshots of action potential propagation at a frame rate of 100000 fps from a neuron. f) Action potentialdelay map. g) Overlay of the action potential delay map with bright field image of the neuron.

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Manuscript received: March 23, 2017Version of record online: May 15, 2017

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