Polymer Skulls With Integrated Transparent Electrode Arrays for Cortex-Wide Opto-Electrophysiological Recordings

Abstract

Electrophysiology and optical imaging provide complementary neural sensing capabilities - electrophysiological recordings have high temporal resolution, while optical imaging allows recording of genetically-defined populations at high spatial resolution. Combining these two modalities for simultaneous large-scale, multimodal sensing of neural activity across multiple brain regions can be very powerful. Here, transparent, inkjet-printed electrode arrays with outstanding optical and electrical properties are seamlessly integrated with morphologically conformant transparent polymer skulls. Implanted on transgenic mice expressing the Calcium (Ca²⁺ ) indicator GCaMP6f in excitatory neurons, these "eSee-Shells" provide a robust opto-electrophysiological interface for over 100 days. eSee-Shells enable simultaneous mesoscale Ca²⁺ imaging and electrocorticography (ECoG) acquisition from multiple brain regions covering 45 mm² of cortex under anesthesia and in awake animals. The clarity and transparency of eSee-Shells allow recording single-cell Ca²⁺ signals directly below the electrodes and interconnects. Simultaneous multimodal measurement of cortical dynamics reveals changes in both ECoG and Ca²⁺ signals that depend on the behavioral state.

Keywords: calcium imaging; cortex-wide recording; electrophysiology; multi-modal recording; transparent electrodes.

Figure 1

eSee‐Shells. a) Schematic diagram of electrode array fabrication process on planar PET substrate. b) Left: magnified photomicrograph of transparent electrode array on PET substrate overlaid on the University of Minnesota logo. Scale bar indicates 1 mm. Right: zoomed‐in photomicrograph of a single electrode, with a dashed line to indicate the border of the transparent electrode and the smaller circle to indicate the contact site. Scale bar indicates 500 µm. c) Schematic of eSee‐Shell components. d) Left: photograph of fully assembled eSee‐Shell. Scale bar indicates 1 cm. Right: zoomed‐in photograph of the transparent electrode array bonded into a frame. Scale bar indicates 2 mm.

Figure 2

Benchtop characterizations of eSee‐Shells. a) Impedance spectra of printed PEDOT:PSS electrodes prior to bonding onto window frames (n = 60 electrodes). Black and red lines show impedance magnitude and phase, respectively, and shaded regions indicate standard deviation. b) Impedance magnitude of electrodes at 1 kHz before and after being bonded onto window frames. Red “X” indicates outlier (n = 60 electrodes). c) Orthogonal sections of the point spread function (PSF) of a 0.1 µm fluorescent bead imaged with a confocal microscope through an electrode. Scale bars indicate 5 µm. d,e) Lateral (n = 22) and axial (n = 12) intensity profiles of fluorescent beads imaged through electrodes. Grey lines indicate individual bead profiles and black lines indicate average profile. f,g) Distributions of lateral and axial PSF full‐widths at half maximum (FWHMs) when imaged through various material stacks. In all box‐and‐whisker plots, “x” indicates mean, horizontal line indicates median, box edges indicate upper and lower quartiles, and whiskers indicate maximum and minimum values.

Figure 3

Chronic implantation of eSee‐Shells: a) Top: bright‐field image of a Thy1‐ GCaMP6f mouse brain implanted with an eSee‐Shell, 7 weeks post‐implantation. Circles and numbers indicate the position of each electrode. Scale bar indicates 2 mm. Bottom: simplified version of the mouse brain atlas for the areas under the brain window. Specified areas are: Retrosplenial (RSC), Visual (VC), Barrel (BC), Somatosensory (SSC), and Motor (MC) Cortices. b) Impedance of all ten electrodes for an implanted eSee‐Shell over time. Black and red circles indicate the impedance of working electrodes and non‐working electrodes, respectively. c) Number of working electrodes for different implanted eSee‐Shells (n = 7) over time. Each color indicates one implant. Black line indicates the average number of working electrodes across all animals. Black stars indicate measurements terminated due to unrelated health issues in two animals. d) Fluorescence image of an electrode on an implanted eSee‐Shell. Each circle indicates a different region of interest (ROI) of the transparent implant with different layers of material analyzed in panels (e–g). Scale bar indicates 250 µm. e) Representative ΔF/F traces for each of the ROIs indicated in panel (d). f–g) Quantification of signal‐to‐noise ratio (SNR) for similar ROIs indicated in (d) for all electrodes at 15 and 103 days after the implantation.

Figure 4

Comparison of ECoG and Ca²⁺ signals in an anesthetized and awake mouse: a) Bright‐field image of a Thy1‐GCaMP6f mouse brain implanted with an eSee‐Shell. Circles show the cortical locations of the ECoG electrodes, identified with numbers. Scale bar indicates 2 mm. b) Simultaneously acquired ECoG from selected electrodes and Ca²⁺ signals from the area under the electrode during Ketamine‐induced oscillations. Signals are recorded from the same implanted brain as in (a). c) Wide‐field oscillations of Ca²⁺ signals over a 1 s period under Ketamine anesthesia. d,e) Power spectral density (PSD) of the ECoG and Ca²⁺ signal from electrode 10 in awake and anesthetized states in the low frequency (1–7 Hz) range. Shaded areas represent 95% confidence intervals. f) PSD of the ECoG from electrode 10 in the range of 1–70 Hz during both the anesthetized and awake state. g) Cross‐correlation between simultaneously recorded ECoG and Ca²⁺ signal under the same electrode in awake and anesthetized states for two different electrodes. The awake/anesthetized legend in (d) applies to panels (d–g).

Figure 5

Measuring sensory evoked responses: a) Spatial plot of the peak Ca²⁺ signal in response to whisker stimulation (n = 141 trials). b) Ca²⁺ signals from ROIs indicated in (a) and ECoG across the cortex. c) ECoG response of contra‐lateral sensorimotor electrodes to whisker stimulation from three mice. d,e) N1 and P2 amplitudes across all trials and mice (n = 272 trials) from contra‐lateral sensorimotor electrodes in response to whisker stimulus. f) Spatial plot of peak Ca²⁺ signal in response to whisker stimuli (n = 68 trials). g) Ca²⁺ signal in ROIs indicated in (f) and ECoG of contra‐lateral electrodes in response to stimuli. h) Spatial plot of peak Ca²⁺ signal in response to visual stimuli (n = 174 trials) in the same mouse as (f). i) Ca²⁺ signal in ROIs indicated in (h) and ECoG on contra‐lateral electrodes in response to stimuli. Vertical dashed lines indicate stimulus onset times. All confidence intervals were calculated using the jackknife standard deviation method. All scale bars indicate 1 mm.

Figure 6

Single‐cell imaging under and near transparent ECoG electrodes: a) Wide‐field image of a Cux2‐CreERT2; Ai163 double transgenic mouse brain implanted with an eSee‐Shell. ROIs around electrodes 7 and 10 are identified with boxes. Scale bar indicates 2 mm. b) Coronal section of a Cux2‐CreERT2;Ai163 double transgenic mouse brain expressing sparse GCaMP6s after being treated with tamoxifen. Scale bar indicates 1 mm. c) Zoomed in region from (b) showing single neurons. Scale bar indicates 200 µm. d) Mean image of a 250 s recording of Ca²⁺ fluorescence in ROI 7 (top) and 10 (bottom), respectively. The same order applies to panels (e,f). Scale bar indicates 300 µm, with the same scale for panels in (e). e) Maximum intensity projection of the ΔF for the recordings in (d), overlaid with the identified cells in the same ROI. Dashed lines show the electrode and interconnect location. Circles and numbers identify selected neurons. f) ECoG signals and ΔF/F traces of selected neurons in the ROI.

Figure 7

Multimodal, multiscale measurement of behavioral state‐dependent cortical response to sensory stimuli: a) Average mesoscale Ca²⁺ signals from a Thy1‐GCaMP6f mouse brain throughout 300 ms in response to a 100 ms whisker puff during quiescent trials (n = 45). b) Average ECoG (black) and Ca²⁺ signal color‐coded from the specified ROIs in the quiescent trials. Averaged trials are the same as those used for panel (a). c) Average mesoscale Ca²⁺ signals in response to the whisker puff stimulus during whisking trials (n = 20). d) Average ECoG and Ca²⁺ signal from the specified ROIs in the quiescent trials. Axis scales are the same for all subpanels in (b,d). e) From top to bottom: position of electrodes 10, 6, and 8. Middle: the ROI around electrode 10. Second to bottom: single neurons detected under electrode 10. Scale bar indicates 300 µm. Bottom: a snapshot of the behavioral camera video used to analyze the whisking state of the mouse. Scale bar indicates 4 mm. f) Top: coherograms of E‐10 with E‐6 and E‐10 with E‐8 from an eSee‐Shell implanted on a Cux2‐CreERT2;Ai163 mouse for a 150 s recording. Middle: spectrogram of E‐10 in the same period as the top. Second to bottom: ΔF/F signals from individual neurons shown on the left. Bottom: optical flow of the whisker video as a measure of the whisking state. Horizontal line indicates the threshold for counting a state as whisking. Gray columns are quiescent trials (middle of the column), while pink columns indicate whisking trials. g,h) Top‐left: position of the electrode (ROI). Scale bar indicates 300 µm. Top‐right: ROI and the identified neurons. Middle and bottom: average ECoG of the specified electrode and Ca²⁺ signals of selected neurons in that ROI segregated based on the whisking state before the whisker puff stimulus. Axis scales are the same for all plots in (g,h).