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Full-field Interferometric Imaging of Propagating Action Potentials


Full-field Interferometric Imaging of Propagating Action Potentials

Tong Ling et al. Light Sci Appl.


Currently, cellular action potentials are detected using either electrical recordings or exogenous fluorescent probes that sense the calcium concentration or transmembrane voltage. Ca imaging has a low temporal resolution, while voltage indicators are vulnerable to phototoxicity, photobleaching, and heating. Here, we report full-field interferometric imaging of individual action potentials by detecting movement across the entire cell membrane. Using spike-triggered averaging of movies synchronized with electrical recordings, we demonstrate deformations up to 3 nm (0.9 mrad) during the action potential in spiking HEK-293 cells, with a rise time of 4 ms. The time course of the optically recorded spikes matches the electrical waveforms. Since the shot noise limit of the camera (~2 mrad/pix) precludes detection of the action potential in a single frame, for all-optical spike detection, images are acquired at 50 kHz, and 50 frames are binned into 1 ms steps to achieve a sensitivity of 0.3 mrad in a single pixel. Using a self-reinforcing sensitivity enhancement algorithm based on iteratively expanding the region of interest for spatial averaging, individual spikes can be detected by matching the previously extracted template of the action potential with the optical recording. This allows all-optical full-field imaging of the propagating action potentials without exogeneous labels or electrodes.

Conflict of interest statement

The authors declare that they have no conflict of interest.


Fig. 1
Fig. 1. Spike-triggered average of the optical phase shift and the electrical signal during an action potential, obtained by averaging 5130 events.
a Propagation of the action potential across the field of view over 6 ms (see Supplementary Video 1). The timing of the frames is shown relative to the electrical spike detected on the electrode indicated by a green arrow. Phase changes can be both positive and negative (shown in false color). The black arrow points to a floating cell, which produced a larger phase shift than the action potential, while the dashed line outlines a semicircular section with detached cells. Scale bar: 25 μm. b Top row: electrical signals recorded on the reference electrode and aligned to the time of maximum deflection. Subsequent spikes exhibit some degree of natural jitter. Bottom row: average optical phase signals extracted from two individual pixels near the reference electrode at the time of an electrical action potential. c Comparison between the electrical signal on the reference electrode (top row) and the time derivative of the optical signal (bottom row). Normalized optical phase signal spatially averaged across the whole FOV (d) and its rising edge (e), with the spike timing corrected by local delays relative to the spike on the reference electrode. Patch clamp recording of the membrane potential (f) and its rising edge (g)
Fig. 2
Fig. 2. Phase changes in a single pixel as a function of the number of averaged spikes and the corresponding cross-correlation with a phase template of the action potential.
Top row (ad): when the number of averaged spikes increases from N = 1 (a) to N = 5130 (d), the SNR of the phase change increases approximately as N. Bottom row (eh): cross-correlation of the phase trace with the spike template shown in Fig. 1d illustrates that a spike can be detected from 50 averages but not from a single trace. Additional averaging marginally increases the SNR of the cross-correlation. In the left two columns, averages can still be performed by frame binning using an ultrafast camera for single spike detection, while the right two columns illustrate high-fidelity detection of the cellular movement based on a larger number of averages using STA
Fig. 3
Fig. 3. All-optical detection of a single action potential by interferometric imaging using a self-reinforcing lock-in algorithm.
a Spatially averaged phase signals in the final iteration of the lock-in algorithm, showing a periodic phase signal (top row). Template matching is implemented by cross-correlating this phase signal with an action potential template obtained in a separate recording, and peaks are identified as spikes (middle row). The timing of the optically and electrically detected spikes is compared in the bottom row. A total of 1584 spikes were recorded electrically in this FOV. b Spiking HEK-293 cells grown on the MEA, as seen in a bright-field microscope. The (c) electrically and (d) optically synchronized spike-triggered average movies cross-correlated with the action potential phase template demonstrate nearly identical spike correlation maps during the action potential. e Histograms of the time lag between the optically and electrically detected spikes after 1, 2, and 7 iterations show convergence to a distribution centered around zero (mean 3.6%, and standard deviation 9.7% of TAP). The analysis is based on 1584 spikes recorded electrically in this FOV. f Convergence analysis. The dashed lines indicate the successive three stages of the algorithm. During the first iteration, an ROI is selected randomly. In the second stage, from the second iteration to steady-state (sixth iteration here), the ROIs are refined and extended to cover > 70% of the FOV. In the final iteration (seventh iteration here), the ROI is further recharted based on SNR optimization for spatial averaging. The area chart (top row) shows the evolution of the ratio of the ROI area to the total area. The bottom row shows the standard deviation of the time lags between the optically and electrically detected spikes, which converges to 9.7% of the action potential period (TAP∼120 ms)
Fig. 4
Fig. 4. System layout.
a Ultrafast QPM synchronized with the MEA recording system. Light from a supercontinuum laser is collimated (C1) and filtered by a dichroic mirror and a bandpass filter (F1). An optical phase image of the sample is obtained from the off-axis interferogram captured by the high-speed camera. b A transparent MEA plated with spiking HEK cells allows simultaneous near-infrared (NIR) optical recording and extracellular electrical recording. c Electrical and optical measurements are synchronized by recording the camera “ready” signal on one of the MEA channels. Trigger signals from the MEA and an external clock control the timing of the captured frames (see Materials and methods)
Fig. 5
Fig. 5. Block diagram of QPM data processing and the self-reinforcing lock-in algorithm for all-optical spike detection.
After QPM processing, frame binning, and background removal, a random region of interest (ROI) is selected for the first iteration of the lock-in detection loop. The phase is spatially averaged across the new spiking ROI, band-stop filtered and correlated with the spike template. This correlation output is used to detect an optical spike trigger, which is applied to the original frames of the phase movie to produce a spike triggered average (STA). Noisy frames are detected and discarded at this step. The STA is then used to threshold a new spiking ROI and the loop repeats. With each iteration, the estimate of the ROI and the resulting STA are improved, and the loop exits when the ROI converges to a stable result

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