Real-time imaging of action potentials in nerves using changes in birefringence

Abstract

Polarized light can be used to measure the electrical activity associated with action potential propagation in nerves, as manifested in simultaneous dynamic changes in their intrinsic optical birefringence. These signals may serve as a tool for minimally invasive neuroimaging in various types of neuroscience research, including the study of neuronal activation patterns with high spatiotemporal resolution. A fast linear photodiode array was used to image propagating action potentials in an excised portion of the lobster walking leg nerve. We show that the crossed-polarized signal (XPS) can be reliably imaged over a ≥2 cm span in our custom nerve chamber, by averaging multiple-stimulation signals, and also in single-scan real-time "movies". This demonstration paves the way toward utilizing changes in the optical birefringence to image more complex neuronal activity in nerve fibers and other organized neuronal tissue.

Keywords: (170.2655) Functional monitoring and imaging; (170.3880) Medical and biological imaging; (260.1440) Birefringence.

Fig. 1

The electronic and optical setup is depicted. On the right is the optical setup used for line-illumination and imaging of nerve birefringence onto a photodiode (PD) array, and using two crossed linear polarizers (LP_i and LP_a). A pulse generator (PG) sends reset (RES) and clock (CLK) signals to the driver circuit to run the PD array. The driver circuit outputs the analog video (VID) signal, as well as TTL pulses for both the end-of-scan (EOS) and each pixel’s individual readout (TRIG). The first data acquisition card (National Instruments, NI DAQ 1) synchronizes the analog input for the video signal with a sample clock from TRIG, so that no pixel readout is skipped. The second data acquisition card (NI DAQ 2) relays a stimulation pulse (Stim. Trig.) to the PG, which outputs a 1-ms, 10-V pulse to the linear stimulus isolator (LSI), which sends a 1-ms, 1-mA stimulus to one end of the nerve; it also records the electrical signal from the other end of the nerve, passed through a bandpass amplifier (Amp). The electronics and data collection are controlled by LabVIEW.

Fig. 2

The average of the XPS over 100 stimulus periods as a function of pixel number are shown at post-stimulus times of 10, 12 17, 32, 70 and 200 ms (A-F, respectively). The onset of the peak resulting from the compound action potential is evident (A-C) and the gradual recovery to baseline (D-F) lasts hundreds of milliseconds. The propagating peak of the XPS takes ~20 ms to travel a distance of ~2 cm. This is demonstrated in Visualization 1.

Fig. 3

A random, single stimulus period is extracted from the data, and a smoothing computational filter with cutoff frequencies of 0.3 Hz and 100 Hz is applied. These data provide a demonstration of a ‘real-time’, fast tracking of the XPS signal. Timepoints of 10, 12, 17, 32, 70 and 200 ms are shown (A-D, respectively). The onset and recovery of the peak can be reliably detected without averaging. This real-time tracking is demonstrated in Visualization 2.

Fig. 4

Traces of the XPS are shown for 100 stimulus-averaged periods at ~6 mm (A) and ~12 mm (B) from the stimulus. Extracted single stimulus periods (C and D) are also shown. The broadening of the peak as a function of distance and the gradual recovery to baseline are evident in both averaged and real-time signals.

Fig. 5

XPS data for averaged stimulus periods at a faster stimulation rate of 14Hz are shown for post-stimulus times of 15, 20, 35 and 65 ms. The recovery of the peak occurs at a faster rate and the gradual recovery is forced to return to baseline just before the initiation of the next stimulus pulse. The temporal width of the peak is reduced. This is demonstrated in Visualization 3.

Fig. 6

A) The electrical recording (black) and XPS (red) at a standard stimulation rate, 2 Hz. The nerve demonstrates an adaptive response to the faster stimulus, and no reversal of polarity, which is evident with a standard stimulation rate. B) The electrical recording (black) and XPS (red) at a faster stimulation rate, 14 Hz. The XPS peak width is reduced with a faster stimulation, which may indicate a reduction in the recruitment of axons to generate action potentials as a result of adaptation to fast stimulation. The peak of the XPS coincides with the peak of the electrical recording for both fast and standard stimulation rates.