Full-field interferometric imaging of propagating action potentials

Abstract

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.

On mechanisms of light-induced deformations in photoreceptors

“…Characterization of the nm-scale deformations in other electrically active cells, such as HEK cells and cortical neurons, has benefited from a thorough understanding of the underlying mechanisms relating the membrane potential change to mechanical deformation [8], [10]. Cellular responses averaged over many trials provide a template for match-filtering temporal signals, thereby significantly boosting the detection performance in future recordings.…”

“…Here, k t is Boltzman's constant and T is the absolute temperature, ϵ w and ϵ " are the relative permeability of water and the permittivity of free space, z is the number of valence ions in the solution, e " is the electronic charge, and C { is the capacitance of the membrane between the Debye layers. The membrane tension increases by about 0.1 / / , or 10 µN • m <= for a 100 depolarization, which is a decrease of trans-membrane voltage [8]. Increased surface tension of the cell membrane leads to deformation of a cell toward minimization of its surface area, i.e.…”

“…To relate the observed mechanical deformations of cells to the underlying physiological processes, the coupling mechanisms must be known. Recently, we demonstrated that nm-scale deformations in neurons and other electrogenic cells can be explained by the voltage dependence of the membrane tension [8], [10]. Changes in the membrane tension necessarily cause a deformation to rebalance forces across the structure, whether it's a whole cell or an organelle.…”

How neurons move during action potentials

“…Quantitative phase microscopy: Quantitative phase microscopy based on common-path interferometer 4,5 was adapted from our previous work 6,7 (Supplementary Fig. S1).…”

“…Minute (nanometer-scale) cellular deformations accompanying the action potential have been observed in crustacean nerves (0.3~10 nm) [1][2][3][4] , squid giant axon (~1 nm) 5,6 and, recently, in mammalian neurons (0.2~0.4 nm) 7,8 . These findings illustrate that rapid changes in transmembrane voltage due to the opening and closing of the voltage-gated ion channels have mechanical consequences accompanying action potentials and other changes in cell potential, which may allow non-invasive label-free imaging of neural signals 9 .…”