Excitation and polarization of isolated neurons by high-frequency sine waves for temporal interference stimulation

The capacity of temporal interference (TI) stimulation to target deep brain regions without affecting nearby surface electrodes remains uncertain. Using artifact-free optical recording, we compare excitation patterns and thresholds in hippocampal neurons stimulated by "pure" and amplitude-modulated sine waves, representing TI waveforms near electrodes and at the target, respectively. We show that pure 2- and 20-kHz sine waves induce repetitive firing at rates that increase up to 60-90 Hz with stronger electric fields. Beyond this limit, action potentials merge into sustained depolarization, resulting in an excitation block. Modulating the sine waves at 20 Hz aligns firing with amplitude "beats" and prevents the excitation block but does not lower excitation thresholds. Thus, off-target TI effects appear unavoidable, though the patterns of neuronal excitation and downstream effects may differ from those at the target. We further analyze membrane charging and relaxation kinetics at nanoscale resolution and confirm an excitation mechanism independent of envelope extraction.