Tunable acoustic rotation for deep biophysical phenotyping of preclinical Alzheimer's disease.

Despite advances in imaging and biomarker-based approaches, early diagnosis of Alzheimer's disease (AD) at single-cell resolution remains challenging. Biophysical signals at single-cell scale such as calcium dysregulation and reduced membrane fluidity may serve as early markers of AD, yet these weak signals are difficult to capture. Here, we characterized both phenomena using sensitive optical readouts that were enhanced by acoustic-induced cell rotation. The photobleaching half-life of the calcium indicator fluorescence signal and the half-recovery time of the readout used to characterize membrane fluidity both varied with the acoustic frequency used to induce cell rotation. Signal enhancement peaked within an intermediate acoustic-frequency window and decreased at both lower and higher frequencies, producing a bandpass-like response. This frequency-dependent behavior guided the selection of optimal operating frequencies for subsequent measurements. We then established an Amyloid-β (Aβ)-induced in vitro cellular model and used multidimensional spatial, temporal, and frequency-domain features to evaluate Aβ-induced cellular stress states. The acoustic-rotation approach distinguished early Aβ-induced cellular stress from untreated controls with 99.0% accuracy and classified five cellular states defined by Aβ exposure duration with 94.8% accuracy. By actively enhancing weak single-cell functional signals, this frequency-dependent acoustic strategy provides a generalizable approach for live-cell optical phenotyping and may support future preclinical studies of AD-related cellular stress.