Acousto-optogenetics bandpass stabilizer: A programmable platform for mapping single-cell phenotypic life trajectories

Modulating cellular phenotypes represents a powerful approach for obtaining an in-depth understanding of cellular functional behaviors, signaling pathways, and disease pathogenesis. Among numerous available strategies, optogenetic stimulation offers precise regulation of cellular activities at the molecular level, with exceptional spatiotemporal resolution. However, the large-scale, nonlinear nature of the resulting data poses considerable challenges for analysis. To address these complexities, our study presents a novel deep learning model for the regulation of cellular phenotypes through optical gating. For the first time, we have successfully integrated optogenetics with acoustofluidics, achieving comprehensive, full-angle observation of cell morphology while significantly reducing photobleaching, thereby prolonging the fluorescence half-life to approximately 4.3 times in HEK293T cells. Furthermore, the use of frequency-division multiplexing in protein activation significantly mitigates fluorescence crosstalk, thereby enabling real-time calcium ion monitoring during optogenetic processes. For data post-processing, we developed FlowMind, an open-source, object-oriented programming, AI-driven application that streamlines the workflow. As a proof of concept, we compared the effects of two calcium channel blockers on calcium ion perturbations induced by optogenetic stimulation, providing new perspectives for drug screening. Our study offers a fully optimized and integrated workflow utilizing a programmable optogenetic-acoustofluidic platform through user-friendly sample handling capabilities. By encompassing hardware configuration, data acquisition, and post-processing, this platform enables precise and high-throughput cellular activity modeling. The innovative approach not only simplifies the experimental workflow but also significantly accelerates the exploration of cellular dynamics, providing a powerful tool for advancing drug discovery pipelines, disease diagnostics, and the development of personalized therapeutic strategies.

Prof. Zhou Yin-Ning currently serves as an Assistant Professor in the Institute of Applied Physics and Materials Engineering at the University of Macau. She leads the High-Tech Intelligent Acoustic Laboratory, focusing on: Novel ultrafast real-time acoustic-impedance microfluidic chips; Acoustic mediated biotissue transfer engineering; automated acoustofluidic single-cell population-control platforms; Intelligent biosensing platforms. These efforts target real-time monitoring of diverse feature cells (with portable applications in oncology and infectious diseases), ultrafast screening of bacterial antibiotic resistance, single-cell analysis and drug screening, and acoustic-transferred tissue organoids with intelligent phenotyping. She has published over 50 papers in high-level journals including Nat.Commun, Adv.Mater. Mater.Today.Bio, Biosens.Bioelectron, Lab Chip, etc..