Wang Yuxin, Quan Yue, Zhou Shizheng, Zhou Yinning
Joint Key Laboratory of the Ministry of Education, Institute of Applied Physics and Materials Engineering, University of Macau, Avenida da Universidade, Taipa, 999078, Macau.
Mater Today Bio. 2025 Jul 15;33:102087. doi: 10.1016/j.mtbio.2025.102087. eCollection 2025 Aug.
Photobleaching-induced signal decay undermines the accuracy of fluorescence-based drug screening across multiple disease models (e.g., HER2/EGFR-targeted therapies). Traditional phototoxicity mitigation strategies, primarily through reduced illumination, inevitably compromise imaging resolution and further providing false positive/false negative. Here, we introduce an innovative active photobleaching intervention strategy to solve these problems with the potential for broad applicability across a wide range of cell species. We employ the ultra-low-frequency (500-2000 Hz) acoustic modulation to control 3D cellular rotation (precisely in x-y/y-z planes) with a linearly tunable rotational speed, establishing the first quantitative framework linking acoustic parameters to photobleaching kinetics. Our results indicate that acoustic frequency scanning exerts a bandpass-filter effect on photobleaching half-life, peaking at 1500 Hz and achieving approximately 30-fold fluorescence preservation compared to static conditions. By harnessing our newly developed Acousto-Optogenetics Bandpass Stabilizer, we transform phototoxicity into a tunable parameter, thereby enabling artifact-free drug response analysis. While maintaining maximal photostability, frequency-division multiplexing further permits real-time calcium flux monitoring during optogenetic activation. Complementing this, we engineer FlowMind, an AI-powered software, to automate single-cell dynamic library processing. As proof of concept, screening calcium channel blockers under optimal photostability conditions reveals significant variations in drug screening sensitivity. By integrating acoustic modulation with optogenetics, this paradigm not only pioneers the programmable control of photostability via acoustics, but also catalyzes a transformative shift in precision oncology, heralding a new era of patient-tailored therapeutic regimens with enhanced clinical predictability.
光漂白诱导的信号衰减会影响基于荧光的药物筛选在多种疾病模型（如HER2/EGFR靶向治疗）中的准确性。传统的光毒性缓解策略主要是通过减少光照，但不可避免地会降低成像分辨率，并进一步产生假阳性/假阴性结果。在此，我们引入一种创新的主动光漂白干预策略来解决这些问题，该策略具有广泛适用于多种细胞类型的潜力。我们采用超低频（500 - 2000 Hz）声学调制来控制3D细胞旋转（精确地在x - y/y - z平面），其转速可线性调节，建立了第一个将声学参数与光漂白动力学联系起来的定量框架。我们的结果表明，声学频率扫描对光漂白半衰期具有带通滤波效应，在1500 Hz时达到峰值，与静态条件相比，荧光保留率提高了约30倍。通过利用我们新开发的声光遗传学带通稳定器，我们将光毒性转化为一个可调节的参数，从而实现无伪影的药物反应分析。在保持最大光稳定性的同时，频分复用还允许在光遗传学激活过程中实时监测钙通量。作为补充，我们设计了FlowMind，一款由人工智能驱动的软件，以实现单细胞动态文库处理的自动化。作为概念验证，在最佳光稳定性条件下筛选钙通道阻滞剂揭示了药物筛选敏感性的显著差异。通过将声学调制与光遗传学相结合，这种模式不仅开创了通过声学对光稳定性进行可编程控制的先河，还催化了精准肿瘤学的变革性转变，预示着一个具有更高临床可预测性的患者定制治疗方案的新时代。
