Bénard Magalie, Chamot Christophe, Schapman Damien, Lebon Alexis, Galas Ludovic
Univ Rouen Normandie, INSERM, CNRS, Normandie Université, HeRacLeS US51 UAR2026, PRIMACEN, Rouen, France.
Bio Protoc. 2025 Feb 20;15(4):e5202. doi: 10.21769/BioProtoc.5202.
Time-lapse fluorescence microscopy is a relevant technique to visualize biological events in living samples. Maintaining cell survival by limiting light-induced cellular stress is challenging and requires protocol development and image acquisition optimization. Here, we provide a guide by considering the quartet , and to obtain appropriate resolutions and information for live cell fluorescence imaging. The pleural mesothelial cell line H28, an adherent cell line that is easy to seed, was used to develop innovative advanced light microscopy strategies. The chosen red and near-infrared probes, capable of passively penetrating through the cell plasma membrane, are particularly suitable because their stimulation from 600 to 800 nm induces less cytotoxicity. The labeling protocol describes the concentration, time, and incubation conditions of the probes and associated adjustments for multi-labeling. To limit phototoxicity, acquisition parameters in advanced confocal laser scanning microscopy with a white laser are determined. Light power must be adjusted and minimized at red wavelengths for reduced irradiance (including a 775 nm depletion laser for STED nanoscopy), in simultaneous mode with hybrid detectors and combined with the fast FLIM module. These excellent conditions allow us to follow cellular and intracellular dynamics for a few minutes to several hours while maintaining good spatial and temporal resolutions. Lifetime analysis in lifetime imaging (modification of the lifetime depending on environmental conditions), lifetime dye unmixing (separation with respect to the lifetime value for the spectrally closed dye), and lifetime denoising (improvement of image quality) provide flexibility for multiplexing experiments. Key features • Cell preservation after labeling with less cytotoxic red, near-infrared dye viable probes. • Determination of lower but efficient probe concentration; adjust good balance between probes concentration and incubation time to achieve multi-labeling. • Long time-lapse acquisition in advanced confocal microscopy with sensitive new-generation detectors. • Confocal image combined with fast FLIM for multi-labeling with spectrally closed dyes, unmixed from lifetime values. • Confocal-STED image acquisition combined with fast FLIM to improve signal-to-noise ratio.
延时荧光显微镜是一种用于可视化活样本中生物事件的相关技术。通过限制光诱导的细胞应激来维持细胞存活具有挑战性，需要制定实验方案并优化图像采集。在此，我们通过考虑四重奏以及来提供指导，以获得适用于活细胞荧光成像的分辨率和信息。胸膜间皮细胞系H28是一种易于接种的贴壁细胞系，用于开发创新的先进光学显微镜策略。所选的能够被动穿透细胞质膜的红色和近红外探针特别合适，因为它们在600至800nm的激发诱导的细胞毒性较小。标记方案描述了探针的浓度、时间和孵育条件以及多标记的相关调整。为了限制光毒性，确定了使用白色激光的先进共聚焦激光扫描显微镜中的采集参数。在红色波长处必须调整并最小化光功率以降低辐照度（包括用于受激发射损耗纳米显微镜的775nm耗尽激光），同时与混合探测器一起使用并结合快速荧光寿命成像模块。这些优异的条件使我们能够在保持良好的空间和时间分辨率的同时，跟踪细胞和细胞内动态几分钟到几个小时。寿命成像中的寿命分析（寿命根据环境条件的改变）、寿命染料解混（相对于光谱封闭染料的寿命值进行分离）和寿命去噪（提高图像质量）为多重实验提供了灵活性。关键特性• 用细胞毒性较小的红色、近红外染料活性探针标记后细胞保存良好。• 确定较低但有效的探针浓度；在探针浓度和孵育时间之间调整良好平衡以实现多标记。• 使用灵敏的新一代探测器在先进共聚焦显微镜中进行长时间延时采集。• 共聚焦图像与快速荧光寿命成像相结合，用于使用光谱封闭染料进行多标记，并根据寿命值进行解混。• 共聚焦-受激发射损耗图像采集与快速荧光寿命成像相结合，以提高信噪比。
