Despite the significant advantages of two-photon excitation microscopy (TPEM) over traditional confocal fluorescence microscopy in live-cell imaging applications, including reduced phototoxicity and photobleaching, increased depth penetration, and minimized autofluorescence, only a few metal ion-selective fluorescent probes have been designed and optimized specifically for this technique. Building upon a donor–acceptor fluorophore architecture, we developed a membrane-permeant, Zn(II)-selective fluorescent probe, chromis-1, that exhibits a balanced two-photon cross section between its free and Zn(II)-bound form and responds with a large spectral shift suitable for emission-ratiometric imaging. With a K_d of 1.5 nM and wide dynamic range, the probe is well suited for visualizing temporal changes in buffered Zn(II) levels in live cells as demonstrated with mouse fibroblast cell cultures. Moreover, given the importance of zinc in the physiology and pathophysiology of the brain, we employed chromis-1 to monitor cytoplasmic concentrations of labile Zn(II) in oligodendrocytes, an important cellular constituent of the brain, at different stages of development in cell culture. These studies revealed a decrease in probe saturation upon differentiation to mature oligodendrocytes, implying significant changes to cellular zinc homeostasis during maturation with an overall reduction in cellular zinc availability. Optimized for TPEM, chromis-1 is especially well-suited for exploring the role of labile zinc pools in live cells under a broad range of physiological and pathological conditions.

中文翻译：

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Chromis-1，一种针对双光子显微镜优化的比例荧光探针，揭示了分化的少突胶质细胞中不稳定的Zn（II）的动态变化

尽管在活细胞成像应用中，双光子激发显微镜（TPEM）优于传统的共聚焦荧光显微镜，包括降低的光毒性和光漂白，增加的深度渗透和最小的自发荧光，但只有少数几种金属离子选择性荧光探针为此技术专门设计和优化。基于供体-受体荧光团的结构，我们开发了一种可透过膜的Zn（II）选择性荧光探针chromis-1，该探针在其自由态与Zn（II）结合形式之间表现出平衡的两光子截面，并且响应时具有适合发射比率成像的大光谱偏移。有K _d它具有1.5 nM的动态范围和很宽的动态范围，非常适合可视化活细胞中缓冲的Zn（II）水平的时间变化，如小鼠成纤维细胞培养所证明的那样。此外，鉴于锌在大脑的生理和病理生理中的重要性，我们在细胞培养发育的不同阶段，采用chromis-1监测少突胶质细胞（大脑的重要细胞成分）中不稳定的Zn（II）的细胞质浓度。 。这些研究表明，分化为成熟的少突胶质细胞后，探针饱和度降低，这意味着在成熟过程中细胞锌稳态的显着变化，同时细胞锌利用率整体下降。针对TPEM进行了优化，