Mitochondrion-Specific Blinking Fluorescent Bioprobe for Nanoscopic Monitoring of Mitophagy
线粒体特异性闪烁荧光生物探针用于纳米监测线粒体自噬(中文译名仅供参考)
作者（Zhongju Ye, Lin Wei, Xin Geng, Xin Wang，Zhaohui Li，and Lehui Xiao）
DOI: 10.1021/acsnano.9b05354
线粒体形态的动态变化在细胞代谢中起重要作用。对线粒体超微结构动力学纳米级别分辨率的实时监测，是对基于线粒体的细胞功能进一步了解的关键。在这工作中，报道了一个荧光碳点（fluorescent carbon dot）（命名MitoCD），MitoCD可以在活细胞中选择性靶向线粒体，无论线粒体的膜电位（MMP）减少或消失，MitoCD都可以有效地在线粒体中积累，以此可以探索MMP-independent的线粒体过程。而且，MitoCD是一种基于硫醇的无反应探针，可靶向线粒体不消耗硫醇基线粒体蛋白。 此外，文章中表明，MitoCD在生理条件下具有良好的光物理性质特别适合于类似以下条件：突发性闪烁，高光子计数和低“on”/“off”比,特别适合与基于定位的纳米成像。作者指出，根据光学显微成像结果，动态裂变与融合在活细胞中观察到了线粒体的过程。在线粒体自噬过程中，发现线粒体逐渐塌陷，然后一部分线粒体分裂并消失。作者认为，由于具有吸引力具有生物学和特殊的光物理特性，该探针在各种超分辨率中显示出广阔的应用前景基于生物学的研究，将为线粒体代谢提供深刻的见解。
Figure 1. Retrosynthesis (a) and synthetic route (b) of MitoCD. (c) Schematic representation of the mitophagy tracking with nanoscopic imaging.
Figure 2. (a) TEM image of MitoCDs. (b) Size distribution of MitoCDs determined by TEM. (c) Absorption spectrum of MitoCDs. (d) Excitation and fluorescence emission spectra of MitoCDs. (e) FT-IR spectrum of MitoCDs. (f) XPS spectrum of the MitoCDs.
Figure 3. (a) CLSM image of MitoCDs (1 μg/mL) in live cells. (b) CLSM image of Rho123 (50 nM) in live cells. (c) Bright-field image of HepG2 cells. (d) Merged image of (a)−(c). (e) Colocalization analysis of (a) and (b). (f) Fluorescence intensity profile of the marked line in (d). (g) Fluorescence images of HepG2 cells stained with MitoCDs (1 μg/mL) and Rho123 (50 nM), and then treated with (right side) and without (left side) CCCP (5 μM). (h) Fluorescence images of HepG2 cells stained with MitoCDs (1 μg/mL) and MTDR (200 nM) after the probes were treated with (right side) and without (left side) mPEG-thiol (1 mg/mL).
Figure 4. Representative fluorescence images of MitoCDs in water (a) and DMEM (b). (c) Representative time-dependent fluorescence intensity track from individual MitoCDs. (d) Histograms of τon from MitoCDs within 100 s. (e) Statistically analyzed duty cycle of MitoCDs. (f) Statistical analysis of the photon counts from individual MitoCDs. (g) Representative fluorescence image of single MitoCDs. (h) Representative 3D intensity profiles from individual MitoCDs. (i, k) Representative conventional fluorescence and reconstructed nanoscopic images of MitoCDs on the cover glass, respectively. (j, l) Corresponding intensity profiles of the blue and green lines in the magnified images from (i) and (k).
Figure 5. Representative conventional fluorescence (a) and reconstructed images (b) of mitochondria. (c, d) Corresponding intensity profiles of the white lines in (a) and (b), respectively. (e, f) Mitochondrial dynamic tracking with nanoscopic imaging.
Figure 6. Fluorescence images and colocalization analysis of HepG2 cells costained with MitoCDs (1 μg/mL) and LTG (50 nM) after being treated with 50 μg/mL rapamycin at different time points.
Figure 7. Conventional fluorescence images (a) and reconstructed images (b) of mitochondria during mitophagy at different time points. (c) Conventional fluorescence images of lysosome during mitophagy at different time points. (d) Merged images of (b) and (c).
CONCLUSIONS
作者成功开发了一种荧光探针， MitoCD，具有良好的生物相容性，低细胞毒性。MitoCD可以有选择地有效地靶向活细胞中的线粒体。探针可以是不管MMP的变化如何，固定在线粒体中并且不消耗线粒体中的巯基（thiol group）。此外，MitoCD表现出爆发性的闪烁（blinking）特性，例如高光子计数和“on”时间的低占空比，这是特别适合于纳米成像。闪烁在生理状态下没有任何行为发生添加剂，可用于长时间跟踪。受启发上述优点，作者可视化并监控了基于活细胞的线粒体动力学变化和线粒体使用纳米成像实时进行。线粒体裂变与融合被超出衍射极限（beyond the optical diffraction limit）清晰地识别，证明其在活细胞中的可行性纳米成像。这些观察将揭示对线粒体依赖性代谢的进一步了解纳米级的过程。作者认为它还提供了对开发和设计具有良好光学性能的探针用于指定的蛋白质或细胞结构成像提供深入视野。
文章链接：https://doi.org/10.1021/acsnano.9b05354
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