Molecular Science Graduate Program, Toronto Metropolitan University, Toronto, Ontario, Canada, M5B 2K3.
Department of Chemistry and Biology, Toronto Metropolitan University, Toronto, Ontario, Canada, M5B 2K3.
Mol Biol Cell. 2023 Sep 1;34(10):ar96. doi: 10.1091/mbc.E22-06-0200. Epub 2023 Jul 5.
The dynamics of living cells can be studied by live-cell fluorescence microscopy. However, this requires the use of excessive light energy to obtain good signal-to-noise ratio, which can then photobleach fluorochromes, and more worrisomely, lead to phototoxicity. Upon light excitation, noble metal nanoparticles such as silver nanoparticles (AgNPs) generate plasmons, which can then amplify excitation in direct proximity of the nanoparticle's surface and couple to the oscillating dipole of nearby radiating fluorophores, modifying their rate of emission and thus, enhancing their fluorescence. Here, we show that AgNPs fed to cells to accumulate within lysosomes enhanced the fluorescence of lysosome-targeted Alexa488-conjugated dextran, BODIPY-cholesterol, and DQ-BSA. Moreover, AgNP increased the fluorescence of GFP fused to the cytosolic tail of LAMP1, showing that metal enhanced fluorescence can occur across the lysosomal membrane. The inclusion of AgNPs in lysosomes did not disturb lysosomal properties such as lysosomal pH, degradative capacity, autophagy and autophagic flux, and membrane integrity, though AgNP seemed to increase basal lysosome tubulation. Importantly, by using AgNP, we could track lysosome motility with reduced laser power without damaging and altering lysosome dynamics. Overall, AgNP-enhanced fluorescence may be a useful tool to study the dynamics of the endo-lysosomal pathway while minimizing phototoxicity.
活细胞的动力学可以通过活细胞荧光显微镜来研究。然而,这需要使用过多的光能来获得良好的信噪比,这可能会导致荧光染料光漂白,更令人担忧的是,导致光毒性。在光激发下,贵金属纳米粒子如银纳米粒子(AgNPs)会产生等离子体,从而可以在纳米粒子表面的直接邻近处放大激发,并与附近辐射荧光团的振荡偶极子耦合,改变它们的发射速率,从而增强它们的荧光。在这里,我们表明,递送到细胞内以在溶酶体中积累的 AgNPs 增强了溶酶体靶向 Alexa488 缀合的葡聚糖、BODIPY-胆固醇和 DQ-BSA 的荧光。此外,AgNP 增加了 GFP 与溶酶体膜蛋白 1(LAMP1)胞质尾部融合的荧光,表明金属增强的荧光可以跨越溶酶体膜发生。AgNP 包含在溶酶体中不会干扰溶酶体的性质,如溶酶体 pH、降解能力、自噬和自噬通量以及膜完整性,尽管 AgNP 似乎增加了基础溶酶体小管化。重要的是,通过使用 AgNP,我们可以在不损坏和改变溶酶体动力学的情况下,用较低的激光功率跟踪溶酶体的运动。总的来说,AgNP 增强的荧光可能是研究内体溶酶体途径动力学的有用工具,同时最小化光毒性。
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