Fluorogenic Dimers as Bright Switchable Probes for Enhanced Super-Resolution Imaging of Cell Membranes
Fluorogenic Dimers as Bright Switchable Probes for Enhanced Super-Resolution Imaging of Cell Membranes
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DOI：
10.1021/jacs.2c07542
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发表时间：
2022-09-26
影响因子：
15
通讯作者：
Klymchenko, Andrey S.
中科院分区：
文献类型：
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作者：
Aparin, Ilya O.;Yan, Rui;Klymchenko, Andrey S.
基于单分子定位显微镜（SMLM）的超分辨率荧光成像能够以纳米级精度可视化细胞结构。然而，它的空间和时间分辨率很大程度上依赖于开/关可切换荧光染料的亮度。此外，在细胞膜中，膜探针的快速横向扩散阻碍了单分子定位。在这里，为了解决这两个基本问题，我们提出了一种基于亮菁染料荧光二聚体的SMLM（纳米级形貌成像点积累，PAINT）的ON/OFF可切换探针的概念。在这些探针中，用低亲和膜锚在其末端修饰与连接器连接的两个菁氨酸单元。由于分子内染料h聚集，它们在水中自猝灭，在与脂质膜的可逆结合上显示发光。连接体中的带电基团进一步降低了探针对脂质膜的亲和力，从而加速了其动态可逆的ON/OFF开关。这个概念在花青素3和5上得到了验证。活细胞的SMLM显示，与参考探针Nile Red和DiD相比，新探针具有更高的亮度，并且在细胞表面的扩散速度降低了10倍，从而将轴向定位精度提高了3倍，降至31 nm。该探针能够以40 s的时间分辨率对活细胞细胞膜上的纳米级纤维突进行前所未有的观察，揭示了它们的快速动态。因此，通过荧光二聚体的协同脱淬和减缓探针在生物膜中的扩散，突破单一可切换染料的亮度限制，为显著增强活细胞的超分辨率荧光显微镜开辟了道路。
Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualiz-ing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. The charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and similar to 10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision >3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40 s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative dequenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of super-resolution fluorescence microscopy of live cells.