A general design of pyridinium-based fluorescent probes for enhancing two-photon microscopy
A general design of pyridinium-based fluorescent probes for enhancing two-photon microscopy
复制标题
用于增强双光子显微镜的吡啶基荧光探针的总体设计
DOI：
10.1016/j.bios.2023.115604
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发表时间：
2023-08-20
影响因子：
12.6
通讯作者：
Sun, Yujie
中科院分区：
文献类型：
--
作者：
Chen, Rui;Qiu, Kangqiang;Sun, Yujie
双光子吸收荧光探针已成为亚细胞水平生物物质和过程监测的强大成像工具，具有深光穿透、最小光损伤、低自身荧光和高空间分辨率等优点。然而，现有的双光子吸收探针仍然面临着双光子吸收截面小、水溶性差、膜透性低以及潜在的高毒性等局限性。本文报道了三种小分子探针，即msp -1臂、lyso -2臂和mito -3臂，它们由一个吡啶中心（电子受体）和各种甲氧基苯乙烯“臂”（电子给体）组成。这些探针具有优异的荧光量子产率和良好的水溶性。利用固有的分子内电荷转移和激子耦合效应，这些配合物在近红外区域表现出优异的双光子吸收。值得注意的是，Lyso-2arm和Mito-3arm分别对溶酶体和线粒体表现出不同的靶向能力。在双光子显微镜实验中，Mito-3arm在2D单层HeLa细胞中优于商用双光子吸收染料，提供更高的分辨率，更宽的近红外光激发窗口和更高的信噪比。此外，三维人类前脑类器官的双光子生物成像证实了Lyso-2arm和Mito-3arm成功的深层组织成像能力。总的来说，这项工作提出了一种合理的设计策略，通过改变用于生物成像应用的共轭“臂”的数量来开发有能力的双光子吸收探针。
Two-photon absorbing fluorescent probes have emerged as powerful imaging tools for subcellular-level monitoring of biological substances and processes, offering advantages such as deep light penetration, minimal photodamage, low autofluorescence, and high spatial resolution. However, existing two-photon absorbing probes still face several limitations, such as small two-photon absorption cross-section, poor water solubility, low membrane permeability, and potentially high toxicity. Herein, we report three small-molecule probes, namely MSP-1arm, Lyso-2arm, and Mito-3arm, composed of a pyridinium center (electron-acceptor) and various methoxystyrene "arms" (electron-donor). These probes exhibit excellent fluorescence quantum yield and decent aqueous solubility. Leveraging the inherent intramolecular charge transfer and excitonic coupling effect, these complexes demonstrate excellent two-photon absorption in the near-infrared region. Notably, Lyso-2arm and Mito-3arm exhibit distinct targeting abilities for lysosomes and mitochondria, respectively. In two-photon microscopy experiments, Mito-3arm outperforms a commercial two-photon absorbing dye in 2D monolayer HeLa cells, delivering enhanced resolution, broader NIR light excitation window, and higher signal-to-noise ratio. Moreover, the two-photon bioimaging of 3D human forebrain organoids confirms the successful deep tissue imaging capabilities of both Lyso-2arm and Mito-3arm. Overall, this work presents a rational design strategy in developing competent two-photon-absorbing probes by varying the number of conjugated "arms" for bioimaging applications.