Liu Jie, Liu Zhihe, Mi Feixue, Yao Zihan, Fang Xiaofeng, Wang Yingjie, Zhao Zhongying, Wu Changfeng
Guangdong Provincial Key Laboratory of Advanced Biomaterials, Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China.
Department of Biology, Hong Kong Baptist University, Kowloon, Hong Kong 999077, China.
Chem Biomed Imaging. 2023 Jun 19;1(6):550-557. doi: 10.1021/cbmi.3c00058. eCollection 2023 Sep 25.
Fluorescence lifetime imaging microscopy (FLIM) has been widely used in cell biology to detect biomolecules and their interactions. However, breaking the diffraction limit remains a challenge in FLIM due to the typically required photon counting method and the limited photon output of conventional dyes. Here, we introduce semiconducting polymer dots (Pdots) for fluorescence lifetime imaging in expansion microscopy by virtue of their tunable lifetime and huge photon budget. We developed three fluorescent Pdots with average lifetimes ranging from 0.4 to 5 ns by varying the polymer species and compositions. Despite their large spectral overlap, distinctive distributions of the Pdots can be resolved in the lifetime domain. The high fluorescence brightness and large photon output offered by Pdots enable multiplex lifetime imaging in photon-starved expansion microscopy, by which subcellular structures were resolved with a spatial resolution of ∼49 nm. This study reveals the potential of the tunable Pdot probes for lifetime multiplex imaging in expansion microscopy.
荧光寿命成像显微镜（FLIM）已在细胞生物学中广泛用于检测生物分子及其相互作用。然而，由于通常所需的光子计数方法以及传统染料有限的光子输出，突破衍射极限在FLIM中仍然是一个挑战。在此，我们引入半导体聚合物点（Pdots）用于扩展显微镜中的荧光寿命成像，这得益于其可调谐的寿命和巨大的光子预算。我们通过改变聚合物种类和组成，开发了三种平均寿命在0.4至5纳秒范围内的荧光Pdots。尽管它们有很大的光谱重叠，但Pdots在寿命域中可以分辨出独特的分布。Pdots提供的高荧光亮度和大量光子输出使得在光子匮乏的扩展显微镜中能够进行多重寿命成像，通过这种方法，亚细胞结构以约49纳米的空间分辨率得到分辨。这项研究揭示了可调谐Pdot探针在扩展显微镜中进行寿命多重成像的潜力。
