Single molecule localization microscopy as an advanced optical imaging technique is capable of super-resolution imaging of biological targets with the size below the optical diffraction limit. It is promising to provide powerful tools for the exploration of occurrence mechanism of severe diseases and precisely therapeutic method at single cell/organelle level, which exhibits wide applications in biomedical field. Generally, stochastic optical reconstruction microscopy (STORM) is prominently dependent on large amount of imaging buffers (Redox enzymes) and thiol-containing reagents for the ideal photoblinking behaviors of optical probes. However, the imaging buffer and thiol-containing reagents are harmful for the live cells, which make it difficult to carry out STORM imaging in live cells. Therefore, it is of significance to exploit new approaches to display STORM imaging in live cells. In this work, we provided a new strategy to facilitate the design of live cell STORM imaging probes with improved photo-blinking mechanism. A new fluorescent pentamethine cyanine probe with a thiol-attachment (SHCH2CH2CH2-) at the N-position of one indoline moiety was synthesized to show spontaneously photoblinking behavior caused by intramolecular ring-closing/-opening processes. The fluorescent probe is biologically compatible with rare cytotoxicity and suitable for the live cell imaging. The probe can exhibit excellent photo-blinking under the direct illumination of a single laser beam (656 nm) with low power density (200 W·cm-2 for solution sample and 100 W·cm-2 for cell sample, respectively), without using any imaging buffer or thiol-chemicals. And the fluorescent probe was used to test cell toxicity with CCK-8, showed almost no cytotoxicity after 24 h incubation. The photo-blinking frames were collected with an electron multiplying charge coupled device (EMCCD, 60 Hz), and different frames were used to pre-treat with
| ImageJ software and then reconstruct STORM images with a Falcon algorithm to show marked imaging resolution enhancement, compared with wide-field images, which provide a new protocol for biomedical imaging.
Key words:
single-molecule localization microscopy,
super-resolution imaging,
pentamethine cyanine,
photo-blinking,
mitochondrial probe
引用此文
杨志刚, 熊佳, 张炜, 李文, 潘文慧, 张建国, 顾振宇, 黄美娜, 屈军乐. 可用于活细胞线粒体随机光学重构超分辨成像的分子内可逆环化五甲川菁染料探针[J]. 化学学报, 2020, 78(2): 130-139.
Yang Zhigang, Xiong Jia, Zhang Wei, Li Wen, Pan Wenhui, Zhang Jianguo, Gu Zhenyu, Huang Meina, Qu Junle. A Reversibly Intramolecular Cyclization Cy5 Optical Probe for Stochastic Optical Reconstruction Microscopy in Live Cell Mitochondria[J]. Acta Chimica Sinica, 2020, 78(2): 130-139.
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