摘要/Abstract
基于单分子定位的随机光学重构超分辨成像作为一种先进的光学成像方法，可用于尺寸小于光学衍射极限的生物结构的超清晰成像，为在单分子层面研究疾病的发病机制及寻找精准的治疗策略提供有力研究工具，在生物医学领域有着广泛的应用前景.随机光学重构超分辨成像技术依赖于标记探针的光物理性质，探针需要在大量缓冲试剂及含巯基试剂存在下才能产生稳定光致闪烁进行超分辨成像，获得理想的超分辨成像结果，但是大量缓冲试剂与巯基试剂对活细胞伤害较大，使得其在活细胞的超分辨成像应用上存在困难，而限制了其在生物医学成像领域的进一步应用，因此，需要开发可用于活细胞的单分子定位超分辨成像的新型光学探针.本工作提出了一种新的可用于单分子定位超分辨成像的五甲川菁染料探针，不需要外加成像缓冲液及巯基试剂就可以产生光致闪烁变化.基于此，开发了一种分子内自发开、关环反应的新型五甲川菁染料探针，具有活细胞膜通透性.探针不需要使用缓冲液体系及对细胞有害的含巯基试剂，在低功率单束激光直接照射下产生光致闪烁，探针对活细胞没有产生明显毒性，适合活细胞的超分辨成像.进入活细胞后探针选择性定位于细胞线粒体上，在激光照射下产生光致闪烁，电子倍增电荷耦合器相机（EMCCD）在采样频率60 Hz下收集不同条件下的光致闪烁图像，设置不同参数进行结果分析，使用ImageJ进行图像预处理后再使用Falcon算法重构获得活细胞线粒体的超分辨成像图像，相比宽场成像，成像分辨率明显提高，为生物医学光学成像提供新的研究手段.
关键词: 单分子定位成像, 超分辨成像, 五甲川菁染料, 光致闪烁, 线粒体探针
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
PDF全文下载地址:
点我下载PDF
