Multicolor re-scan super-resolution imaging of live cells.
Multicolor re-scan super-resolution imaging of live cells.
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DOI：
10.21037/qims.2019.05.05
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发表时间：
2019-05
影响因子：
2.8
通讯作者：
Jiuling Liao;Longchao Chen;Xianyuan Xia;Jia Yu;Tingai Chen;Hui Li;Wei Zheng
中科院分区：
文献类型：
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作者：
Jiuling Liao;Longchao Chen;Xianyuan Xia;Jia Yu;Tingai Chen;Hui Li;Wei Zheng
背景多色荧光显微镜在生物学研究中已被证明是必不可少的。然而，传统的显微镜成像亚细胞器的应用受到其衍射有限的空间分辨率的限制。重扫描共焦显微镜（RCM）是一种新型的超分辨成像技术，可以有效地解决这一问题。然而，以往的双波长RCM成像方法通常会导致图像的空间失配，这是由于使用多个激发激光对样品进行顺序扫描。方法提出了一种新的RCM系统，实现了二维超分辨成像。采用光谱仪作为荧光检测系统，采用线性光谱分解算法分离光谱图像中的不同荧光团。此外，由于图像重建过程中引入了人为的分辨率提高，伽马校正被引入到恢复的超分辨率图像。结果通过对乳腺癌细胞中鬼笔环肽标记的F-actin进行成像，我们发现我们的系统的横向分辨率约为171 nm，比宽视场成像提高了1.8倍。三种类型的荧光珠的成功鉴定表明，我们的荧光RCM可以解决不同的荧光团，其光谱很大程度上相互重叠。最后，我们证明了我们的方法适用于成像多色标记的活细胞的细胞器。结论新型RCM系统能够获得无空间失配、无明显光漂白或光损伤的活体细胞超分辨图像。该系统可能提供一种新的成像工具，用于监测涉及细胞中多个分子和细胞器之间相互作用的动态事件。
Background Multicolor fluorescence microscopy has proved essential in biological studies. However, the application of conventional multicolor microscopy to imaging subcellular organelles is restricted by its diffraction-limited spatial resolution. Re-scan confocal microscopy (RCM), a novel super-resolution imaging technique, can effectively address this problem. However, previous multicolor RCM imaging methods usually led to spatial mismatch in images due to the sequential scanning of the sample with multiple excitation lasers. Methods We present a new RCM system to achieve multicolor super-resolution imaging. A spectrograph was used as the multicolor detection system, and a linear spectral unmixing algorithm was applied to separate different fluorophores in the spectral image. Moreover, since the image reconstruction process induced an artificial resolution improvement, a gamma correction was introduced to restore the multicolor super-resolution image. Results By imaging phalloidin-labeled F-actin in breast cancer cells, we found that the lateral resolution of our system is approximately 171 nm, which is a 1.8-fold improvement over that of wide-field imaging. The successful identification of three types of fluorescent beads indicated that our multicolor RCM can resolve different fluorophores whose spectra largely overlap with each other. Finally, we demonstrated that our method is suitable for imaging multicolor-labeled organelles of live cells. Conclusions Our novel RCM system can acquire multicolor super-resolution images of live cells without spatial mismatch, obvious photobleaching or photodamage. This system may provide a new imaging tool for monitoring dynamic events involving interactions between multiple molecules and organelles in cells.