Chromatin Labeling and Imaging group, Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
ACS Chem Biol. 2021 Nov 19;16(11):2130-2136. doi: 10.1021/acschembio.1c00538. Epub 2021 Nov 4.
Here we report a small molecule tubulin probe for single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy and MINFLUX nanoscopy, which can be used in living and fixed cells. We explored a series of taxane derivatives containing spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR) and found that the linker length profoundly affects the probe permeability and off-targeting in living cells. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and the 6'-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameter of ≤50 nm was routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin allows a complementary use of different nanoscopy techniques for investigating microtubule functions and developing imaging methods. For the first time, we resolved the inner microtubule diameter of 16 ± 5 nm by optical nanoscopy and thereby demonstrated the utility of a self-blinking dye for MINFLUX imaging.
在这里，我们报告了一种用于单分子定位显微镜（SMLM）、受激发射损耗（STED）显微镜和 MINFLUX 纳米显微镜的小分子微管蛋白探针，该探针可用于活细胞和固定细胞。我们探索了一系列含有自发闪烁远红染料羟甲基硅罗丹明（HMSiR）的紫杉烷衍生物，并发现连接体长度对探针在活细胞中的通透性和脱靶性有很大的影响。表现最好的探针 HMSiR-tubulin 由卡巴他赛和 HMSiR 的 6'-非对映异构体通过 C6 连接体桥接而成。在活细胞和固定细胞的 SMLM 实验中， routinely 测量了直径≤50nm 的微管。HMSiR-tubulin 允许互补使用不同的纳米显微镜技术来研究微管功能并开发成像方法。我们首次通过光学纳米显微镜解析了 16±5nm 的内微管直径，从而证明了自闪烁染料在 MINFLUX 成像中的实用性。
