Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes.
Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes.
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DOI：
10.7554/elife.63129
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发表时间：
2021-12-03
期刊：
影响因子：
7.7
通讯作者：
Brenker C
中科院分区：
文献类型：
--
作者：
Kierzek M;Deal PE;Miller EW;Mukherjee S;Wachten D;Baumann A;Kaupp UB;Strünker T;Brenker C
荧光探针在与小生物分子、离子结合或膜电位（Vm）变化时改变其光谱特性，是研究细胞信号传导途径的宝贵工具。在这里，我们介绍了一种新的技术，同时记录多个探头在毫秒级的时间分辨率：频率和频谱调谐复用（FASTM）。与目前的多路复用方法不同，FASTM使用相敏信号检测，这使得用于Vm和离子的常见探针的各种组合可用于多路复用。使用动力学停流荧光法，我们表明，FASTM允许同时记录快速变化的Ca 2+，pH值，Na+，和Vm具有高灵敏度和最小的串扰。FASTM也适用于使用单细胞显微镜和基因编码的FRET生物传感器的多路复用。此外，FASTM与光化学工具兼容，可以研究使用光的信号。最后，我们表明，特殊的时间分辨率的FASTM也可以解决快速化学反应。总之，FASTM为询问细胞信号提供了新的机会。
Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (Vm) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FASTM). Different from present multiplexing approaches, FASTM uses phase-sensitive signal detection, which renders various combinations of common probes for Vm and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FASTM allows simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity and minimal crosstalk. FASTM is also suited for multiplexing using single-cell microscopy and genetically encoded FRET biosensors. Moreover, FASTM is compatible with optochemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FASTM also allows resolving rapid chemical reactions. Altogether, FASTM opens new opportunities for interrogating cellular signaling.