Covalently Tethered Rhodamine Voltage Reporters for High Speed Functional Imaging in Brain Tissue
Covalently Tethered Rhodamine Voltage Reporters for High Speed Functional Imaging in Brain Tissue
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DOI：
10.1021/jacs.9b12265
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发表时间：
2020-01-08
影响因子：
15
通讯作者：
Miller, Evan W.
中科院分区：
文献类型：
--
作者：
Deal, Parker E.;Liu, Pei;Miller, Evan W.
电压敏感的荧光团能够直接观察生命系统中的膜电位变化。为了将化学合成荧光指示剂的速度和灵敏度与细胞类型特定的遗传方法相结合，我们在这里开发了基于罗丹明的电压报告基因（RhoVR），它可以与基因编码的自标记酶共价连接。这些化学-遗传杂交体的特征在于光诱导电子转移触发的RhoVR电压敏感指示剂通过长的水溶性聚乙二醇接头（RhoVR-Halo）与氯代烷烃HaloTag配体偶联。当应用于细胞时，RhoVR-Halo染料选择性地共价结合到转基因细胞上表面表达的HaloTag酶。RhoVR-Halo染料保持高电压灵敏度-高达34% Delta F/F/100 mV-和非靶向RhoVR典型的快速响应时间，同时获得遗传编码电压指示剂的选择性。我们表明，RhoVR-Halos可以记录从培养的大鼠海马神经元的动作电位在单次试验中，并可用于与绿色荧光钙离子指示剂，如GCaMP，以提供同时电压和钙离子成像。在脑切片中，RhoVR-Halos提供了对特定细胞的精确标记，并可使用落射荧光、共聚焦或双光子显微镜进行成像。使用高速落射荧光显微镜，RhoVR-Halos提供了大鼠脑切片中标记的皮层神经元的动作电位读数，而不需要进行试平均。这些结果证明了混合化学-遗传电压指示剂的潜力，其将小分子发色团的光学性能与遗传编码系统的固有选择性联合收割机相结合，从而允许单独使用任一技术无法获得的成像模式。
Voltage-sensitive fluorophores enable the direct visualization of membrane potential changes in living systems. To pair the speed and sensitivity of chemically synthesized fluorescent indicators with cell-type specific genetic methods, we here develop Rhodamine-based Voltage Reporters (RhoVR) that can be covalently tethered to genetically encoded, self-labeling enzymes. These chemical-genetic hybrids feature a photoinduced electron transfer triggered RhoVR voltage-sensitive indicator coupled to a chloroalkane HaloTag ligand through a long, water-soluble polyethylene glycol linker (RhoVR-Halo). When applied to cells, RhoVR-Halo dyes selectively and covalently bind to surface-expressed HaloTag enzyme on genetically modified cells. RhoVR-Halo dyes maintain high voltage sensitivities-up to 34% Delta F/F per 100 mV-and fast response times typical of untargeted RhoVRs, while gaining the selectivity of genetically encodable voltage indicators. We show that RhoVR-Halos can record action potentials in single trials from cultured rat hippocampal neurons and can be used in concert with green-fluorescent Ca2+ indicators like GCaMP to provide simultaneous voltage and Ca2+ imaging. In a brain slice, RhoVR-Halos provide exquisite labeling of defined cells and can be imaged using epifluorescence, confocal, or two-photon microscopy. Using high-speed epifluorescence microscopy, RhoVR-Halos provide a read-out of action potentials from labeled cortical neurons in a rat brain slice, without the need for trial averaging. These results demonstrate the potential of hybrid chemical-genetic voltage indicators to combine the optical performance of small-molecule chromophores with the inherent selectivity of genetically encodable systems, permitting imaging modalities inaccessible to either technique individually.