All-optical crosstalk-free manipulation and readout of Chronos-expressing neurons.
All-optical crosstalk-free manipulation and readout of Chronos-expressing neurons.
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Chronos 表达神经元的全光学无串扰操纵和读出。
DOI：
10.1088/1361-6463/aaf944
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Soor NS
中科院分区：
文献类型：
--
作者：
Soor NS
所有光学神经生理学允许操作和读取单细胞空间分辨率和毫秒时间分辨率的神经网络活动。神经元可以表达在光吸收时驱动跨膜电流的蛋白质，从而实现对膜电位和动作电位信号的光学控制。此外，神经元可以通过基因或合成荧光报告细胞内钙浓度或膜电位的变化来标记。因此，为了光学操纵和并行读出神经活动，涉及两个光谱：致动器的动作光谱和荧光报告的吸收光谱。由于这些光谱的重叠，以前的全光神经生理学范式被读出光引起的神经元活动的虚假激活所阻碍。在这里，我们将蓝绿色吸收光遗传致动器Chronos与深红色荧光钙报告细胞CaSiR-1配对。我们发现转染了Chronos的中国仓鼠卵巢细胞在适合激发单光子CaSiR-1荧光的波长和强度照射下不表现出跨膜电流。然后，我们展示了无串扰、高信噪比的CaSiR-1红色荧光成像，在100帧s−1的频率下，以高达20 Hz的蓝光脉冲在神经元中诱发chronos介导的钙瞬态。这些结果表明，红光激发的荧光团（在640 nm或以上有效激发）与蓝绿吸收视蛋白（如Chronos）之间的光谱分离足以避免成像波长对视蛋白的虚假驱动，从而实现无串扰全光神经元操作和读出。
All optical neurophysiology allows manipulation and readout of neural network activity with single-cell spatial resolution and millisecond temporal resolution. Neurons can be made to express proteins that actuate transmembrane currents upon light absorption, enabling optical control of membrane potential and action potential signalling. In addition, neurons can be genetically or synthetically labelled with fluorescent reporters of changes in intracellular calcium concentration or membrane potential. Thus, to optically manipulate and readout neural activity in parallel, two spectra are involved: the action spectrum of the actuator, and the absorption spectrum of the fluorescent reporter. Due to overlap in these spectra, previous all-optical neurophysiology paradigms have been hindered by spurious activation of neuronal activity caused by the readout light. Here, we pair the blue-green absorbing optogenetic actuator, Chronos, with a deep red-emitting fluorescent calcium reporter CaSiR-1. We show that cultured Chinese hamster ovary cells transfected with Chronos do not exhibit transmembrane currents when illuminated with wavelengths and intensities suitable for exciting one-photon CaSiR-1 fluorescence. We then demonstrate crosstalk-free, high signal-to-noise ratio CaSiR-1 red fluorescence imaging at 100 frames s− 1 of Chronos-mediated calcium transients evoked in neurons with blue light pulses at rates up to 20 Hz. These results indicate that the spectral separation between red light excited fluorophores, excited efficiently at or above 640 nm, with blue-green absorbing opsins such as Chronos, is sufficient to avoid spurious opsin actuation by the imaging wavelengths and therefore enable crosstalk-free all-optical neuronal manipulation and readout.