While calcium imaging has become a mainstay of modern neuroscience, the spectral properties of current fluorescent calcium indicators limit deep-tissue imaging as well as simultaneous use with other probes. Using two monomeric near-infrared (NIR) fluorescent proteins (FPs), we engineered an NIR Förster resonance energy transfer (FRET)-based genetically encoded calcium indicator (iGECI). iGECI exhibits high levels of brightness and photostability and an increase up to 600% in the fluorescence response to calcium. In dissociated neurons, iGECI detects spontaneous neuronal activity and electrically and optogenetically induced firing. We validated the performance of iGECI up to a depth of almost 400 µm in acute brain slices using one-photon light-sheet imaging. Applying hybrid photoacoustic and fluorescence microscopy, we simultaneously monitored neuronal and hemodynamic activities in the mouse brain through an intact skull, with resolutions of ~3 μm (lateral) and ~25–50 μm (axial). Using two-photon imaging, we detected evoked and spontaneous neuronal activity in the mouse visual cortex, with fluorescence changes of up to 25%. iGECI allows biosensors and optogenetic actuators to be multiplexed without spectral crosstalk.

虽然钙成像已成为现代神经科学的支柱，但当前荧光钙指示剂的光谱特性限制了深层组织成像以及与其他探针的同时使用。我们使用两种单体近红外 (NIR) 荧光蛋白 (FP)，设计了一种基于 NIR Förster 共振能量转移 (FRET) 的基因编码钙指示剂 (iGECI)。iGECI 表现出高水平的亮度和光稳定性，并且对钙的荧光响应增加高达 600%。在分离的神经元中，iGECI 检测自发的神经元活动以及电和光遗传学诱导的放电。我们使用单光子光片成像验证了 iGECI 在深度近 400 µm 的急性脑切片中的性能。应用混合光声和荧光显微镜，我们通过完整的头骨同时监测小鼠大脑中的神经元和血流动力学活动，分辨率为~3 μm（横向）和~25-50 μm（轴向）。使用双光子成像，我们检测到小鼠视觉皮层中的诱发和自发神经元活动，荧光变化高达 25%。iGECI 允许在没有光谱串扰的情况下复用生物传感器和光遗传学执行器。