Aggarwal Abhi, Sunil Smrithi, Bendifallah Imane, Moon Michael, Drobizhev Mikhail, Zarowny Landon, Zheng Jihong, Wu Sheng-Yi, Lohman Alexander W, Tebo Alison G, Emiliani Valentina, Podgorski Kaspar, Shen Yi, Campbell Robert E
University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada.
Allen Institute for Neural Dynamics, Seattle, Washington, United States.
Neurophotonics. 2024 Apr;11(2):024207. doi: 10.1117/1.NPh.11.2.024207. Epub 2024 Apr 4.
Genetically encoded calcium ion () indicators (GECIs) are powerful tools for monitoring intracellular concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for and fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.
We describe the development and applications of T-GECO1-a high-performance blue-shifted GECI based on the -derived mTFP1.
We use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance using one-photon excitation in cultured rat hippocampal neurons, using one-photon excitation fiber photometry in mice, and using two-photon imaging in hippocampal slices.
The -bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of , a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The -dependent fluorescence increase is 15-fold, and the apparent for is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.
T-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.
基因编码钙离子（Ca²⁺）指示剂（GECIs）是监测活细胞和模式生物细胞内Ca²⁺浓度变化的强大工具。特别是，GECIs在监测与神经元动作电位相关的Ca²⁺浓度瞬时增加方面具有特殊用途。然而，用于神经元活动成像的高度优化的GECIs种类仍然相对有限。扩大可用GECIs的选择范围以包括新颜色和独特的光物理特性，可能为神经元活动的Ca²⁺和荧光成像创造新机会。特别是，GECIs的蓝移变体预计具有增强的双光子亮度，这将有助于多光子显微镜检查。
我们描述了基于源自mTFP1的T-GECO1——一种高性能蓝移GECI的开发和应用。
我们使用蛋白质工程和广泛的定向进化来开发T-GECO1。我们对纯化的蛋白质进行表征，并在培养的大鼠海马神经元中使用单光子激发评估其性能，在小鼠中使用单光子激发光纤光度法评估其性能，并在海马切片中使用双光子成像评估其性能。
T-GECO1的Ca²⁺结合状态具有最大激发峰为468nm、最大发射峰为500nm、消光系数为、量子产率为0.83，并且双光子亮度约为EGFP的两倍。Ca²⁺依赖性荧光增加为15倍，并且Ca²⁺的表观解离常数为82nM。在850nm的双光子激发条件下，与晚期一代的GCaMP变体相比，T-GECO1始终能够以更高的信噪比（SNR）检测动作电位。
T-GECO1是一种高性能蓝移GECI，在双光子激发条件下，相对于晚期一代的GCaMP变体具有优势。
