Nanomolar-range fluctuations of intracellular [Ca²⁺] are critical for brain cell function but remain difficult to measure. We have advanced a microscopy technique to monitor intracellular [Ca²⁺] in individual cells in acute brain slices (also applicable in vivo) using fluorescence lifetime imaging (FLIM) of the Ca²⁺-sensitive fluorescent indicator Oregon Green BAPTA1 (OGB-1). The OGB-1 fluorescence lifetime is sensitive to [Ca²⁺] within the 10-500 nM range but not to other factors such as viscosity, temperature, or pH. This protocol describes the requirements, setup, and calibration of the FLIM system required for OGB-1 imaging. We provide a step-by-step procedure for whole-cell OGB-1 loading and two-photon FLIM. We also describe how to analyze the obtained FLIM data using total photon count and gated-intensity record, a ratiometric photon-counting approach that provides a highly improved signal-to-noise ratio and greater sensitivity of absolute [Ca²⁺] readout. We demonstrate our technique in nerve cells in situ, and it is adaptable to other cell types and fluorescent indicators. This protocol requires a basic understanding of FLIM and experience in single-cell electrophysiology and cell imaging. Setting up the FLIM system takes ∼2 d, and OGB-1 loading, imaging, and data analysis take 2 d.

中文翻译：

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使用荧光寿命成像监测细胞内纳摩尔钙。


细胞内 [Ca ²⁺ ] 的纳摩尔范围波动对于脑细胞功能至关重要，但仍然难以测量。我们先进的显微镜技术可使用 Ca ²⁺敏感荧光指示剂 Oregon Green BAPTA1 (OGB-1) 的荧光寿命成像 (FLIM) 监测急性脑切片中单个细胞的细胞内 [Ca ²⁺ ]（也适用于体内） ）。 OGB-1 荧光寿命对 10-500 nM 范围内的 [Ca ²⁺ ] 敏感，但对粘度、温度或 pH 等其他因素不敏感。该协议描述了 OGB-1 成像所需的 FLIM 系统的要求、设置和校准。我们提供全细胞 OGB-1 加载和双光子 FLIM 的分步程序。我们还描述了如何使用总光子计数和门控强度记录来分析获得的 FLIM 数据，这是一种比率光子计数方法，可提供高度改进的信噪比和更高的绝对 [Ca ²⁺ ] 读数灵敏度。我们在神经细胞中原位展示了我们的技术，并且它适用于其他细胞类型和荧光指示剂。该协议需要对 FLIM 的基本了解以及单细胞电生理学和细胞成像方面的经验。设置 FLIM 系统需要 ∼2 天，OGB-1 加载、成像和数据分析需要 2 天。