We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.

中文翻译：

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使用Quin-2对钙的荧光寿命成像。

我们描述了基于钙指示剂的荧光寿命的一种新的成像技术，荧光寿命成像（FLIM），用于钙浓度的成像。已显示Quin-2的荧光寿命对[Ca2 +]高度敏感。我们使用Quin-2的相移和调制响应强度调制光来创建二维寿命图像。二维相位和调制值是使用增益调制图像增强器和慢速扫描CCD相机获得的。使用标准频域测量来验证2D图像中的寿命值。重要的是，FLIM方法不需要探头显示激发或发射光谱的变化，由于缺少光谱偏移，这可能允许使用当前未广泛使用的其他Ca2 +探针进行Ca2 +成像。荧光寿命成像可以优于固定（稳态）成像，因为寿命与局部探针浓度和/或强度无关，因此应广泛应用于使用荧光显微镜的化学成像。