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首页> 外文期刊>Journal of Neuroscience Methods >A simple method for quantitative calcium imaging in unperturbed developing neurons.
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A simple method for quantitative calcium imaging in unperturbed developing neurons.

机译：在未受干扰的发育中神经元中定量钙成像的简单方法。

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摘要

Calcium imaging has been widely used to address questions of neuronal function and development. To gain deeper insights into the actions of calcium as a second messenger, but also to measure synaptic function, it is necessary to quantify the level of calcium at rest and during calcium transients. While quantification of calcium levels is straightforward when using ratiometric calcium indicators, these dyes have several draw-backs due to their short wavelength excitation spectra, such as light scattering and cytotoxicity. In contrast, many single-wavelength indicators exhibit superior photostability, low phototoxicity, extended dynamic ranges and very high signal to noise ratios. However, quantifying calcium levels in unperturbed neurons has not been performed with these indicators. Here, we explore a new approach for determining the calcium concentration at rest as well as calcium rises during evoked and spontaneous neuronal activity in unperturbed developing neurons using a single-wavelength calcium indicator. We show that measuring the maximal fluorescence at the end of an imaging experiment allows determining calcium levels with high resolution. Specifically, we assessed the limits of calcium measurements with a CCD camera in small neuronal processes and found that even in small diameter dendrites and spines the intracellular calcium concentration and its changes can be estimated accurately. This approach may not only allow mapping patterns of neuronal activity quantitatively with the resolution of single synapses and a few tens of milliseconds, but also facilitate investigating the role of calcium as a second messenger.
机译：钙成像已广泛用于解决神经元功能和发育的问题。为了更深入地了解钙作为第二信使的作用，并测量突触功能，有必要对静止时和钙瞬变过程中的钙水平进行量化。尽管使用比例钙指示剂时钙含量的定量很简单，但由于它们的短波长激发光谱（例如光散射和细胞毒性），这些染料有一些缺点。相比之下，许多单波长指示器显示出优异的光稳定性，低光毒性，扩展的动态范围以及极高的信噪比。但是，尚未使用这些指标对未受干扰的神经元中的钙水平进行定量。在这里，我们探索了一种使用单波长钙指示剂确定静息的发育中神经元在诱发和自发神经元活动过程中静息钙浓度以及钙升高的新方法。我们显示，在成像实验结束时测量最大荧光可以确定高分辨率的钙水平。具体来说，我们评估了在小型神经元过程中用CCD相机进行钙测量的极限，发现即使在小直径的树突和棘突中，也可以准确估算出细胞内钙的浓度及其变化。这种方法不仅可以用单个突触的分辨率和几十毫秒来定量地绘制神经元活动的模式图，而且还有助于研究钙作为第二信使的作用。

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