A simple method for quantitative calcium imaging in unperturbed developing neurons

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.