LAUSR

Graphical abstract How to get error bars on the ratiometric calcium estimator? The two measurements areas (region of interest, ROI, on the cell body and background measurement region, BMR, outside of the cell) are displayed on the frame corresponding to one actual experiment. Two measurements, one following an excitation at 340 nm and the other following an excitation at 380 nm are performed (at each ’time point’) from each region. The result is a set of four measures: adu340 (from the ROI), adu340,B (from the BMR), adu380 and adu380,B.. The fact that the measurements as well as the subsequent quantities derived from them are random variable realization is conveyed throughout the figure by the use of Gaussian probability densities. The densities from the BMR are ’tighter’ because there are much more pixels in the BMR than in the ROI (the standard deviations of the densities shown on this figure have been enlarged for clarity, but their relative size has been preserved, the horizontal axis in black always starts at 0). The key result of the paper is that the standard deviation of the four Gaussian densities corresponding to the raw data (bottom of the figure) can be reliably estimated from the data alone, egσ380≈Gadu380+V, where V is the product of the CCD chip gain squared by the number of pixels in the ROI by the CCD chip read-out variance. The paper explains how to compute the standard deviation of the derived distributions obtained at each step of the calcium concentration estimation.