Figure 2
From: CyberSco.Py an open-source software for event-based, conditional microscopy
From simple to advanced MDA. (A) Example of a classic Multi-Dimensional Acquisition (MDA) protocol to observe yeast proliferation in a microfluidic chamber, with two imaging channels (brightfield and RFP) imaged every 6 min for several hours. The HTB2 protein of the yeast cells is tagged with a mCherry fluorescent reporter. A sketch of the program (nested loops) is shown on the left side: the imaging parameters are identical for every position and timepoint. (B) An advanced MDA, in which the user has defined several positions, but set different illumination settings in the blue channel (LED intensity: 0%, 5%, 10% and 20%). This programming was done without scripts, by just using the drag and drop interface (see Supplementary Materials). Yeast cells bearing an optogenetic gene expression system (pC120-venus) were imaged for 15 h. Each position is exposed to a different level of light stimulation, which alters the expression of a yellow fluorescent reporter both in terms of cell–cell variability, the maximum level of expression and dynamics. Thus, in one experiment, it was possible to quantitatively calibrate the pC120 optogenetic promoter using our settings without any requirement for coding (objective × 20). Fluorescence levels are averaged across the field of view and the error values are the standard deviation of pixel intensity.
