Figure 3

Synchronization of the acquisition framerate with dynamic perturbations to capture yeast cell signaling dynamics. (A) Time course of nuclear accumulation of Hog1p in yeast cells growing as a monolayer in a microfluidic chamber subjected to an osmotic stress (1 M sorbitol). The insets show localization of Hog1-GFP before and after the osmotic stress. The acquisition framerate (orange bars) is automatically adjusted from one frame every 5 min to one frame every 25 s (12 times faster) just before the cells are stressed osmotically. The autofocus was turned off during the first 4 min of rapid Hog1 nuclear import. Recovery of the cells was then monitored at one frame every minute for 20 min, and finally the framerate was set back to its initial value (one frame every 5 min) until the next stress. The grey area represents the ± standard deviation of nuclear localization across 13 tracked cells from one microfluidic chamber. (B) The adaptive sampling rate used in (A) was repeated three times to demonstrate that cells exhibit reproducible dynamics in response to every stress. This experiment allowed the timescales of activation (fast) and deactivation (slow) of the HOG cascade to be measured in an unsupervised manner. (C) Sketch of the adaptive sampling MDA, which consists of three MDA experiments: one with a fast acquisition rate (nuclear import dynamics), one with a medium acquisition rate (nuclear export dynamics), and one with a slow acquisition rate (cell division after recovery). The switch from MDA#1 to MDA#2 is synchronized with the activation of an electrofluidic valve that delivers an osmotic stress of 30 min duration (repeated every 60 min). Nuclear localization is computed as the mean of GFP fluorescence in the nucleus normalized to the mean of GFP fluorescence in the entire cell.