Evaluation of ARCOS sensitivity to iterative image and time series degradation with additive Gaussian white noise. (A) Sample images of the sum of donor and acceptor channels of the EKAREV-NLS FRET biosensor in MDCK cells with an increasing amount of Gaussian white noise. The amount of added noise corresponds to a target SNR in dB according to Eq. 2 (Materials and methods). (B) Segmentation with the stardist python package. (C) Track-length histograms of respective iterations. (D) Spaghetti plots depicting CEs detected at a specific SNR. (E) Sample single-cell ERK trajectories of MDCK cells measured using the EKAREV-NLS FRET biosensor. An increasing amount of Gaussian white noise was added to the time series. The noise amplitude is calculated according to Eq. 2 (Materials and methods) to reach a target SNR in dB. (F) Spaghetti plots depicting CEs detected at a specific SNR. (G) Total number of false positives (FP), false negatives (FN), and precision and recall for detecting CEs with ARCOS for progressive image degradation from panels A–D. Calculated by checking if a specific cell was detected as part of a CE at a specific time point. (H) Same as panel G but for progressive time series degradation from panels E and F. (G and H) Shaded regions represent ±1 SD of 10 random noise additions.