Figure 1. Data acquisition and analysis for GCaMP6 and brightfield signal.

(A) For the brightfield method, images are processed by an optical flow algorithm to generate vectors that represent the motion of the iPS-CMs. The blue vector arrows represent the average motion of 20 × 20 pixel regions, while the red vector arrows represent 50 × 50 pixel regions. The convergence of the vector arrows toward a center visually represents an iPS-CM cluster in the contraction phase. (B) Based on the vectors, a contractile profile is derived from the 1^st PCA of the norm of the x- and y-vectors. Positive PCA values indicate contraction phase, while negative values signify the relaxation phase. (C) GFP signal produced by the contraction of cardiomyocytes derived from GCaMP6 iPS cells. (D) GCaMP6 signal recorded over time for 3 sequential acquisitions at baseline. The signals show a rapid photobleaching effect within the first 5 seconds, but also show a photobleaching effect between the different acquisitions. (E) The starting points and peaks of the calcium transients are identified using the detrended GCaMP6 signal. (F) A graphical explanation of CTD₉₀. The SR₉₀ is defined as the ratio of CTD₉₀ to the amplitude of a given calcium transient. (G) A longitudinal experiment shows an artificial decrease in CTD₉₀ due to photobleaching. This is corrected by normalizing by the amplitude in SR₉₀.