Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Flow chart of methodology Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Zebrafish embryos and imaging equipment. (a) Zebrafish embryos next to a United States penny for scale. (b) Ventral view of embryonic zebrafish heart. Blood cells can easily be seen through the transparent tissues surrounding the heart. As blood flows through the heart, it proceeds through the inlet (i), atrium (a), atrioventricular junction (AVJ), and finally through the ventricle (v). Scale bar indicates 100 μm. (c) Experimental setup, consisting of bright field stereomicroscope, high speed camera, and computer. Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Image Processing. A comparison is made between the unprocessed data (top row) and the processed data (bottom row). For each frame, a sliding average of the intensity of the previous frames is subtracted from the current frame. This removes static portions of the image and creates a black background. Moving portions of the image are easily seen as gray or white pixels. The upper ST plot segment (top middle) reveals notable background artifacts, which result in multiple inaccurate variance peaks (top right). Background artifacts are greatly reduced in the processed images (bottom left and middle), resulting in significantly more accurate variance peaks (bottom right) and, consequently, more accurate velocity estimation. Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Creation of spatiotemporal plots. Part (a) shows a series of three frames with mock cells moving in the vertical direction. The vertical gray column marks the pixels that lie along an arbitrary reference line. The pixels along this line are plotted side by side to create the spatiotemporal plot in (b). As cells move along the reference line, their changing position is seen as an angled streak in the ST plot. The vertical axis of the ST plot represents position along the reference line, while the horizontal axis represents time. The velocity of the cells along the reference line can be determined by calculating the slope (dy/dt) of the streaks in the ST plot. Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / ST plot analysis with Radon transforms. Part (a) shows a cartoon of an embryonic heart. ST plot reference lines are placed as shown at the atrial inlet and AVJ. A sample section of a typical ST plot is shown in (b). The ST plot must be analyzed in smaller sections, called bins. One such bin is outlined in (b). Part (c) shows the Radon transform analysis at 0 deg, 90 deg, and 138 deg. The projection of this bin is summed along each of these projection angles (θ) to produce a corresponding plot. When the projection angle matches the streak angle (138 degree sign (°) in this case), the resulting plot contains prominent peaks. A complete Radon transform contains an analysis of the ST plot bin from every angle, where Radon transform values are represented by intensity, as seen in (d). Part (e) shows a plot of the Radon transform variance for each angle. The projection angle with the highest variance is perpendicular to the streak angle in the ST plot. The velocity (pixels/frame) is then tan(θ–90 deg). Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Measurement of lumen diameter. ST plots were created from reference lines which spanned the width of the lumen at the atrial inlet (a) and AVJ. The lines in (b) mark the flow boundary used for measuring orifice diameter, ‘D’. Measurements were used to plot diameter versus time. Time was normalized such that one complete cardiac cycle spans from 0 to 1. Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Cardiac physiological parameters. Plots were created for orifice diameter (a), blood velocity (b), flow rate (c), and accumulated blood volume (d) versus time at each orifice. Results are shown for C2. Time was normalized such that one complete cardiac cycle spans from 0 to 1. Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Changes in flow rate and volume at the atrial inlet and AVJ. Plots of the estimated flow rate versus time (a) and (c) and accumulated orifice volume versus time (b) and (d) for each experimental case. Time was normalized such that one complete cardiac cycle spans from 0 to 1. Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Bin size optimization. Bin size was optimized based on an ST plot segment with changing velocity. The local ST plot streak angle was manually measured as a baseline. Bin size was varied and results were compared to the baseline. Results are shown (from left to right) of the ST plot bin, the resulting Radon transform, and the variance of the derivative of the intensity of the Radon transform. Figure Legend:

Date of download: 6/3/2016 Copyright © ASME. All rights reserved. From: Quantifying Function in the Early Embryonic Heart J Biomech Eng. 2013;135(4): doi: / Span size optimization. Span size was optimized at low ‐ moderate velocities (top) and peak velocities (bottom). Span size was varied and results were shown of (from left to right) the processed image, the resulting ST plot, the resulting Radon transform, and the variance of the derivative of the intensity of the Radon transform. Figure Legend: