Image analysis
This protocol is extracted from research article:
Rapid and inefficient kinetics of sickle hemoglobin fiber growth
Sci Adv, Mar 13, 2019; DOI: 10.1126/sciadv.aau1086

Additional DIC enhancement performed after collection included local background subtraction, followed by sequential image averaging and 2 × 2 binning. For local background subtraction, an average of the first 500 frames (20 s) was used as a reference and subtracted from all frames in the movie. An offset of 100 was added to avoid negative values. Performing local background subtraction increased the DIC signal contrast of growing fibers specifically, beyond that accomplished by subtracting the illumination background alone. Frames used as the local background reference were not included in all subsequent analyses. Image averaging and binning to reduce the experimental noise of tracking were performed within the semi-automated tracking program (described below). Kymographs and cross-sectional scans of individual HbS fibers were performed in ImageJ with a linewidth of 3 pixels. Cross-sectional pixel values were fit to Eq. 7 below using MATLAB R2015b (The MathWorks Inc., Natick, MA). Reported DIC signal values are the values of IF obtained from fitting of Eq. 7. Unless otherwise stated, all analyzed images were an average of 25 frames.

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