Glyph Imaging

  Materials and Methods for tracking:

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  An arbitrary number was given to each sperm tracked, 1-6 are +/+ and 7-12 are -/-. Sperm 1-2 are +/+, early time point (2 minutes). Sperm 3-4, +/+ , middle time point (ten minutes). Sperm 5-6, +/+ , late time point (90 minutes). Sperm 7-8, -/- early time point, Sperm 9-10, -/- middle time point, Sperm 11-12, -/- late time point. Thus, there were 12 sperm tracked for this data, two from each time per genotype. The total frames tracked for sperm 1-2=152, total frames tracked for sperm 3-4= 84, total frames tracked for sperm 5-6= 132, total frames tracked for sperm 7-8= 118, total frames tracked for sperm 9-10=207, total frames tracked for sperm 11-12=161. The number of frames was fairly even from +/+ and -/-.

  The sperm were selected based on viability and focus. The video was first digitized to an uncompressed Quicktime movie file. Raw video footage was contrast enhanced using Adobe AfterFX to facilitate computer tracking sperm features. Tracking and data output was done in Alias|wavefront Maya4.

  An irregular polygon (perimiterCurve) was created which encased the entire tortuous sperm head path using lines drawn from peaks along the path of the sperm head beginning and ending at the last video frame (Fig..) An "amplitude meaurement module" was designed to take amplitude measurements across this perimeter from one side to the other, traversing the perimiter and measuring that distance once per frame for as many frames as the sperm was tracked, (30/sec). The amplitude module was scripted to create a line perpendicular to one side of the perimeter curve pointing inward, and measure the distance to the perimeter curve on the opposite side. Output from the amplitude module consisted of floating point numerical data, one point for each frame of the video, for which a mean value was determined using SigmaPlot. The means were then put into a SAS database and analyzed using the General Linear Model, and plotted using Jandel SigmaPlot 2000. Because the perimeter boundary is closed on each end, the values for amplitude at the ends of the sperm path created an measurement error, at two to three points (of > 100 points). This error was nullified somewhat by having all video clips be of similar size (not necessarily identical), however, deleting these two or three values from the hundreds of amplitude measurements did not change the overall mean values significantly, and therefore no adjustment for this effect was made. A second check for the validity of using a scripted amplitude module was performed on hard copy from the video tracing of the sperm head path, where a perpendicular line was digitized using a Summagraphics digitizing tablet and Jandel Sigma Scan Pro software from one side of the perimeter path to the other. Hand digitized data were in close agreement with the data derived from the amplitude module. Parameters collected for analyzing the paths of individual sperm were: Original video time, the number of video frames, genotype, cycles per second (hz) (one cycle = complete plus and minus phases of a wave), amplitude of the sperm head path, the length of the longest distance traveled by the sperm head (meandering), the distance traveled through the “center” of the meandering sperm head path, and linear distance from the start and end points of the track.




   -/- (knockout) group:

2.09.57 - 2.10.23, track number 2

AVI video of the above measurement module - 1.52 mb
AVI video of the corresponding sperm footage and tracked point - 5.44 mb

QUICKTIME video of the above module (increased magnification) - 2.70 mb
QUICKTIME video of the above module + calibration (increased magnification)- 3.09 mb

Additional calibration reference image for this track


  +/+ (control) footage, same magnification:

AVI video of measurement tools for a +/+ track - 2.65 mb
AVI video of the +/+ example sperm footage with tracked point - 2.40 mb

QUICKTIME video of the +/+ example - 2.40 mb


  All measurements in these videos are arbitrary units, but were calibrated later for real world units.
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