Materials
and Methods for tracking:
DOWNLOAD ALL RELEVANT
FILES IN A WINZIP ARCHIVE HERE
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.
please allow sufficient loading time for the videos, especially
over a phone dialup connection.
