The plugin Speckle_Velocity calculates the velocity of retrograde flow from tracks of speckle position generated in Speckle TrackerJ. It also requires a snake file generated in JFilament to indicate the position of the cell's leading edge and the output of LE_Velocity. When Speckle_Velocity is run
it will prompt the user to input parameters:
xCM: the x-coordinate of the cell center, measured in pixels
yCM: the y-coordinate of the cell center, measured in pixels
dx: image resolution, measured in nm/pixel
qmax: the maximum angle over which to analyze data, in degrees
qmin: the minimum angle over which to analyze data, in degrees
dt: the time per frame, in seconds
M: the number of time frames used for velocity calculations
S: scaling factor for the velocity vector flow maps.
The user will then be prompted to select 3 data files:
A velocity matrix (the output of LE_Velocity)
A snake file (the output of JFilament)
A speckle file (the ouput of Speckle TrackerJ)
Output data file: Contains the
calculated speckle velocity data in two formats. The first, a duplicate stack displaying vector flow maps of speckle velocities. A value of S=1 yields velocity vectors that scale 1nm/s per pixel. The second is a data file formatted: speckle
number, frame number, speckle lifetime (s), angle (degrees), distance from the leading
edge (µm),
local leading edge speed (nm/s), net speckle speed (nm/s), x-component
of the speckle velocity (nm/s), y-component of the speckle velocity
(nm/s).
A second plugin, Speckle_Velocity_NoSnakes, calculates speckle velocities as a function of time and lifetime. When Speckle_Velocity_NoSnakes is run
it will prompt the user to input parameters:
dx: image resolution, measured in nm/pixel
dt: the time per frame, in seconds
M: the number of time frames used for velocity calculations
S: scaling factor for the velocity vector flow maps.
The user will then be prompted to select a speckle file.
Output data file: Contains the
calculated speckle velocity data in two formats. The first, a duplicate stack displaying vector flow maps of speckle velocities. A value of S=1 yields velocity vectors that scale 1nm/s per pixel. The second is a data file formatted: speckle
number, frame number, speckle lifetime (s), net speckle speed (nm/s), x-component
of the speckle velocity (nm/s), y-component of the speckle velocity
(nm/s).
References:
G. L. Ryan, N. Watanabe, D. Vavylonis,
"Image Analysis Tools to Quantify Cell Shape and Protein Dynamics near the Leading Edge," Cell
Structure and Function (2013).
G. L. Ryan, H. Petroccia, N. Watanabe, D. Vavylonis,
"Excitable Actin Dynamics in Lamellipodial Protrusion and Retraction,"
Biophys. J. 102:1493-1502 (2012).