Measure velocity of cells or nuclei using tracking

Parameters:

Name	#	Type	Description
Analyze only the frame range below	1	boolean	Yes executes output #1, No executes output #2.
Analysis start frame	2	integer	Frame numbering starts at 1.
Analysis end frame	3	integer	Frame numbering starts at 1.
Number of parameters measured (1-5)	4	real	Select how many parameters do you want to measure, and enter what do you want to measure below.
Plot histogram (use None for means)	5	string	This option supersedes other normalization, mean and median calculations. See histogram parameters below.
Tracking parameter 1	6	string	Plots and saves time course of selected tracking data
Tracking parameter 2	7	string	Plots and saves time course of selected tracking data
Tracking parameter 3	8	string	Plots and saves time course of selected tracking data
Tracking parameter 4	9	string	Plots and saves time course of selected tracking data
Tracking parameter 5	10	string	Plots and saves time course of selected tracking data
Number of tiles in x	11	integer	The image consists of this number of equal sized tiles in x dimension.
Number of tiles in y	12	integer	The image consists of this number of equal sized tiles in y dimension.
Subtract reference image background	13	boolean	Yes executes output #1, No executes output #2.
Background level (percentile)	14	real
Scaling top level (percentile)	15	real	Brighter details of the image than this will be ignored.
Largest object size (pixels times number of tiles)	16	real	Cut on of the band pass Butterworth filter
Approximate cell diameter	17	integer	Diameter of the cell in pixels. Diameter of the nucleus in pixels. A range of cell size from about half to double will be selected.
Minimum roundness (shape factor)	18	real	The shape factor is 0-1 larger values mean rounder. Nuclei less round than this threshold will be rejected.
Minimum cell fluorescence (%)	19	real	Cells dimmer than this will be rejected. Increase this value if debris dimmer than the cells is detected.
Maximal distance that a cell travels between consecutive frames (pixels)	20	real	Largest distance in pixels that objects may move between frames
Minimum track length (frames)	21	integer	Removes cells that cannot be tracked longer than this number of frames.
Use cut off to reject erroneous velocity determinations	22	boolean	Calculates a mean value of all segments.
Velocity cut off value	23	real	Discards values larger than cutoff. Zero means no cutoff applied
Calibrate velocities to microns/seconds	24	boolean	Calibrates data to um or um/s based on the calibration information stored in the image. If set No, velocities are shown as pixels/sec.
Drift Correction	25	boolean	Subtracts the vectorial sum of dislocations or velocities.
Plot only mean values in this range of frames	26	string	If left empty, all time points are shown, or give ranges to calculate means e.g. 1-3;7-9. Try also copy/paste marks in the plot window context menu.
Export Tracks, Filename	27	string

Description:

This pipeline measures cellular motility as nuclei move frame to frame. The input is fluorescence time series with nuclei or cell bodies stained. Images are segmented first, then objects are tracked through the time lapse. There is no requirement for frame-to-frame overlap between nuclei, therefore image sequences recorded by longer acquisition intervals can be also used. The output data are velocities as microns/sec if calibrated or pixels/sec if not calibrated. Because mistakes in tracking may lead to excessive velocities, objects traveling too fast can be excluded from the mean velocity calculation using the “Use cut off to reject erroneous velocity determinations” switch. The drift if the whole view field can be corrected with the “Drift Correction” switch that assumes that cells move randomly therefore the mean dislocation vector is due to stage drift, and this mean vector is subtracted before velocity calculation.
Tiled images: the background tiling pattern is efficiently removed by spatial filtering if the recording was performed without overlap and image registration. Provide the number of tiles in x and y direction.

Tracking Parameters:

"dislocation": Time course of frame to frame movement of objects. The cutoff is defined as distance / frame.

"velocity": Time course of frame to frame velocities of objects (dislocation divided by frame interval). The cutoff is defined as velocity in the same units.

"average path velocity": Time course of time-averaged velocities of objects (dislocation divided by frame interval). A Savitzky-Golai weighed running average with a time window given by "Window width for average path" in frames is used for averaging. The cutoff is defined as velocity in the same units.

"average path acceleration": Same as "average path velocity", but acceleration is calculated. This value is higher for unsteady or zigzaggy motion. The unit of cutoff is acceleration.

"curvilinear velocity": Mean velocity for the entire track, calculated frame-by-frame and then averaged. The cutoff is defined as velocity in the same units.

"straight-line velocity": Mean velocity for the entire track, calculated between the start and end point of the track, or until a velocity cutoff is encountered. The cutoff is defined as velocity in the same units.

"mean average path velocity": Mean velocity for the entire track, calculated by temporal averaging as for "average path velocity". The cutoff is defined as velocity in the same units.

"mean-square displacement" : Square of the straight line displacement an object during a time period indicated on the x-axis of the graph. Time periods with the indicated length are averaged using all possible starting an ending points within the time course. The average for all objects is given. Important: this assumes even acquisition intervals. The cutoff is distance/frame.

"diffusion coefficient": Diffusion coefficient calculated from the slope of the "mean-square displacement", representing all objects in the time course. The cutoff is distance/frame. The unit of the diffusion coefficient is pixel^2/s or um2/s, if the "Calibrtated" option is set to yes.

"track length": The total distance that an object travels. The cutoff is distance/frame and if dislocations greater than the cutoff encountered, the track is truncated at that point.

"duty cycle": Fraction of frames where the object moves faster than the velocity provided in "Parameter 1". The cutoff is distance/s.

"linearity": A fraction, being 1 for linear or completely straight motion, less for other, e.g. curved or zigzaggy, zero if the track ends in the starting point, i.e. is a loop. Calculated as "straight-line velocity"/The cutoff is distance/s.

"straightness": A fraction describing how straight or linear is the overall motion not counting local excursion`s (or wobble) around the average path. The value is 1 for linear motion and less for curved trajectories. Calculated as "straight-line velocity"/"mean average path velocity". The average path velocity is calculated by temporal averaging and "Window width for average path" frames are used for averaging. The current implementation overestimates irregular motion when large changes in velocity resulting greater than 1 values.

"wobble": A fraction describing excursions from (or wobbling around) the average path. Calculated as 1-"mean average path velocity"/"curvilinear velocity". A value of 0 indicates smooth motion and greater values indicate more wobbly motion.

"tracks (time course)": (t,x,y) coordinates of tracks. To export tracks to Excel the "Show, copy and save errors of mean" in the Preferences dialog Misc tab must be check marked. y-coordinates are exported in the SEM columns.

"tracks (xy)": (x,y) coordinates of tracks as scatter plot. Not that the y-axis of the plot is flipped compared to the image.

"tracks (xy, relative)": (x,y) coordinates of tracks as scatter plot. All tracks start at the origin.

Automatic file naming:
To automatically generate file names use the following example:
=%LoadFolderName%_%LoadBaseName%%BACK%%LoadPositionNumber:2%w1.txt
Use any string after = and include any of the variables below between % marks; :n pads to n digits
%BACK% or %BACK:n% is (n times) backspace
Variables referring to last opened image set: %LoadFolderName%, %LoadFileName%, %LoadFileExt%, %LoadBaseName%, %LoadPositionNumber%, %LoadPositionName%, %LoadPositionCount%, %LoadPlateRow%, %LoadPlateCol%, %DocumentsFolder%, %PipelinesFolder%, %MyPipelinesFolder%
%LoadBaseName% refers to the common part of filenames within all opened files, with trailing zeros removed.
%LoadFolderName% refers to the name of the folder, that contains all of the opened files, not the path.
%LoadFolder%, %DocumentsFolder%, %PipelinesFolder%, %MyPipelinesFolder% refer to full path to the given location.
When only filename is given after the = the default folder is used. Relative path (e.g. =folder\file.txt) is appended to the default path. If no filename is given for saving, the Image Window name and default extension are used. Set default folders in the File/Set Folder Locations... menu point. Importantly, if no = is used in the beginning of the filename, then the full path needs to be entered.
Characters incompatible with the Windows files system are automatically removed from the save name. Variables and commands are case sensitive.

Keywords: cell motility, cell movement, cell migration, cellular motility, cellular movement, cellular migration, straightness, linearity, wobble, straight-line velocity, curvilinear velocity, average-path velocity, diffusion coefficient, mean-square dislocation, tracks

Version history:
V2
Image background is optional
Improved segmentation and tracking
V3
Disabled tracking cell divisions (Group size diameter = 0 for the Track Objects function)
Added options for plotting up to 3 tracking parameters
V4
Fixed image re-scaling
Added background level option
Increased number of options for plotting up to 3 tracking parameters
Added description of tracking parameters
V5
Histogram option added
Plate worksheet output in Excel tables turned off