Plot Tracking Parameters ( IAPlotTracking )

Plots and saves time course or values of the selected tracking parameter. The image series must be first segmented, then tracked to calculate these parameters.
When constrained to ROIs, for each ROI only those segments are calculated which have their center point within the ROI. Parameters of the whole segments are counted, even if the segment is partially outside of the ROI.
To enable quality controlling tracking errors, a velocity or dislocation cutoff may be used. If cut off is used, values larger than the cut off will be discarded as missing or MASKed data point, not counted into means.The unit of cutoff is either dislocation/frame or velocity (dislocation/second). Calibration by setting the "Calibrtated" option to yes, uses the image resolution embedded into the opened image recording or set in the Image Properties window. Non-calibrated velocities are shown in pixels and pixels/s. Calibrated values are shown in um and um/s.
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.

Cell division and death parameters:

"Multiplets": numbers of cells for each tracked progeny. The "Group recursion" must be >1 in the Track Objects function to detect multiplets.

"Divisions": split (+1) and join (-1) events for each track.

"Total divisions": total number of divisions for the whole time course. Detection of divisions is blocked for each track for "Cutoff" seconds after each event. Optionally a division event must be followed by the presence of a doublet (multiplet) in the track at least "Parameter 1" frames. The "Group recursion" must be >1 in the Track Objects function to detect multiplets. The time axis value is the total time that is available for observation of divisions (depending the on the length of the observation window set by parameter 1 and 2, a sum for all cells) in seconds. If cell death has been previously labeled using the "Count Division and Cell Death" function, than dead cells are omitted from this count.

"Doubling time (hours)": the total time that is available for observation divided by the above defined division count.

"Cell death rate: fraction of cells dies in an hour."


Saving overwrites existing file if "Confirm saving" is set to No.
Automatic file naming convention:
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.

*ΔF/F0: ΔF/F0 normalization is performed on the data (the baseline will be set to 0. Alternatively see Baseline normalization below, *Resample: resamples along the time axis to result even time points, facilitating mean calculations between different experiments. The resampling interval can be set in the Preferences Dialog/(Data/Loading/ROIs)/Resample/X data resample interval (sec).where the baseline is set to 1). F0 is calculated as the mean of first n data points, where n is set in the Preferences Dialog/Misc/Normalization: Frames to average...
*Mean of all traces: Mean values of all traces are calculated for each time point. Mean calculation happens after optional normalization and rate calculation (see below). Mean+/-Standard Error is calculated.
*Median of all traces: Median values of all traces are calculated for each time point. Overrides the "Mean of all traces" switch. Median calculation happens after optional normalization and rate calculation (see below).
*Normalization: Each trace is normalized individually according to chosen way of normalization: None, Baseline, Endpoint, Minimum, Maximum , Percents Min to Max, Percents Baseline to Endpoint.
Baseline and endpoint are calculated as the mean of first, or last n data points, where n is set in the Preferences Dialog/Misc/Normalization: Frames to average... Minimum and Maximum is the single lowest or highest value for each time lapse. The normalization is performed by dividing all data points with the respective normalization factor for each trace independently. Normalization disables ΔF/F0 calculation. The normalization is performed before mean, median or rate calculation.
*Markers for means or rates: Enter ranges, e.g. 1-3;7-9 in frame numbers to calculate mean values or rates of traces within these ranges. The mean values will be shown in the plot or exported to tables. This calculation happens after normalization, but before trace-to-trance mean or median calculation.
*Calculate rates at markers: if ranges are set at "Markers for means or rates" enabling this will return rates calculated by linear regression on the set range for each trace and range independently. Rate calculation happens after normalization, but before trace-to-trance mean or median calculation.

Parameters affecting export and saving only:
*Y-values only: The time values are omitted during tabular export (text file or Excel).

Optionally use the Excel Data Window to record output. The following parameters control the appearance of the data in the Excel Data Window:
*Place channels into columns (former MD output): Output is organized according to channel number. Multiple runs on the same stage position will overwrite output.
*Processing different stage positions results data appearing below the previously calculated data, unless "Place positions into columns" is set "Yes". Data will be organized according to the highest channel number present among the linked images.
*Plate worksheet output: Information on plate row and column numbers are included, and data are additionally organized into plate view in additional worksheets. Data are organized on separate sheets for each channel, and separate plates for each time point and ROI.
*Expect only single channel: Only one pair of column (or one column if "Y-values only" is set "Yes") is entered into the Excel Data Window, regardless of the actual channel number and the numbers of open channels. Use this for time lapse processing of large numbers of stage positions.
*New plot always entered in a new row: When "Place channels into columns" is set to yes repeated plotting in a Multi-Dimensional open position will enter data in the same row to make data from different channels appear next to each other. Set this to yes to override this behavior and force each plot command to create a new row in the Excel Data Window. Use this switch to enable to gather stage position, row and column information using the "Place channels into columns" switch while creating multiple plots of the same channel.
Mathematica usage:
Result={{t1,t2,t3,...},{{ROI1t1,ROI1t2,ROI1t3,...},{ROI2t1,ROI2t2,ROI2t3,...},...}}