Plots and saves time course of selected morphological data for the whole image as a mean, sum or variance of all objects or corresponding to ROIs.
When constrained to ROIs, for each ROI only those segments are calculated which have (any partial) overlap with the ROI. Parameters of the whole segments are counted, even if the segment is partially outside of the ROI.
Supports Mean, Sum and Variance calculation with the exception of "numbers" morphological parameter. These statistics represent the population of segments in the image or bounded by constraining ROIs.
Alternatively use the "Plot Type"=Each to record the selected morphological parameter for each segment.
This function plots evaluated segment data, not the visible image. If changing segmented image please use "Reevaluate Segments" before plotting segment data.
Constrain to ROIs: every segment that is at least partially within a ROI will be evaluated in whole.
Morphological parameter:
"number": total number of segments in the image or within each ROI.
"area": average pixel area of segments in the image or averaged for each ROI.
"perimeter": average perimeter of segments in the image or averaged for each ROI.
"diameter": average diameter of bounding boxes of segments in the image or averaged for each ROI.
"filament length": is a deprecated parameter, returns fiber length instead.
"branch points": for skeletonized images numbers of pixels with >2 neighbors. Average value for all segments in the image or averaged for each ROI.
"shape factor": circularity 1 for circles (could be >1 for small objects), less than 1 for non-circular, elongated or jagged objects. Average value for all segments in the image or averaged for each ROI.
"fiber length": average, approximate length of filamentous segments in the image or averaged for each ROI.
"fiber breadth": average, approximate width of filamentous segments in the image or averaged for each ROI.
"distance from active ROI": distance between the center of the segments and the boundary of the active ROI, zero for segments bounded by ROIs. Average value for all segments in the image or averaged for each ROI. Note: using a point ROI allows faster calculation.
"distance from ROI#1": distance between the center of the segments and the boundary of ROI #1, zero for segments bounded by ROIs. Average value for all segments in the image or averaged for each ROI. Note: using a point ROI allows faster calculation.
"distance from any ROI": distance between the center of the segments and the boundary of the nearest ROI, zero for segments bounded by ROIs. Average value for all segments in the image or averaged for each ROI.
"depth inside ROI": distance between the center of the segments and the boundary of the bounding ROI. Average value for all segments in the image or averaged for each ROI.
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,...},...}}