The Plot Spectrum function calculates intensity or power terms as a function of spatial frequencies (frequencies of respective sine wave components) for each frame of an image series. This can be used for texture or granularity analysis of whole frames. Each frame results in one spectrum (one trace in the graph). Spectral components are calculated by radial averaging in Fourier space, and normalized to pixel counts. For the “Magnitude spectrogram” intensities of the absolute value of complex Fourier space terms are taken. The first component (FREQ1 column in Excel) is the average intensity of the image. The Power Density spectrogram averages the square of these terms, therefore larger components are overrepresented. Optionally use the dB (20*Log10) scale. Use windowing if objects at edges cause problems in high frequency components. The Fourier transformation is performed on the original image size, and it will work faster with images with width and height of power of 2. Non-quadrangular images may lead to distortions in the spectra. Export to Excel is one spectrum per row.
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.
Histograms and counting:
Use the "Histogram: number of bins" and the "Histogram: range" parameters, or the Histogram Properties menu point of the Plot Window context menu to adjust how the histogram appears.
"Histogram: range or threshold"=All will use all data
"Histogram: range or threshold"=low-high will plot numbers or percents in the range of low to high (low inclusive, high exclusive) divide to "Histogram: number of bins" equal bins, where 100% is all the data.
"Histogram: range or threshold"=threshold will count or calculate percents above (or below depending on the value of "Histogram and counts") this threshold, where 100% is all the data. This is equivalent to a single-bin histogram.
"Histogram: range or threshold"=percentile with "Histogram and counts"=Value at Percentile will calculate a single value at the percentile given here. This value is directly calculated from the data without binning.
"Histogram: range or threshold"=low,high will count or calculate percents in the low to high range (low inclusive, high exclusive), where 100% is all the data. This is equivalent to a single-bin histogram.
Reference based counting: first use "Histograms and counting"="Store reference value at percentile". This will associate the channel number of the image with the plotted value. This reference value will be live until the program is closed. The current plot and image windows can be closed. Then use the "Histograms and counting"=Count or Percent above/below reference modes to count objects or ROIs based on this stored reference value in other Image Windows with the same channel number. For 2-operand plots both channel numbers must match.
Mathematica usage:
Result={{t1,t2,t3,...},{{ROI1t1,ROI1t2,ROI1t3,...},{ROI2t1,ROI2t2,ROI2t3,...},...}}
Functions
Glossary
