Image processing functions in Image Analyst MKII
Image Analyst MKIIFunctions Glossary
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2D DFT Filter
2D DFT Filter Butterworth BP
2D DFT Filter Butterworth BP Tiled
2D Kernel Convolution
2D Median
2D Morphological Operator
2D Nonlinear Filter
2D Savitzky-Golay filter
Absolute Value
Affine Transformation
Align Channels
Align Series (Image Stabilizer)
Align Tiled Channels
Align Tiled Series (Image Stabilizer)
Anisotropic Diffusion Filter
Attach Intensity Gating Image
Attach Overlay Image
Automatic ROI drawing
Band Pass filter Optimization
Blind Spectral Unmix with NMF
Calculate Simple Crossbleed
Calculate Spatial Moments
Calibration Wizard Parameters
Clear Segmentation Classifiers
Close Image Window
Copy Image Window
Copy ROIs from Other Image
Correct Intensity Jump
Correct Lens Distortion
Count Division and Cell Death
Count Object Colocalization
Create ROI
Create ROIs from Segments
Crop
Crop Image in Place
Crop Image to Segments
Cross-correlation data
Cross-correlation image
Detect Nuclei Convolution
Differential Evolution Optimizer
Distance from Segments
Draw Model Mitochondrion
Draw Random Position Model Mitochondria
EndIf
Erase All ROIs
Excel Window Command
Export
Fill Mask
Fill or Mask Active ROI
FLIPR Calibration with [K+]ec steps and known [K+]ic and known kP
FLIPR Complete Calibration
FLIPR Complete Calibration with known kP
FLIPR Complete Calibration with known kP - Goldman
FLIPR Complete Iterative Calibration
FLIPR Estimate PN
FLIPR Short Calibration based on known potential during MDC and CDC
FLIPR Short Calibration between known baseline and CDC
FLIPR Short Calibration between known baseline and MDC
FLIPR Short Calibration between known baseline and separately measured fP0
FLIPR Short Calibration from Zero with fx=0
FunctionOptions
Get Image Information
Get Linked Channel
If
If Greater Than Zero
Image Arithmetic (image-image math)
Image Arithmetic In Place
Image Arithmetic Single Frame
Inpaint Mask
Input
Invert
Lens Correction Optimization
Link Image Windows
Load and Run Pipeline
Load ROIs
Mask Borders
Mask Frames by Plot Values
Mask Images
Measure Object Intensity
Measure Object Morphology
Mirror or Rotate (new image)
Mirror or Rotate in Place
Multi-Dimensional Open Information
Multi-Dimensional Open Stage Position
Multi-Dimensional Reload Channel
New Image
New Time Scale
Onset Image
Open File
Optical Flow
Options
Pipeline
Pipeline Optimization
Pipeline Optimization Parameter
Plot
Plot Correlation (Colocalization)
Plot Intensities Corresponding to Segments
Plot Morphological Parameters of Segments
Plot Ratio
Plot ROI Dimensions
Plot Tracking Parameters
Potential calibration constants
Potential calibration error propagation
Potential calibration expert overrides
Projection of Vectors from a Point
Ratio
Ratiometric ROI Classifiers
ReCount Division and Cell Death
Reevaluate Segments
Remove Blank Frames
Rename
Resample Image
ROI Classifiers
Run Membrane Potential Calibration
Save ROIs
Scalar Arithmetic (image-value math)
Scalar Arithmetic Multi
Secondary Watershed Segmentation
Select
Select by Number
Sensor Noise Characteristics
Set Reference Image
Set Scaling/LUT
Set Segmentation Classifiers
Set Segmentation Intensity Classifiers
Shift Time Scale
Simple 2D Cross-correlation
Simple Segmentation
Skeletonize
Spectral Unmix
Strip to Well Cell Count
Substitute Poisson Noise
Subtract Background or Normalize
T or Z-project
Template Matching
Temporal Average Filter
Temporal Block Filter
Temporal Median Filter
Temporal Rolling Projection
Temporal Savitzky-Golay Filter
Thinness Ratio Optimization
Threshold
Time Stamp and Scale Bar
TMRM Complete Calibration
TMRM Complete Calibration with known kT
TMRM Complete Calibration with known kT and K-steps
TMRM Short Calibration between known baseline and MDC or CDC
Track Objects
Truncate or Cut
Wait for All Inputs
Watershed Segmentation
Wiener filter
Window Menu Command
Write Back Scaled Values
ΔF/F0

Plot Ratio ( IARatioPlot )

Parameters:
Name Short Name Type Description
ΔF/F0 dfperf boolean Calculates ΔF/F0 normalization of the trace
Resample resample boolean Resamples the trace by the time interval given in Preferences.
Mean of all traces allMean boolean Calculates a mean value of all traces.
Median of all traces allMedian boolean Calculates a median value of all traces for each time point. If "Yes" then mean calculation is disabled.
Time course normalization advnorm string This option supersedes ΔF/F0 normalization.
Histogram and distribution histogram string This option supersedes other normalization, mean and median calculations. See histogram parameters below.
Save name savename string
Save save boolean
Confirm saving saveconfirm boolean Overwrites automatically is No, pops up dialog if Yes.
Range markers for means or rates ranges string Leave markers field empty to show all time points, or give ranges to calculate means e.g. 1-3;7-9. Use "All" to select all time points. Hint: Try copy/paste marks in the plot window context menu.
Calculate rates at markers rates boolean Plots slopes of traces for each range, calculated after normalization, if any set above. This is performed before mean of all traces calculation, if selected.
Normalize rates ratesnorm boolean Normalization is performed after rate calculation if Yes, or before rate calculation if No.
Y-values only yonly boolean Copies y-data only to data table
Place channels into columns mdoutput boolean Organizes data in table according to Multi-Dimensional Open parameters.
Range of channel numbers to lay out channelrange string This will set up the header of the output, when channels are laid out as columns. Set All to use all channels present in images linked to the one that is processed first. If plotting data from unlinked images, provide a range of channel numbers here, e.g. 1-3.
Plate worksheet output plateoutput boolean Organizes data in table according to microplate well associations in additional worksheets.
Place positions to columns stagetocolumn boolean Data from multiple stage positions will appear in new columns instead of being appended below the existing data. This option requires the "Place channels into columns" to be turned on.
Expect only a single channel singlechannel boolean Discards channel information and a single channel is recorded. Use this to suppress empty columns in output.
New plot always entered in a new row forcenewrow boolean 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.
Truncate traces at low denominator values mindenominatorcheck boolean To avoid excess noise, traces are truncated at low denominator values defined by either criteria below. In addition points with negative numerator are also discarded.
Minimal percentile of denominator mindenominatorperc real To avoid excess noise, traces are truncated at denominator values equal or lower than this image percentile (calculated for the whole time series).
Minimal value of denominator mindenominatorval real To avoid excess noise, traces are truncated at denominator intensity values equal or lower than this value.
Repeat previous valid ratios instead of truncating repeatvalid boolean Instead of truncating traces at low denominator values, the last valid values are repeated.
Histogram: number of bins histogrambins integer When the output is histogram, the histogram is calculated using this number of equal sized bins, where the lowest bin starts at the smallest data point and the highest bin ends at the largest data point. If explicit range definition is used below, the bins will bound the given range. Use zero bin size for automatic binning.
Histogram: range histogramrange string If left empty or set to "All", a histogram is generated where the lowest bin starts at the smallest data point and the highest bin ends at the largest data point. If a range is given here e.g. 100-500, then the lowest bin starts at the beginning of the range and the highest bin ends at the end of the range.
Description:
Plots the ratio of ROI mean intensities (Image A / Image B).
Calculation of fluorescence ratio from mean ROI intensities is preferred over calculating ROI means in ratio images because of noise and misalignment between channels. This function provides normalization, rate, ROI mean+/-SE and median calculation, and a flexible output to Excel, supporting work in microplates or multiple stage positions recordings.
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 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.
*Truncate traces at low denominator values: if a ratiometric probe fluorescence gets close to the noise floor invalid ratio values are calculated due to near-zero or negative denominator values or negative numerator values.
This option and the following values allow to remove (mask) these traces in the time course. Use the "Repeat previous valid ratios instead of truncating" option to fill in these gaps with the previous valid value.
Mathematica usage:
Result: results are transferred to Mathematica