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>Open Image sequences

>Multi-Dimensional data sets

>Image Window

>Set Image Linkage

> Parametering image processing functions

>Dialogs

>>Multi-Dimensional Open

>>Setup DFT Filter

>>Attach IMD Intensity

>>Attach Overlay Image

>>Attach Overlay Text

>>Pipeline Optimizer

>>Membrane Potential Calibration Wizard

>>Interactive Ion Calibration

>>Crossbleed correction

>>Preferences

>Fixing imaging artifacts

>Smoothing

>Pipeline

>Mathematica

>Annotate, overlay

>Define ROIs

>Background subtraction

>Plotting intensities, ratios

>Masking images

>Thresholding

>Segmenting images

>Plot morphological parameters

>Save or copy plot data in tabular format

>Export RGB images or movies

>Evaluate expressions

Defining ROIs
Next

Manual Management of ROIs:

  • To draw ROIs use the following toolbar buttons: point ROI: drawROI line ROI: drawROI area ROI: drawROI
  • To select and move ROIs use the drawROI in the toolbar
  • To erase the selected ROI use DEL, the context menu of the Image Window or drawROI  in the toolbar.
  • ROIs cannot be modified, just repositioned once finished drawing.
  • Copy ROI copyROI copy All ROIs to clipboard and past ROI pasete ROIs between non-linked Image Windows
  • Save ROI save or load ROI load ROIs for later use. Use the FSave ROIs and FLoad ROIs functions to save and load ROIs from the Pipeline, or to use automatic file naming.
  • When pasting or loading ROIs into a different sized image ROIs can be proportionally resized based on the settings in Preferences/'Data Loading / ROIs'/Scale ROIs proportionally at pasting.
  • The active ROI is marked by the dashed outline. To switch between ROIs use the < > buttons. ALT enables a cross-hair to show the active ROI.
  • ROIs are shared between linked  Image Windows.
    • When a ROI is moved or deleted it will affect all linked Image Windows, and Plots calculated from these windows.
    • When image windows are unlinked ROIs are copied.
    • When new image windows are linked ROIs are merged.

Automated ROI drawing:

Magic wand tool: ROI wand

  • Click once the middle of bright objects to circle the brighter region, or click closer to the edge of the object to circle a larger region. The magic wand tool uses a random process to find the boundaries of the object, so repeated trial-error (use DEL) can be used to draw a properly sized region.
  • Use it on fluorescence images (bright) object or in transmitted light images focused below the focal plane, where cells appear bright with a dark ring around them.
  • The magic wand tool calculates mean intensity in the vicinity of the pointer upon left click, then extends the selection for the similarly bright pixels, until the variance of the pixel intensities does not reach a threshold given by the Magic Wand tool Coefficient.
  • Set the parameters of the magic wand tool in the Preferences/'Data Loading / ROIs'/Magic Wand Cell Circling Tool:
    • Coefficient: determines the sensitivity of the magic wand. The coefficient is a real number between 0 and 1. Smaller coefficients allow greater deviation between intensities, so larger region is circled
    • Progressive: when checked the sensitivity is iteratively changed until a region in between the Min. Size. and Max. Size. in the number of pixels covered.
    • Gamma: gamma correction is applied on intensities (not affecting image data) before detection. Use a value <1 to detect brighter details, or a value>1 to enhance detection of dim details next to bright ones.
    • Start size: the selection is at least this sized in pixels, the mean intensity of these pixels is the basis of extension of the ROI around this initial region
    • Dilate: the ROI is finally dilated (grown) by this diameter given in pixels (half of the given number in any direction)

FPlotting/Automatic ROI Drawing: is a function for repeated application of magic wand tool on the brightest objects on the image. In addition to Dilation this function supports Exclusion diameter to prevent ROIs getting too close together.

Typical use:

  • Use area ROIs drawROI , the Magic Wand tool ROI wand or FAutomatic ROI Drawing to generate plots of intensity over a polygonal shaped area. Select  drawROI in the toolbar and left click image to place points. Alternatively keep left button down to hand draw an outline. Double click to finish ROI. If the ROI is not proper use DEL to erase it and redraw.
  • Use line ROI  drawROI to measure intensity over membranes or to generate kymograms (line scans).
  • Use point ROIs drawROI to measure intensity over single pixels or to designate positions in kymograms.