Measure the signal in a channel in areas defined by the stained nuclei in another channel. Remove the bright spots when measuring the signal.

The source code in git-hub can be found here.

Getting started

You must have MorphoLibJ and BaSiC installed.

To install the tool download the file measure_intensity_without_spots.ijm and put it into the folder macros/toolsets of your FIJI installation. In FIJI, select the measure_intensity_without_spots-toolset from the more tools button >>. On the toolset, click on the Install or Update Tool (the rightmost tool of the toolset).

Note: In case you do not have jython installed the first time you run the installation, only jython will be installed. You then have to retsart FIJI and click on the Install or Update Tool again.

After the installation finished, restart FIJI. Select the measure_intensity_without_spots-toolset from the >>-button of the ImageJ launcher.

• The first button (the one with the image) opens this help page
• The c-button exports the images from a .htd-file to a tif-series with each well as a stack
• The r-button runs the BaSiC-plugin on the tif-stacks, to correct the background
• The m-button measures the signal in the active image
• The j-button positions the image around the label whose line is selected in the results table.
• The br-button run the export and background correction on a batch of htd-images
• The b-button runs the processing and measuring for a batch of htd-images

Options

Remove Background

Right-click the "r"-button to open the options-dialog:

• Nuclei Channel: The number of the channel containing the nuclei
• Signal Channel: The number of the channel in which the measurements will be made
• Lambda Flat: Regularization parameter for the flat-field estimation (augment when images are correlated)
• Lambda Dark: Regularization parameter for the dark-field estimation (augment when images are correlated)

Measure Without Spots

Right-click the "m"-button to open the options-dialog:

• Nuclei Channel: The number of the channel containing the nuclei
• Signal Channel: The number of the channel in which the measurements will be made
• Min. area nucleus: Smaller objects will be removed from the analysis
• Method: The method used to segment the nuclei.
• sigma: The sigma of the Gaussian-blur filter used in the nuclei segmentation method.

Jump To Label

Right-click the "j"-button to open the options-dialog:

• Label; The label to which the display will be centered, if a line in the results table is selected the corresponding label will be used instead
• Zoom out: The number of zoom-outs after the zoom has been set to the label.