The tool counts the number of spots (foci) per nucleus and measures the intensity, form, size and position of the spots. It also optionally measures the intensity in the membrane of the nucleus.

You can download some example images here: example_images.zip

To install the tool save the file measure_border_and_spots_tool.ijm into the folder macros/toolsets of your FIJI installation.

Select the "measure_border_and_spots_tool.ijm" toolset from the >> button of the ImageJ launcher.

• the first button opens the help page
• the a-button runs the analysis on the current image
• the b-button runs a batch-analysis on the images in a folder

The tool expects separate files representing the different channels. The files must be in the same folder and have the same name except for the part of the name that represents the channel. For example:

Open the nuclei-channel file and press the a-button to run the analysis on the opened file. Modify the parameters (right-click on the a-button) if necessary. Right-click on the b-button to set the file-extension of your input files, then press the b-button to run the batch-analysis. Select the folder containing the input images. The result-measurements and control-images will be written into a sub-folder out of the input-folder.

A difference-of-Gaussian (DoG) filter is applied to the nuclei channel and an auto-threshold is applied. Holes in the resulting mask are filled with the Fill Holes command. Small objects are eliminated using the particle analyzer.

The nuclei borders are transformed into band-selections using the Enlarge and Make Band... commands.

The background is subtracted either using the Rolling ball-method or by subtracting a blurred version of the image. An auto-threshold is applied and small objects are eliminated. A seeded watershed can optionally be applied to separate touching spots.

The Exact Euclidean Distance Transform (3D) command is used on the nuclei-channel. The pixel value in the EDT-image at the center of the spot is the distance of the spot to the border of the nucleus.

Right-click the a-button to open the options dialog:

nuclei channel

The name of the nuclei-channel (must be part of the filename)

min. sigma dog

The smaller sigma-value for the Difference-of-Gaussians-Filter

max. sigma dog

The bigger sigma-value for the Difference-of-Gaussians-Filter

nuclei auto-thresholding method

The auto-thresholding method used for the nuclei segmentation

min. area nucleus

Objects with a smaller area are eliminated

border channel

The name of the channel in which the nuclei-border intensities are measured

measure border

When selected the intensity in the nuclei borders are measured

border radius

The radius of the band that will be created around the nuclei contours

spots channel

The name of the channel in which the spots (foci) are measured

min. spot area

Objects with a smaller area are eliminated

use rolling ball

If selected rolling-ball background correction is used, otherwise a blurred version is subtracted from the image

rolling-ball radius

The radius of the rolling ball (radius of the smallest feature that shall remain in the image)

sigma gaussian filter>

The sigma of the Gaussian-filter for the image that will be subtracted from the input image

foci auto-thresholding method

The auto-thresholding method used for the segmentation of the spots (foci)

do seeded watershed

If selected a seeded watershed is applied to separate touching spots

proeminence of max.

Tolerance for the maxima that will be detected as seed points for the watershed

threshold 1

Lower area-threshold for the spot groups. Spots will be reported in three groups: below t1, between t1 and t2 and above t2.

threshold 2

Upper area-threshold for the spot groups. Spots will be reported in three groups: below t1, between t1 and t2 and above t2.

name of table

The name of the table in which the global results are reported