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Measure_Border_And_Spots_Tool

Volker edited this page May 31, 2021 · 1 revision

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.

output.png measurements.png

You can download some example images here: example_images.zip

Getting started

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.

toolbar.png

  • 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

Usage

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:

files.png

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.

Method

Nuclei segmentation

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.

Membrane selection

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

Spot segmentation

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.

Distances of spots to the nucleus border

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.

Options

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

options2.png

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
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