Volker Baecker, uses the Watershed plugin from Christopher Mei.
The application counts the number of objects in an image and writes the number per image into a spreadsheet-file. When the application is started the list-editor is opened. Press the add button and select the files you want to measure. Press close on the list editor to start. You will be asked where to save the result spreadsheet file. The first image is loaded and displayed. The application has selected a number of objects. You can modify the selection by activating the point-selection tool from ImageJ and using shift+click to add objects and (control-) alt-click to remove objects. When you finished correcting the selection, press accept on the “accept or skip” dialog. The measurements will be saved and the next image will be loaded. Skip allows to not take into account an image and exit will stop the application before the last image is reached.
Important options are smoothing diameter, and the min. size and max. size.
The smoothing diameter is the radius of the kernel of a smoothing filter that is applied before the watershed. It needs to be big enough to make disappear internal structures of the objects you want to count, since otherwise objects will be split too much. It should not be bigger than necessary since otherwise the contours of the objects become imprecise and touching objects may not be split.
The min. and max. size can be used to eliminate wrong objects that are either too small or too big. The area of the objects you want to measure must be between the min. and the max. value.
For each image a control-image showing the nuclei that have been taken into account is written into the sub-folder named count-nuclei-control of the folder containing the input images. The nuclei that have been taken into account are marked with a point. Note that the point-selection is saved with the image. If you open the control-image with ImageJ the selection will be displayed. The number of objects per image and the coordinates of all objects are written into a spreadsheet-file.