ImageJ macro for obtaining sperm perimeters from Hoechst 33342 or propidium iodide stained sperm nuclei and it has been tested with up to ImageJ 1.49o:

Download parrish_021515_comp.txt and place in macro folder. Alternatively if you wish to use this every time you open imageJ, you can download startup_version_021015_comp.txt and rename as StartupMacros.txt and place in the macro folder replacing the file with that name that comes with ImageJ. Options exist for using Multi Thresholder Selector, Laplacian Edge Subtraction and FFT Bandpass for thresholding specific species of sperm. Currently bull works best with Multi Thresholder Selector, boar works with Lapalcian Edge Subtraction, and human works with the FFT Bandpass. Use the action bar windows 'Create p h all' when recording only phase and fluorescent images of all sperm nuclei. Use 'Create p h y' when recording phase, a hoechst image for all sperm and a yoyo-1 image for dead sperm. An addition action bar window also appears to examine morphology of sperm already evaluated in the prior selected sperm (ie the above create action bar windows) with a psuedo-DIC image or a phase image.

To run this version of the parrish macros you also need specific version of some ImageJ plugins previously developed by others. You will need:

1. action_bar201a.jar
2. the zip file for the folder ActionBar - unzip this file and then put in plugins
3. FeatureJ_.jar
4. imagescience.jar
5. Multi_Thresholder.jar

Credits for Jar files

1. action_bar201a was obtained from the ImageJ Wiki and is from 2013 with the authors: Jerome Mutterer, Johannes Schindelin , Stephan Saalfeld , Michael Schmid , Serge Mazères , Olivier Burri , Simon Roussel , Gabriel Landini
2. FeatureJ was obtained from the FeatureJ website but the version we are using 1.6.0 is no longer on the website and so the above copy is essential. The author is Erik Meijering.
3. Imagescience.jar is version 2.4.1, December 21, 2011 with the author Erik Meijering. This also was obtained previously from the FeatureJ website but is no longer available and so you must use this version.
4. Multi_Thresholder.jar was downloaded from the ImageJ Plugins site. It was downloaded before this was the default within ImageJ and may be required due to changing of methods for calling the specific thresholding algorithms. The authors are: Kevin (Gali) Baler, Gabrial Landini, Wayne Rasband

The general approach was to obtain a deblurred image using an actual point spread function image (PSF) and 2D deconvolution. The deblurred image was then assessed for sperm nuclear texture after sperm objects were identified with the sperm perimeter extraction approach above. The exact procedures are described in the methods of Megan Krautkramer.

Specific plugins and macros required are:

Parallel Interative Deconvolution 1.9 was downloaded from the Fiji.sc website. Newer versions can be found in the at the google site by the author Piotr Wendykier but it is unkown if this would work with other macros we developed to run in conjuction with this software.

Texture_62915c.txt is a macro file written by Megan Krautkramer and John Parrish to run Texture analyzer (version 0.4; 200; Julio Cabrera). The texture analyzer is a ImageJ plugin that computes several of the texture parameters described by Haralick (Haralick, R. M., Shanmugam, K., and Dinstein, I. (1973). Texture parameters for image classification, IEEE Trans SMC 3, 610-621).