Volumetric measurements (how to mask binary volume and to translate to measure in volume from different channel)


#1

Hello. My goal is to learn how to select binary volume and to measure signal (fluorescence) intensity outlined by binary volume in volume with fluorescent intensity (from another channel of the same volume). I want to measure average volumetric intensities (I guess in voxels). I would appreciate if anyone can give me a clue how to do it? Also if you know such a link with presentation - please let me know. Thank you!


#2

Hi @1timur1,

I would advise you to first try to understand the basic principles of image analysis first in 2D, you can go trough the ImageJ user guide, you will find in Analyse the Set Measurements command that will do what you want : select a binary mask but perform intensity measurement in another image. For 3D a similar plugin that does the same is 3D Object Counter .

Thomas


#3

Hi Thomas,
Now I know how to do everything that you describe in 2D. The problem is 3D now. Can you please send me a macro for how to do it in 3D for sample “flybrain”? How to make volume first, and then to make red signal as a mask of voxels, and then to measure intensities of green inside of masked voxels 3D partilcels only (measuring size of voxels and their mean intensities)?
Thank you!!
Timur


#4

Hi @1timur1,

The procedure is exactly the same in 2D or 3D, just instead of Analyze particles you need to use 3D Objects Counter. Here a simple macro done with Record :

run("Fly Brain (1MB)", "statusservice=[org.scijava.app.DefaultStatusService [priority = 0.0]] log=[org.scijava.log.StderrLogService [priority = -100.0]] uploaderservice=[net.imagej.updater.DefaultUploaderService [priority = 0.0]] commandservice=[org.scijava.command.DefaultCommandService [priority = 0.0]]");
run("Split Channels");
selectWindow("flybrain.tif (blue)");
close();
selectWindow("flybrain.tif (green)");
setAutoThreshold("Otsu dark stack");
setOption("BlackBackground", true);
run("Convert to Mask", "method=Otsu background=Dark black");
run("3D OC Options", "volume surface nb_of_obj._voxels nb_of_surf._voxels integrated_density mean_gray_value std_dev_gray_value median_gray_value minimum_gray_value maximum_gray_value centroid mean_distance_to_surface std_dev_distance_to_surface median_distance_to_surface centre_of_mass bounding_box dots_size=5 font_size=10 store_results_within_a_table_named_after_the_image_(macro_friendly) redirect_to=[flybrain.tif (red)]");
run("3D Objects Counter", "threshold=128 slice=28 min.=100 max.=3735552 objects statistics");
run("Enhance Contrast", "saturated=0.35");

If you have problem with the 3D Objects Counter, you may want to try the original plugin 3D Object Counter.

Best,

Thomas


#5

Thank you Thomas. Kind of strange thing happened again. Even after downloading original plugin I still have “Console” window where it is written that “3D OC Options not up-to-date becasue 1 soruce files are not up-to-date”. I will try it on another computer soon. May it it will help.