Unable to use ClassifyObjectsByTwoMeasurements




I’m trying to use the ClassifyObjectsbyTwoMeasurements module to quantify the number of nuclei with both FITC and TRITC label, either FITC or TRITC and without any label.
The module works fine provided there are measurements below the two threshold values, however if for example all the intensity measurements in the FITC channel are above the threshold (eg all nuclei FITC positive) the following error message pops up and the pipeline stops. Is there something I’m doing wrong here or is that a bug in the module, and if it is not a bug how else could I do that counting ?



There was a problem running the analysis module ClassifyObjectsByTwoMeasurements which is number 18.
Image processing was canceled in the ClassifyObjectsByTwoMeasurements module because the threshold 0.04 you selected for the first set of data is invalid. The threshold should be a number in between the minimum and maximum measurements of the data, which are 0.043171 and 0.054518, respectively.

ClassifyObjectsByTwoMeasurements in C:\Program Files\CellProfiler_105811\CompiledCellProfiler\CellProfiler_mcr\Modules\ClassifyObjectsByTwoMeasurements.m (199)
AnalyzeImagesButton_Callback in C:\Program Files\CellProfiler_105811\CompiledCellProfiler\CellProfiler_mcr\CellProfiler\CellProfiler.m (10424)
gui_mainfcn in C:\Program Files\CellProfiler_105811\CompiledCellProfiler\CellProfiler_mcr\CellProfiler\CellProfiler.m (12164)
CellProfiler in C:\Program Files\CellProfiler_105811\CompiledCellProfiler\CellProfiler_mcr\CellProfiler\CellProfiler.m (57)


Hi Dirk,

This is indeed a bug; there is no reason that the threshold needs to be constrained in this way and the module proceeds successfully without this check.

If you are using the developers version of CellProfiler, you can edit the ClassifyObjectsbyTwoMeasurements module .m file by finding and commenting out/removing the following lines:

[quote]if Separator{FeatNum}<LowerBinMin(FeatNum) || Separator{FeatNum}>UpperBinMax(FeatNum)
error(blah,blah, blah…)

If you are using the compiled version, then you can’t edit the module but you can use two ClassifyObjects modules, one for one feature of interest and one for the other. If you are outputing to an Excel file, you can then check which objects are represented in all, some, or no bins for both measurements.

Hope this helps!