I am hoping to get some help with some basic usability/transition issues in analyzing metamorph/metafluor images. I have read the manual in detail, and hope that I have not missed the answers to the following questions:
pixel values - I am collecting 12 and 16 bit data, yet the data all seem to be floating point data well less than 1. What conversion is being made, and is this optional?
image files names - I have a large number of tif files produced by metafluor that have the structure red.001, red.002, green.001, etc. Is there any way for cell profiler to recognize these files as images or do I have to rename them all as tifs?
Object count information - my first application with this program is to count living and cells using 3 stains: all nuclei (Hoechst), living cells (calcein) and dead nuclei (propidium). Two issues arise from my efforts so far:
a. where is the object count information from IdentifyPrimary and IdentifySecondary stored and how do I get at it?
b. In my images. there are often nuclei (hoechst image) from dead cells that have no surrounding cytoplasm (calcein image). the nuclei are being correctly identified with IdentifyPrimary, but IdentifySecondary on the calcein image is incorrectly creating an object around every primary object. How can I stop this happening?
Finally (sorry for the long post), I am getting an error message with IdentifySecondary that I cannot solve: “the object outlines were not calculated by the IdentifySecondary modue, so these images were not saved to the handles structure…”. What am I setting incorrectly?
thanks for everyone’s help.
University of Chicago