Quantifying cells in 4 channel fluorescent

First, thank you for making this very capable software available.
I need guidance on building a pipeline for analyzing 4-channel fluorescent images. I usually acquire images (*.ZVI or *.LSM) of tissue or culture stem cells stained with different probes or expressing 4 different fluorescent tags (BFP, GFP, dsRed, Cy5). I managed to build a pipeline for counting cells in 3 channels using one of the posted examples, but I am not sure how to analyze 4 channels.
I would like to (1) count the number of cells in 4 channels separately (i.e. # of BFP cells, #GFP cells, etc…) (2) quantify cells expressing 2 or more tags (BFP+/GFP+, BFP+/RFP+, RFP+/GFP+, etc…).
I am not sure what steps to combine to perform these computations.

You should be able to use LoadImages for this. Under file type to be loaded, select “tif,tiff,avi or zvi files”, enter the text that identifies each .zvi file under “Text that these images have in common” and then click the “Add channel” for as many channels as you need, giving a unique name to each channel. All channels will be load simultaneously for each image file.

Once the channels are loaded, you can apply IdentifyPrimaryObjects to identify the cells in each channel, and then use a combination of the RelateObjects and FilterObjects module to find cells that are positive for each stain; have a look at the PercentPositive example pipeline from our Examples page for more details on this.

Hope this helps!
-Mark

This looks like what I need. Thanks for your quick response.
~A