I am trying to segment out membrane, cytoplasm and nuclei from a
confocal image set. This is so we can measure translocation of a protein
from the membrane to the cytoplasm/nucleus over time. I can do this in
MetaMorph but would like to be able to do it in 3D in Imaris if
possible.

The sample is stained as follows
-	Nuclei - DAPI
-	Whole Cell - CellMask (Invitrogen)
-	Protein of Interest (Alexa Antibody)

Since there is not a specific stain from membrane or cytoplasm i have
been doing the following
-	Segment out whole cells and create a binary mask
-	Erode that mask by 4 or so pixels, this represents the cytoplasm
and nuclei of the cell
-	Subtract the eroded mask from the whole cell mask. This leave a
ring that represents the membrane of the cell.
-	Segment and subtract the nuclei from the combined cytoplasm and
nuclei mask to give a cytoplasm mask.
-	The end result is three masks; one each representing membrane,
nuclei and cytoplasm.

I can create these masks for each slice of a confocal set and get
intensity etc information out from MetaMorph. I have tried exporting the
mask stacks out to Imaris to create 3D masks but it doesn't work very
well, especially for the membrane mask, as the slices don't necessarily
overlap so there are gaps in the mask. My 3D model of the membrane masks
looks more like a badly piled up lot of rubber bands. The 3D masks for
the whole cell or nucleus work fine. 

So i guess the main question is: is it possible in Imaris to do
subtractive image mathematics?



Thanks


Cam



Cameron J. Nowell
Microscopy Manager 
Centre for Advanced Microscopy 
Ludwig Institute for Cancer Research 
PO Box 2008 
Royal Melbourne Hospital 
Victoria, 3050 
AUSTRALIA 
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Fax: +61 3 9341 3104 
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