Hi Cameron,


On Jul 16, 2009, at 7:03 AM, CONFOCALMICROSCOPY automatic digest  
system wrote:

> Date:    Wed, 15 Jul 2009 15:52:27 +1000
> From:    Cameron Nowell <[log in to unmask]>
> Subject: 3D Eroded Object Masks
>
> 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.

why not use ImageJ (or rather even Fiji - is just image|J batteries  
included)?
... its free....

>
> 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.

sounds logical!

>
> 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.=20

i would expect that to me the case if your z resolution / sampling is  
not high enough.
This is a 3d case so you could probably sacrifice some xy resolution
by opening the pinhole to 2 airy units,
and making more z slices, so that the membrane masks overlap better.

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

who knows, maybe you shouldf ask them.

In Fiji - imageJ you can do things like that with a bit of Jython  
scripting.

for free, and once you learn how to do it, you could share that info  
in a tutorial on the Fiji Wiki.
Then other people profit from your efforts.

if you need help, just ask

Dan



Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

+49 (0)15114966933 (German Mobile)
+49  (0)351 210 2627 (Work phone at MPI-CBG)
+49  (0)351 210 1078 (Fax MPI-CBG LMF)

http://www.bioimagexd.net 	BioImageXD
http://pacific.mpi-cbg.de		Fiji is just ImageJ - Batteries Included
http://www.chalkie.org.uk
[log in to unmask]
( [log in to unmask] )