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Re: RGB weights and fluorescence intensity

Posted by Daniel James White on Jul 27, 2009; 11:10am
URL: http://imagej.273.s1.nabble.com/RGB-weights-and-fluorescence-intensity-tp3691651p3691653.html

Hi Sinead,

On Jul 24, 2009, at 10:47 AM, Sinead Roberts wrote:

> That is a great help, thank you very much :).
>
> I have one question now following on from the tutorial you posted-  
> about watershed algorithms to
> recognise touching objects. If I have stained cells with DAPI and a  
> plasma membrane stain, where it
> is at the plasma membrane the cells are touching, can I apply some  
> sort of watershed that will
> recognise and outline as cells those regions that contain the nuclei?

not quite sure what you mean?

do you mean use information from the plasma membrane stain to help  
determine the outline of single nuclei?


> Basically I mean a way for the
> programme to recognise as objects the real cells as opposed to the  
> background regions that also
> theoretically are 'enclosed' by overlapping membrane?

maybe you can explain better by sending me a sample image and an  
explanation of what you really want to measure.

> And moreover, if I have a z-stack of images, can I go about linking  
> as being from one 'cell' the
> corresponding cell outlines in each slice? Or, will this be  
> something I have to do manually?

you can use imageJ in 3D and count objects in 3D
in the  Fiji - imageJ distribution ,
see
Analyse - 3D objects counter.

first you need to segment the nulclei in 3D (in fact you can do it per  
z slice with a global or local threshold)
then use the 3d objects counter plugin to measure the objects in 3D

if you nuclei seem to touch each other, and you need to separate them,
you can use the watershed separation  method in ImageJ to separate  
them before counting.

cheers

dan


>
> Thanks again for you help,
> Sinead





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
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