Re: flattening a layer from z-stacks of thick uneven tissue

Posted by dpoburko on Oct 13, 2009; 5:03pm
URL: http://imagej.273.s1.nabble.com/flattening-a-layer-from-z-stacks-of-thick-uneven-tissue-tp3690504p3690505.html

Sorry to beat a dead horse, but did you try the 3D-modeling version of
Extended depth of field? I will also reduce your stack to single plain,
but it does a pretty solid job of removing out of focus fuzz. I'm using
this to gather fluorescent puncta on multiple plain into a single image.
Deconvolution before had might also be helpfull, as per Glen's suggestion.

Damon

Mark Krebs wrote:

> Hi All,
>
> I'm a relatively new ImageJ user.  I have confocal fluorescence microscopy
> z-stacks of a 50-um thick epithelial tissue stained for nuclei (blue
> channel.  The tissue is not completely flat; the z-position of its topmost
> surface varies gradually within the xy plane by 5-10 um.   I would like to
> analyze the cell nuclei of the topmost ~10-um thick cell layer (number,
> area, volume, etc.), but I am unable to merge the z-slices without
> fluorescence contributions from nuclei in other layers.  This makes it
> much more difficult to identify nuclei and analyze them properly.
>
> I am wondering whether there is any ImageJ macro or plugin that will
> isolate the nuclei of the topmost cell layer from underlying nuclei by
> calculating a topmost nuclear surface (S), which follows the uneven
> surface of the tissue.  I imagine several steps to the analysis.  
>
> First, there would be an algorithm to define S by threshold criteria,
> which should be feasible since the top of the stack is black background.  
> At each xy location of the image, the z-stack would be examined from the
> top, defining the z-position of the surface as the first pixel encountered
> that exceeds the threshold blue value.  Since nuclei are separated by ~20-
> 30 um, there would need to be a mechanism to ignore z positions calculated
> at xy locations where there are no nuclei in the topmost cell layer.  This
> might be achieved perhaps by analyzing a limited z-range that is provided
> as input, or by "rolling a ball" of defined radius over the image surface
> and selecting only the topmost positions.  Finally, S would be calculated
> from the coordinates of the topmost nuclear positions.
>
> Second, S would be used to collect a slab of z-pixels (with an input value
> slightly exceeding the nuclear diameter) downwards from the topmost
> nuclear surface.  This slab of z-pixels would be used to create a new,
> flattened z-stack, which can be used as input for merging and analysis.
>
> Does anyone know of an ImageJ application for this purpose?  Or is there a
> simpler approach?  If there is no such application (we DON'T have an app
> for that!), can someone offer advice on how to go about creating such an
> application?
>
> Thanks everyone!
>
> Cheers,
> Mark
>