Posted by
Glen MacDonald-2 on
Oct 12, 2009; 5:10pm
URL: http://imagej.273.s1.nabble.com/flattening-a-layer-from-z-stacks-of-thick-uneven-tissue-tp3690504p3690517.html
Have you tried the 'Extended Depth of field' plugin?
Regards,
Glen
Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]
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The box said "Requires WindowsXP or better", so I bought a Macintosh.
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On Oct 12, 2009, at 7:26 AM, 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