Three contrast levels in stack

[gssjr]

I have a stack of hundreds of images (x,y,time) acquired from a microscope. Ideally there should only be three levels of contrast: hi, mid, lo (or -1,0,1 etc however you prefer)

I wrote an app to track how each pixel changes over time between the three states but the contrast levels change between each frame (and sometimes within a given frame) so I can't properly set a threshold (e.g. less than 80 is lo, greater than 120 is high, and everything in between is mid).

If I look at a histogram for any given slice, I can clearly see three Gaussian-like peaks, but the positions and shapes of those peaks change every frame.

Knowing that there should only be three states, how can I best normalize the stack so I can properly track each pixel?

I attached an image showing two example frames and their corresponding histograms.tri-level.tif

[Jan Eglinger-3]

Hi,

On 22.07.2011 3:08 AM, gssjr wrote:

> I have a stack of hundreds of images (x,y,time) acquired from a microscope.
> Ideally there should only be three levels of contrast: hi, mid, lo (or
> -1,0,1 etc however you prefer)
>
> [...]
>
> If I look at a histogram for any given slice, I can clearly see three
> Gaussian-like peaks, but the positions and shapes of those peaks change
> every frame.
>
> Knowing that there should only be three states, how can I best normalize the
> stack so I can properly track each pixel?

Have you tried to use the IsoData Classifier? It might be of use in your
case.
http://fiji.sc/IsoData_Classifier

Cheers,
Jan