Re: Sensitive Thresholding?

Posted by Gabriel Landini on Nov 30, 2009; 12:09pm
URL: http://imagej.273.s1.nabble.com/Sensitive-Thresholding-tp3690288p3690291.html

On Monday 30 November 2009  11:46:47 Wilson R S (AT) wrote:
> I have tried using the plugins "stack focuser" and "extended depth of
> focus", however both result in a "fuzzy" image.

Those procedures work fine if the stack is properly registered (eg some
microscopes do not move vertically orthogonal to the stage and so the image
seems to move while going down the stack) and that illumination does not
change from frame to frame. Without correction, there is no chance of getting
a good result.

But more importantly, one has to consider that for semi transparent samples
(for instance in bright field, phase contrast), the problem of extending the
depth of focus does not have a single solution. For example if you embed *semi
transparent* objects in gelatine, take photos at various depths and then run
the algorithm. You are likely to find several "good focus" points at the same
x,y position at various z. If the objects were opaque, occlusion would prevent
this to occur.
 
> My initial idea was to use a thresholding method, however this seams to
> pick out the out of focus sections first. I have tried a few different
> thresholding methods, colour and greyscale (on green channel seems to be
> the best), but nothing I get is useful.

There is extensive literature on this and (the related problem) autofocus.
Reading what has been done so far and what works, will save you a lot of time.

Cheers,

G.