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Have you tried ImageJ for stitching?  Fiji ImageJ (http://fiji.sc/)
comes pre-loaded with s stitching plugin where you can specify the
relative location of tiles to prime the autostitching algorithm
(Plugins > Stitching > Grid/Collection stitching).  It has worked very
well for me, the priming made a big difference and even stitched some
low-contrast brightfield images well.

-Esteban


On Thu, Aug 16, 2012 at 12:30 AM, David Johnston <[log in to unmask]> wrote:
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>
> Hi all,
>
> I have a user who is assessing bacterial biofilm clearance by ciliated airway
> epithelial cells grown on air liquid interface cultures. He then fixes the cultures,
> cuts out and divides the membrane and does FISH for the bacteria, immuno for
> the cilia plus nuclear counterstain. The filter slices are mounted in mountant
> between 2 coverslips and imaged on SP5 in tile scan mode, imaging a long, thin
> radial strip of approx 45x1 fields at 512x512 resolution and multiple Z levels (up
> to 50ish) It is impossible to get the filters perfectly flat - they tilt and buckle
> so we need to set a large Z range to encompass absolute highest and lowest
> points and many Z slices. Ultimately he wants to calculate an stimate of
> biofilm volume
>
> My 2 problems, seeking advice and solutions:
> (1) because of the wide Z range, some slices on some fields cut into the filter
> (autofluorescence and non specific binding at holes) or close to coverslip
> (flash). Because of the undulations of the membrane, this varies from field of
> view to field of view. Therefore I need a way to crop or blank Z slices off the
> data set in a manner which is field of view specific, rather than global, but still
> retains the relative Z position in the overall stack.
>
> (2) Despite the fields forming an absolute linear strip, Leica's autostitcher
> sometimes makes a complete hash of it. Is there any easy way to get a stitch
> of Z stacks based simply on relative location.
>
> We are talking reasonably large datafiles here (several GB)
>
> Thanks in Advance,
> Dave Johnston,
> Biomedical Imaging Unit, Southampton, UK.
>
> PS, learned the hard way, if doing tile sets, make sure scan head rotation is
> set to 0 :-)