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Hi Dave,

Contact your Leica confocal sales rep and have them visit to demo the 
Leica MATRIX module(s) to do what you need. They might also want to 
bring with them a Leica MATRIX application(s) expert. A quote for a new 
64-bit PC might also be useful.

I've already complained to Leica that their LAS AF stage tiling 
acquisition and image stitching is bad (I probably complained about it 
being bad in TCS and LCS when I managed an SP1 - accomplished nothing). 
Not that Zeiss LSM710 / ZEN software is any better.

George



On 8/16/2012 3:30 AM, David Johnston wrote:
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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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 :-)
>
>