Posted by
daj1u06 on
Aug 16, 2012; 7:30am
URL: http://confocal-microscopy-list.275.s1.nabble.com/processing-and-Z-cropping-tiled-Leica-images-tp7578835.html
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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 :-)