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Hi Timothy,

like Kees, we're happy with NIS-Elements stitching, overall. Automatic
calibration works best with a high-contrast, continuous sample - tissue
sections (even just HE) work fine, e.g. dog ileum (but not, say, breast
tissue). Stitching overlap between 5-15% is fine. I did run into problems
with too large samples though. A coral I took was ~5k images
(x14*y4*z30*ch3), resulting in around a 5GB nd2 file. NIS-Elements
subsequently crashed trying to put it together, and the file was corrupted.
Luckily, I always save them separately as tifs, and stitched those
together. For ease of handling, I collapsed z and stitched maximum
intensity projections (and extended depth of field for DIC, using the BIG
Lausanne's plugin) together for the big picture, using 3D stitching for
smaller ROIs. MosaicJ works fine, as does Stephan Preibisch's.
StackReg (BIG Lausanne) often works ok (I only stick with Translation or
rigid body, never scaled or affine for obvious reasons). Especially useful
is a version using StackReg, called MultiStackReg or MultiStackRegFix (Brad
Busse/Ping Fu & Jennifer Staab, resp.), allow saving the transformation
matrix for one channel and aligning the next using those parameters. I'd
like to do the same for the SIFT-based registration plugin (Stephan
Saalfeld), which mostly works really well (parameters sometimes need
adjusting) and often gets the job done where StackReg fails.

Kind regards

Philippe

_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[log in to unmask]
privatewww.essex.ac.uk/~plaissue <http://privatewww.essex.ac.uk/%7Eplaissue>




_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[log in to unmask]
privatewww.essex.ac.uk/~plaissue


On 3 April 2014 09:05, Straatman, Kees (Dr.) <[log in to unmask]> wrote:

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> *****
>
> Dear Timothy,
>
> We recently had some problems with stitching using NIS-Elements which were
> resolved by calibrating the stage.  The info I received from our Nikon
> product specialist was:
>
> "Go to the Calibration menu and objectives, there is an option on the
> right to recalibrate objective (ensure the correct objective is selected).
> In the wizard select the auto option, ensure you have a sample on the stage
> in focus with good detail. The software will run an autocalibrate and
> should then stitch correctly."
>
> This solved the problem for us. We also noticed that after the camera was
> slightly turned by accident the stitching did not work properly anymore and
> we needed to recalibrate.
>
> Best wishes
>
> Kees
>
>
> Dr Ir K.R. Straatman
> Senior Experimental Officer
> Centre for Core Biotechnology Services
> University of Leicester
>
> http://www2.le.ac.uk/colleges/medbiopsych/facilities-and-services/cbs/lite/aif
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Feinstein, Timothy
> Sent: 02 April 2014 19:11
> To: [log in to unmask]
> Subject: Aligning large stacks
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>
> Hello all,
>
> We have a number of folks who routinely test the limits of our A1-RS
> system by collecting for example large and high-resolution atlases of
> clarified brains (at least the 300 um -1 mm depth that we can reach with an
> inverted scope).  Our coded stage and a software feature in Elements lets
> us create 3D stacks of very large images stitched together from 16 to 100
> individual images per slice.
>
> We have found that the alignment can go slightly off from one slice to the
> next, screwing with our deconvolution and analysis results.  This is not by
> itself shocking; these jobs are asking an awful lot of the system.  We have
> the system pretty well isolated from vibration and air currents and nobody
> goes near it while collecting.
>
> Elements has an alignment feature built in but it does not produce great
> results from very large images (e.g. 4096 ^2 and up).  I have tried the
> registration plugins menu in Fiji and my current favorite, StackReg, can
> produce solid results.  Rigid body registration seems to do the job most of
> the time.  On the other hand its progress bar is not very helpful and some
> alignments can take overnight to all weekend, if they work.
>
> So I thought it would help to throw this out to those of you who do this
> more often - do you have a favorite workflow for big-job registrations?  Do
> you have a favorite freeware, Matlab plugin or paid option for these big
> jobs?  Should I stop stitching the images and let the coded stage handle
> alignment on its own?
>
> We use a workstation with a decent graphics card and 36 GB of RAM.  We can
> put in a lot more if that will help.
>
> Any advice on or off list would be appreciated.
>
> Thanks and all the best,
>
>
> TF
>
> Timothy Feinstein, Ph.D. | Confocal Manager
> 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
> Phone: 616-234-5819 | Email: [log in to unmask]<mailto:
> [log in to unmask]>
>