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Thanks again to everyone who replied on and off list. We calibrated the
objectives as Kees recommended and still found some misalignment. We next
tried the same with stitching turned off and the alignment was perfect.
Blurring of fine details was a little worse at image borders but not by
very much. It turns out that the coded stage really is very good at
maintaining a position when imaging 100+ tiled images repeatedly in a
large number on z sections.
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]
On 4/3/14, 9:36 AM, "phil laissue" <[log in to unmask]> wrote:
>*****
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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]
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>
>
>
>
>_____________________________________
>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]
>http://scanmail.trustwave.com/?c=129&d=3eu904ma0h6a25H5kaVFITC-PSzasnHZIGe
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>
>
>On 3 April 2014 09:05, Straatman, Kees (Dr.) <[log in to unmask]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>>http://scanmail.trustwave.com/?c=129&d=3eu904ma0h6a25H5kaVFITC-PSzasnHZID
>>LC32Z3pg&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm
>>icroscopy
>> Post images on
>>http://scanmail.trustwave.com/?c=129&d=3eu904ma0h6a25H5kaVFITC-PSzasnHZIG
>>fDjWwj9g&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your
>>posting.
>> *****
>>
>> 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://scanmail.trustwave.com/?c=129&d=3eu904ma0h6a25H5kaVFITC-PSzasnHZID
>>aV3z8n9A&u=http%3a%2f%2fwww2%2ele%2eac%2euk%2fcolleges%2fmedbiopsych%2ffa
>>cilities-and-services%2fcbs%2flite%2faif
>>
>>
>>
>>
>> -----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://scanmail.trustwave.com/?c=129&d=3eu904ma0h6a25H5kaVFITC-PSzasnHZID
>>LC32Z3pg&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm
>>icroscopy
>> Post images on
>>http://scanmail.trustwave.com/?c=129&d=3eu904ma0h6a25H5kaVFITC-PSzasnHZIG
>>fDjWwj9g&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your
>>posting.
>> *****
>>
>> 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]>
>>
|
