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Hi Stan,
Plucking one idea from Jim Pawley's post - the objective lens should be
very well corrected in the center of the field - zoom up to 2x (or more)
and use motorized scanning stage tiling if you need a larger field of
view. If the stitching software does not not do seamless stitching,
complain to the manufacturer until they fix it.
Sincerely,
George
p.s. For 2D stitching, Adobe Photoshop CS5 (and 3 and 4) have a very
nice multi-file stitching command (File menu - Automate, or something
like that). Carl Boswell and/or Jerry Sedgewick had mentioned this to me
several years ago (CS3), but I only recently upgraded from 2 to 5.
On 1/13/2011 4:28 PM, Stanislav Vitha 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
> *****
>
> Hi Dan,
> The translational correction is very useful, but what if the shift varies
> across the field?
> Do you know if there is a plugin or a script to perform affine transform in
> 3D? The goal would be to "warp" one channel in XYZ to match the second channel.
> I have imaged 100 nm multi-color beads on our confocal (FV1000 with a
> hand-picked 100x/1.4 Plan Super Apo lens), and besides a moderate z-shift
> between blue and other channels (as expected), I have a lateral chromatic
> aberration between the "DAPI" channel (405 nm ex, 430-460 em) and the longer
> wavelength channels (green, orange, red fluorescence). So for instance, the
> colors are colocalized in top left corner of the image, but are several
> pixels off in XY plane in bottom right of the image. The UnwarpJ plugin for
> ImageJ works well to correct this in individual XY images, but 3D correction
> may be needed, since the plan correction for the blue channel (405 nm laser
> illumination) is not as good as for the rest of the spectrum.
>
>
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M University
>
>
> On Tue, 11 Jan 2011 11:23:02 +0100, Daniel James White<[log in to unmask]> wrote:
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Martin,
>>
>> On Jan 11, 2011, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:
>>
>>
>>> Date: Mon, 10 Jan 2011 17:18:29 -0600
>>> From: Martin Wessendorf<[log in to unmask]>
>>> Subject: Re: Glycerol Objectives - experience with
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> On 1/10/2011 4:32 PM, Rosemary White wrote:
>>>
>>>
>>>> I've found that the red and blue emission aren't quite lined up vertically,
>>>> at least with our "blue" 63x objective, so have to cut the top one or two
>>>> slices off a blue vertical series (depending on depth of slice), and the
>>>> bottom slices off a red series and reassemble to get the emissions aligned -
>>>> i.e. from the same depth in the tissue. The objectives do vary a bit,
>>>> perhaps ours isn't as well corrected as some.
>>>>
>>> Can anyone explain where the difficulty arises in correcting for axial
>>> chromatic aberration? I've consistently seen serious axial chromatic
>>> aberration in good quality oil objectives from one very reputable
>>> manufacturer, between red, green and far-red (1 um off in the z-axis
>>> between green and red, and between red and far-red; 2 um between green
>>> and far-red). I have always heard that axial chromatic aberration was
>>> easy to correct for and would've thought that a solution for 3-color
>>> correction would've been found 100 years ago. However, I don't know
>>> enough optics to understand the subtleties. Are there trade-offs to
>>> trying to obtain good correction, besides the cost in scattering of
>>> adding additional lens elements?
>>>
>> Sadly, not all objective lenses were made equal.
>>
>> In our hands on point scanning confocals, even when you align the
>>
> pinhole(s) as best you can
>
>> there can still be a micron of misalignment in between "green", "red" or
>>
> "far red",
>
>> with DAPI etc. often being way off, as it comes through a different optic
>>
> fiber and collimator anyway (eg on an LSM 510)
>
>> I have tested different lenses of the same spec from the same manufacturer,
>> and found that they all have their own personality.
>> Good correction is very possible, but "good" is a relative thing.
>>
>> I send back ones that are too "bad", and keep the "good" ones.
>> I take z stacks of 1 micron multi colour beads to measure the remaining error
>> (dont need sub resolution bead images for this measurement)
>>
>> There will always be a significant error, even in the very best
>> "Super-dooper Mega Extra Apo-Chromat" lens you can get from any
>>
> manufacturer for less than 100 k dollars.
>
>> They simply can not be made perfect at a reasonable cost (as explained to
>>
> me by a Zeiss lens guru)
>
>> If you want to do precise colocalization studies at the highest optical
>>
> resolution (everyone does, even if they don't immediately realize it)
>
>> then you simply MUST measure the error, then correct/shift the images (in
>>
> 3D) before colocalization/correlation analysis.
>
>> This can be done in 3D in ImageJ/ Fiji
>> using nice interpolation methods from Erik Meijering, for sub pixel shifts
>>
> - TJ-Translate :
>
>> http://pacific.mpi-cbg.de/wiki/index.php/TransformJ
>> leading to
>> http://imagescience.org/meijering/software/transformj/translate.html
>>
>> or in the great colour shift corrector of Huygens Professional (no
>>
> commercial interest - just a happy customer)
>
>> http://www.svi.nl/ChromaticShiftCorrector
>>
>> or roughly in the Zeiss AIM 510 software in xy only (whole pixel shifts),
>>
>> The effect on the 2 channel scatterplot / 2D histogram / fluorogram is very
>>
> significant.
>
>> A ugly poorly correlated cloud turns into tight correlated populations of
>>
> pixels,
>
>> and the coloc. coefficients jump much higher.
>>
>> more info here:
>> http://ifn.mpi-cbg.de/wiki/ifn/index.php/Chromatic_aberration_measurement_and_correction
>>
>> cheers
>>
>> Dan
>>
>>
>>
>>
>>> Thanks--
>>>
>>> Martin
>>> --
>>> Martin Wessendorf, Ph.D. office: (612) 626-0145
>>> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
>>> University of Minnesota Preferred FAX: (612) 624-8118
>>> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
>>> Minneapolis, MN 55455 e-mail: [log in to unmask]
>>>
>> Dr. Daniel James White BSc. (Hons.) PhD
>> Senior Microscopist / Image Visualisation, Processing and Analysis
>> Light Microscopy and Image Processing Facilities
>> Max Planck Institute of Molecular Cell Biology and Genetics
>> Pfotenhauerstrasse 108
>> 01307 DRESDEN
>> Germany
>>
>> +49 (0)15114966933 (German Mobile)
>> +49 (0)351 210 2627 (Work phone at MPI-CBG)
>> +49 (0)351 210 1078 (Fax MPI-CBG LMF)
>>
>> http://www.bioimagexd.net BioImageXD
>> http://pacific.mpi-cbg.de Fiji - is just ImageJ (Batteries Included)
>> http://www.chalkie.org.uk Dan's Homepages
>> https://ifn.mpi-cbg.de Dresden Imaging Facility Network
>> dan (at) chalkie.org.uk
>> ( white (at) mpi-cbg.de )
>>
>
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