http://confocal-microscopy-list.275.s1.nabble.com/Re-Objective-lens-chromatic-aberration-Color-shift-correction-for-colocalization-analysis-was-Glycerh-tp5919806p5929266.html
view. If the stitching software does not not do seamless stitching,
p.s. For 2D stitching, Adobe Photoshop CS5 (and 3 and 4) have a very
like that). Carl Boswell and/or Jerry Sedgewick had mentioned this to me
> *****
> 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<
[hidden email]> wrote:
>
>
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>>
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>>
>> 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<
[hidden email]>
>>> 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:
[hidden email]
>>>
>> 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 )
>>
>