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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 <[log in to unmask]>
> Subject: Re: Glycerol Objectives - experience with
>
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>
> 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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