Re: Objective lens chromatic aberration - Color shift correction for colocalization analysis - was Glycerol Objectives - experience with

Posted by Daniel James White on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-Objective-lens-chromatic-aberration-Color-shift-correction-for-colocalization-analysis-was-Glycerh-tp5919806p5921609.html

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Hi Stan,

On Jan 14, 2011, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system wrote:

>
> Date:    Thu, 13 Jan 2011 15:28:40 -0600
> From:    Stanislav Vitha <[hidden email]>
> Subject: Re: Objective lens chromatic aberration - Color shift correction for colocalization analysis - was Glycerol Objectives - experience with
>
> Hi Dan,
> The translational correction is very useful,  but what if the shift varie=
> s
> across the field?

then the assumption of the shift being space invariant is clearly false,
and you need a more clever way to correct it, that IS spatially variant... read on.

> Do you know if there is a plugin or a script to perform affine transform =
> in
> 3D?

Oh yes!

> The goal would be to "warp" one channel in XYZ to match the second ch=
> annel.

There are options here...

In Fiji (i just ImageJ) there is Ignacio's nice bUnwarpJ
http://pacific.mpi-cbg.de/wiki/index.php/BUnwarpJ
points to
http://biocomp.cnb.csic.es/~iarganda/bUnwarpJ/index.html

dont think it can also warp z though...

> 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 lon=
> ger
> wavelength channels (green, orange, red fluorescence). So for instance, t=
> he
> 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 f=
> or
> ImageJ works well to correct this in individual XY images,

not the same as bUnwarpJ, but similar....

> but 3D correct=
> ion
> may be needed, since the plan correction for the blue channel (405 nm las=
> er
> illumination) is not as good as for the rest of the spectrum.=20=20=20

thats often the case,
as well as the UC laser coming in through a different collimator, so its often 0.5 microns or so off the vis lines.

cheers


Dan


>
> =20
>
> Stan Vitha
> Microscopy and Imaging Center
> Texas A&M University

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

[phone] (German Mobile)
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