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Hi Emannuel,

Adjusting the distance between the camera and the W1 slightly might fix
this issue without getting out of focus. The image-side numerical aperture
is very small and the depth of focus is ~ 1 mm (at least with
high-magnification lenses).

But it's very difficult to achieve ~ 1 pixel registration between
two cameras. In fact, our two-camera systems show worse alignment than your
W1. At some point the emission filters (thickness, tilt) and lens
aberrations will limit the precision of alignment you can get.

In cases where alignment is critical (e.g. when measuring colocalization) I
have some Fiji macros for image registration using calibration images
(fluorescence beads imaged the same day with the same lens).

Best, zdenek

-- 
Zdenek Svindrych, Ph.D.
Research Scientist - Microscopy Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth



On Mon, Apr 15, 2024 at 9:30 AM Emmanuel Levy <[log in to unmask]>
wrote:

> *****
> To join or leave the confocal microscopy listserv or to change your email
> address, go to:
> https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear All,
>
> We have a W1 spinning disk with dual cameras. We recently had them aligned,
> and upon close inspection, we see that the GFP channel image is about 1 to
> 2 pixels larger than the RFP image in every corner (we used a 60x
> objective, and the cameras are primeBSI express with 6.4um pixels, so the
> image is off by 100-200nm in every corner). you can download the two images
> as a composite here if you are curious:
>
> https://drive.google.com/file/d/1TWIxwiZF6uf3FspyA96AX6r092PB_102/view?usp=sharing
>
> What would be the best way to solve this issue? Shouldn't the standard
> camera mounts enable us to correct this?
>
> Thanks for your help and comments,
> Best wishes,
>
> Emmanuel
>