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Hi Emmanuel, 

The reality is that tuning the hardware perfectly is impossible. Effects at this level are objective dependent (ir “identical” objective might have magnification differences of 0.1% between colours (ie 2 pixels over 2,000). In fact if you do super resolution imaging and are locking at small distance measurement between colours you have to ensure you correct for non-linearities between colours, where the images might align perfectly at both edges but have shifts in other regions. 

With the reality that you cannot align perfectly in hardware you should always take calibration data and develop a workflow that includes a (3D) alignment step, or distance correction. To pay particular attention to Z shifts as these are often larger than xy shifts. Dispersion in your sample will linearly scale the image in Z with refractive index change. The oil/coverslip component should be mostly taken account of by the lens, but the sample component cant be solved in the objective. Any small distance measurement must also include a Z component to be realistic.

Ian


> On Apr 16, 2024, at 5:22 PM, Emmanuel Levy <[log in to unmask]> wrote:
> 
> *****
> To join or leave the confocal microscopy listserv or to change your email address, go to:
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> *****
> 
> Dear Zdenek and Cătălin,
> 
> Thank you for your replies! Indeed, the "distance" of the camera relative
> to the camera-port lens sounds like an intuitive parameter to adjust.
> 
> So the question is, have you adjusted that distance before and achieved a
> 1-2 pixel zoom in/out in this manner? I assume that there should also be an
> optimal distance for proper focus
> 
> The picture I attached was imaged with beads. We do a lot of
> co-localization experiments, so it would be good if we managed to tune the
> hardware flawlessly.
> 
> Thank you for sharing your experience.
> Best wishes,
> 
> Emmanuel
> 
> 
> On Mon, 15 Apr 2024 at 17:31, Cătălin Pavel <[log in to unmask]> wrote:
> 
>> *****
>> To join or leave the confocal microscopy listserv or to change your email
>> address, go to:
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>> *****
>> 
>> Hi Emmanuel,
>> It looks that one of the camera is closer to the microscope than the
>> other. Try to check that all the connections are flush and tight.
>> 
>> Catalin
>> 
>>> On Apr 15, 2024, at 09:30, Emmanuel Levy <[log in to unmask]>
>> wrote:
>>> 
>>> *****
>>> To join or leave the confocal microscopy listserv or to change your
>> email address, go to:
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>>> 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
>>