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Dear Emmanuel,

We wrote a paper about this phenomenon, and how it can be used to align microscopes. It's not quite published yet, but there's a preprint here: https://arxiv.org/abs/2307.04940

In short, for an infinite conjugate system, if the cameras have different optical path lengths from their sensor plane to the sample, then incorrect positioning of the sample with respect to the working distance of the objective will lead to incorrect magnifications. We provide software in the paper to model this and correct the alignment of the system. The procedure already described in this thread sounds like the fastest way to correct your current problem, but hopefully the paper will give some more insight and tools for the future!

All the best

Brian


Brian Patton (He/Him)
Senior Lecturer
Department of Physics, University of Strathclyde,
107 Rottenrow East,
Glasgow, G4 0NG,
Scotland
Phone: +44 (0) 141 548 3474

________________________________
From: Confocal Microscopy List <[log in to unmask]> on behalf of Emmanuel Levy <[log in to unmask]>
Sent: 16 April 2024 22:47
To: [log in to unmask] <[log in to unmask]>
Subject: Re: Yokogawa W1 with two identical cameras : one image is larger than the other

CAUTION: This email originated outside the University. Check before clicking links or attachments.

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Dear Gary,

I didn't think of that; it is much simpler than using beads, thank you for
this great tip!

In terms of the "zoom" effect, do you think there is room for improvement?
If so, is the camera port the only element that could/should be tuned?

Thank you!

Best wishes,

Emmanuel



On Tue, 16 Apr 2024 at 23:31, Gary Laevsky <[log in to unmask]> wrote:

> *****
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> *****
>
> Hi Emmanuel,
>
> If you stop the disc, while imaging, you will see the pinholes.
>
> Adjust the camera, sharpest image/intensity while looking at this image.
>
> Hope this helps.
>
>
> Best,
>
> Gary
>
> > On Apr 16, 2024, at 5:23 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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> > Post images on https://eur02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=05%7C02%7Cbrian.patton%40STRATH.AC.UK%7C9237bfea29274936371008dc5e5ef20d%7C631e0763153347eba5cd0457bee5944e%7C0%7C0%7C638489009491737811%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C60000%7C%7C%7C&sdata=KosRr05km%2Fih6rgxCq3PdVtcGBVGY6BlJVhtpLfDf2U%3D&reserved=0<http://www.imgur.com/> and include the link in your
> posting.
> > *****
> >
> > 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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> >> Post images on https://eur02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=05%7C02%7Cbrian.patton%40STRATH.AC.UK%7C9237bfea29274936371008dc5e5ef20d%7C631e0763153347eba5cd0457bee5944e%7C0%7C0%7C638489009491745758%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C60000%7C%7C%7C&sdata=v4E2iCownLcOhY9s2ecZmy8mo2rAc9YXB1RrHMUK%2Fxc%3D&reserved=0<http://www.imgur.com/> and include the link in your
> posting.
> >> *****
> >>
> >> 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 https://eur02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=05%7C02%7Cbrian.patton%40STRATH.AC.UK%7C9237bfea29274936371008dc5e5ef20d%7C631e0763153347eba5cd0457bee5944e%7C0%7C0%7C638489009491754255%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C60000%7C%7C%7C&sdata=%2BoRb758j36E%2B%2BdVqdlvARlV6jD3EPiavcSgLprTJldc%3D&reserved=0<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://eur02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fdrive.google.com%2Ffile%2Fd%2F1TWIxwiZF6uf3FspyA96AX6r092PB_102%2Fview%3Fusp%3Dsharing&data=05%7C02%7Cbrian.patton%40STRATH.AC.UK%7C9237bfea29274936371008dc5e5ef20d%7C631e0763153347eba5cd0457bee5944e%7C0%7C0%7C638489009491758319%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C60000%7C%7C%7C&sdata=bzux6IydL2S0FmrV33UpLJCMT0GmAbbE0Ye%2F158Xn2Q%3D&reserved=0<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
> >>
>