***** 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 Emmanuel, The description of your issue (/"the GFP channel image is about 1 to 2 pixels larger than the RFP image in every corner"/) suggests that it origins from lateral chromatic aberration, which can be seen as a dependence of the magnification of your system with wavelength (emphasized in the corners), rather than from misalignment between your two cameras. Either way, I agree with Ian: tuning the hardware perfectly is impossible. However, improvements can be obtained via software image processing. The correction of co-registration inaccuracies, either between two cameras or two colors, can be achieved using suitable quality control solutions. Argolight provides such solutions. In particular, a fluorescence pattern called "field of rings" allows to measure the "lateral co-registration inaccuracy" of fluorescence microscopes within the entire field of view and provides linear transformation parameters to correct for it. Protocols and guidelines can be found in the related documentation: https://argolight.notion.site/Lateral-co-registration-accuracy-f20381d9231144d7b8e07827f3ac1de0 Hope this helps, Best regards, Arnaud *Arnaud ROYON, Ph.D.* Argolight Cité de la Photonique, Bat. Elnath 11 avenue de Canteranne 33600 Pessac, FRANCE Email: [log in to unmask] Tel: (+33) 5 64 31 08 50 Web site: www.argolight.com <http://www.argolight.com> Le 18/04/2024 à 15:44, Ian Dobbie a écrit : > ***** > 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 onhttp://www.imgur.com and include the link in your posting. > ***** > > 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: >> https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1 >> Post images onhttp://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: >>> https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1 >>> Post images onhttp://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:https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1 >>>> Post images onhttp://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