***** 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. ***** I've also observed the measured PSF is larger in the y direction than the x direction with our Nikon A1 confocal. I thought this was because confocal scans the image line by line in the x direction, which might cause a little bit shorter pixel-pixel distance in the x direction than in the y direction. This will lead to worse resolution in the y direction than the x direction. But I haven't validated this in some way. Best, Jianjiang Jianjiang Hu | PhD Department of Biosciences and Nutrition | Karolinska Institutet Blickagången 16 | SE-141 52 Huddinge +46 8 524 811 22 (office) [log in to unmask] | ki.se Karolinska Institutet – a medical university ________________________________ From: Confocal Microscopy List <[log in to unmask]> on behalf of Mark Cannell <[log in to unmask]> Sent: Tuesday, 6 February 2024 09:12 To: [log in to unmask] <[log in to unmask]> Subject: Re: Asymmetric PSF on LSM980 Confocal ***** To join or leave the confocal microscopy listserv or to change your email address, go to: https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FSUBED1%3Dconfocalmicroscopy%26A%3D1&data=05%7C02%7Cjianjiang.hu%40KI.SE%7C798fcbd55b99477043e808dc26eb6f49%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C638428039967159325%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C80000%7C%7C%7C&sdata=sp%2BMivvBmzm6CMEEofsCOd59bJqIe2Y6Q7kKYrNAhhc%3D&reserved=0<https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1> Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=05%7C02%7Cjianjiang.hu%40KI.SE%7C798fcbd55b99477043e808dc26eb6f49%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C638428039967167796%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C80000%7C%7C%7C&sdata=Fz%2BtxiPoihay3R1eSR31az0Pw%2Bb%2FBvQ3RssMXVr%2FfVM%3D&reserved=0<http://www.imgur.com/> and include the link in your posting. ***** How big is the psf -could it be the effect of polarization of the input beam? Mark B. Cannell. Ph.D. FRSNZ FISHR Department of Physiology, Pharmacology & Neuroscience School of Medical Sciences University Walk Bristol BS8 1TD [log in to unmask] On 06/02/2024, 02:57, "Confocal Microscopy List on behalf of Cammer, Michael" <[log in to unmask] on behalf of [log in to unmask]> wrote: ***** To join or leave the confocal microscopy listserv or to change your email address, go to: https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FSUBED1%3Dconfocalmicroscopy%26A%3D1&data=05%7C02%7Cjianjiang.hu%40KI.SE%7C798fcbd55b99477043e808dc26eb6f49%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C638428039967173428%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C80000%7C%7C%7C&sdata=5atiSXpbL%2FzSJ6DCRQ%2BoNMk%2BVGAAo75so83vPXv8EZE%3D&reserved=0<https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1> Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=05%7C02%7Cjianjiang.hu%40KI.SE%7C798fcbd55b99477043e808dc26eb6f49%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C638428039967177937%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C80000%7C%7C%7C&sdata=Tjee6Em4PDeb9bQcoYHAfG6HWIiv45NWqvZhJoZVBAU%3D&reserved=0<http://www.imgur.com/> and include the link in your posting. ***** We had astigmatism in our Zeiss 710 which I found back in 2011 or so when characterizing the system with 100 nm beads. It wasn't the objective lens as rotating it did not change the orientation of the astigmatism. I tried to get Zeiss to address this and they were not helpful. I pushed and pushed and then gave up because because it was the type of thing that bothered me but none of the biologists noticed, even when I pointed out slight skewing in some of their Z series. Seems like ancient history now, but history does repeat; curious if you can get more traction now. As you are doing FCS, this is definitely something they should address. Regards- Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, New York, NY 10016 [log in to unmask]<mailto:[log in to unmask]> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fnyulmc.org%2Fmicros&data=05%7C02%7Cjianjiang.hu%40KI.SE%7C798fcbd55b99477043e808dc26eb6f49%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C638428039967181868%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C80000%7C%7C%7C&sdata=4xHrczoZP5C95fpgcUx%2BfSuUTKsb5CPa2lm0hWC4qtk%3D&reserved=0<http://nyulmc.org/micros> https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2F&data=05%7C02%7Cjianjiang.hu%40KI.SE%7C798fcbd55b99477043e808dc26eb6f49%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C638428039967185659%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C80000%7C%7C%7C&sdata=7YeIFghqsEUrWQ3ddIuBHIeZQI605Kwz5VN%2B0pgxtoc%3D&reserved=0<http://microscopynotes.com/> Cell voice (no text) 1-914-309-3270 Office 1-646-501-0567 ________________________________ From: Confocal Microscopy List <[log in to unmask]> on behalf of Claire Brown, Dr. <[log in to unmask]> Sent: Monday, February 5, 2024 8:38:21 PM To: [log in to unmask] Subject: Asymmetric PSF on LSM980 Confocal [EXTERNAL] ***** To join or leave the confocal microscopy listserv or to change your email address, go to: https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FSUBED1%3Dconfocalmicroscopy%26A%3D1&data=05%7C02%7Cjianjiang.hu%40KI.SE%7C798fcbd55b99477043e808dc26eb6f49%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C638428039967189286%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C80000%7C%7C%7C&sdata=qFxmT%2BTMIOR%2F8mkGmiUju0R%2FS4CPwySnQqoNGpmYdtM%3D&reserved=0<https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1> Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com%2F&data=05%7C02%7Cjianjiang.hu%40KI.SE%7C798fcbd55b99477043e808dc26eb6f49%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C638428039967193166%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C80000%7C%7C%7C&sdata=O38GpUlT1cd6eDfL9SQD%2BmZLy1Tb0AWkFjqA5H4dlfk%3D&reserved=0<http://www.imgur.com/> and include the link in your posting. ***** Hello All, I'm posting this for a colleague who is not on the list and is trying to do FCS on a Zeiss LSM980. "When constructing the PSF to measure its dimensions in the Zeiss LSM 980 using 100 or 20 nm diameter fluorescent beads, there is large astigmatism in the y dimension compared to the x dimension where y is up to 30% larger than x. The same problem is witnessed for the Airyscan PSF on the same microscope. Has anyone else witnessed this? Is that normal? Can that be fixed without impacting the sensitivity or the resolution limits? And how does that impact FCS measurements where the 3D fitting diffusion model assumes x, y symmetry. " All the best, Claire När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>. Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.
