Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This is something I've wanted for a long time, the ability to take large fields of view (2k X 2k pixels) at high resolution instead of having to do tiling. Also, with the new cameras that have oodles of small pixels... I'm considering replacing our 63X with this new 40X. Any experience with this, other than the battle of having to explain to other scope users why this is not really lower magnification? Regards, Michael =========================================================================== Michael Cammer, Microscopy Core & Dustin Lab , Skirball Institute, NYU Langone Medical Center Cell: 914-309-3270 Lab: 212-263-3208 http://ocs.med.nyu.edu/microscopy & http://www.med.nyu.edu/skirball-lab/dustinlab/ ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
 
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Ryan Schreiner |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Michael, The 40X/1.4 is a good objective for dim samples on a widefield system, I have used it quite a lot. The 40X/1.4 has: 24.0 brightness 161nm sampling on a 6.5µm pixel (ie Flash 4.0 CMOS) 333x333µm field of view on the Flash4.0 The 63X/1.4 has: 9.7 brightness 102nm sampling on a 6.5µm pixel (ie Flash 4.0 CMOS) 211x211µm field of view on the Flash4.0 If you are going to deconvolve the image you will find you are undersampling quite a bit with the 40X. When you look through the eyepiece you will really appreciate the 2.5X improvement in brightness. Another note is that the 1.4NA 40X and 63X have ~80% transmission from 450-750nm and the new 100X/1.4 from Zeiss has ~90%. Also, if you are using a spinning disk you will see your axial resolution suffer with the 40X at least with the CSU-X1 pinholes. -Ryan Schreiner Weill Cornell Medical College On Fri, Jul 11, 2014 at 9:53 AM, Cammer, Michael <[hidden email] > wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This > is something I've wanted for a long time, the ability to take large fields > of view (2k X 2k pixels) at high resolution instead of having to do tiling. > Also, with the new cameras that have oodles of small pixels... > > I'm considering replacing our 63X with this new 40X. Any experience with > this, other than the battle of having to explain to other scope users why > this is not really lower magnification? > > Regards, > > Michael > > =========================================================================== > Michael Cammer, Microscopy Core & Dustin Lab , Skirball Institute, NYU > Langone Medical Center > Cell: 914-309-3270 Lab: 212-263-3208 > http://ocs.med.nyu.edu/microscopy & > http://www.med.nyu.edu/skirball-lab/dustinlab/ > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the > intended recipient(s) and may contain information that is proprietary, > confidential, and exempt from disclosure under applicable law. Any > unauthorized review, use, disclosure, or distribution is prohibited. If you > have received this email in error please notify the sender by return email > and delete the original message. Please note, the recipient should check > this email and any attachments for the presence of viruses. The > organization accepts no liability for any damage caused by any virus > transmitted by this email. > ================================= > |
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simon walker (BI)-2 |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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In reply to this post by mcammer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hello, We have this objective on our new 780 and initial impressions are very good. I haven't quantitatively compared the performance with the 63x 1.4, but there's no obvious drop in performance for high resolution imaging and the signal is excellent. I'm trying to push confocal users on to this as their default lens, which can be difficult ...'but I need higher magnification to see my small cells!' etc. The only downside I've noticed so far is that the lens barrel and top surface area are considerably larger than the 63x 1.4, so imaging samples mounted at the edge of a slide can be problematic (one user has already scratched the top edge of the lens by impacting on the stage insert whilst trying to focus on a coverslip mounted at the edge of a slide). As we have both lenses I'm happy to try any tests people would like to see to compare performance. Simon > On 11 Jul 2014, at 16:47, "Cammer, Michael" <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This is something I've wanted for a long time, the ability to take large fields of view (2k X 2k pixels) at high resolution instead of having to do tiling. Also, with the new cameras that have oodles of small pixels... > > I'm considering replacing our 63X with this new 40X. Any experience with this, other than the battle of having to explain to other scope users why this is not really lower magnification? > > Regards, > > Michael > > =========================================================================== > Michael Cammer, Microscopy Core & Dustin Lab , Skirball Institute, NYU Langone Medical Center > Cell: 914-309-3270 Lab: 212-263-3208 > http://ocs.med.nyu.edu/microscopy & http://www.med.nyu.edu/skirball-lab/dustinlab/ > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. > ================================= The information transmitted in this email is directed only to the addressee. If you received this in error, please contact the sender and delete this email from your system. The contents of this e-mail are the views of the sender and do not necessarily represent the views of the Babraham Institute. Full conditions at: www.babraham.ac.uk<http://www.babraham.ac.uk/terms> |
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George McNamara |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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In reply to this post by mcammer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, why 2kx2k? If the 40x lens has a 250x250 um field of view, this would be undersampling, pixel size 125x125 nm. If even larger field of view, undersampling even more. I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step 200 nm, maybe closer), George On 7/11/2014 8:53 AM, Cammer, Michael wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This is something I've wanted for a long time, the ability to take large fields of view (2k X 2k pixels) at high resolution instead of having to do tiling. Also, with the new cameras that have oodles of small pixels... > > I'm considering replacing our 63X with this new 40X. Any experience with this, other than the battle of having to explain to other scope users why this is not really lower magnification? > > Regards, > > Michael > > =========================================================================== > Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU Langone Medical Center > Cell: 914-309-3270 Lab: 212-263-3208 > http://ocs.med.nyu.edu/microscopy& http://www.med.nyu.edu/skirball-lab/dustinlab/ > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. > ================================= > > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42 |
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Miroslav Varecha |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hello, I would like to react to George. In my humble opinion, best lateral optical resolution for confocal microscope is around 130 nm, so going to resolution 60x60 nm is overshoot and it is just wasting your drive space as you are not collecting any new real information. Resolutions of 50x50nm and such are area of superresolution microscopy. Our confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k. High quality CCDs have even less, but sCMOS can get higher than 2k x 2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you that it is the most used objective (usually we observe stem cells) even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger field of view of 40x. You still get great detail and many cells in one image. But if you want to observe really up close organels and such I would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible objective for vast majority of observations. Miroslav 2014-07-14 1:31 GMT+02:00 George McNamara <[hidden email]>: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Michael, > why 2kx2k? > If the 40x lens has a 250x250 um field of view, this would be undersampling, > pixel size 125x125 nm. If even larger field of view, undersampling even > more. > I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step 200 > nm, maybe closer), > George > > On 7/11/2014 8:53 AM, Cammer, Michael wrote: >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This >> is something I've wanted for a long time, the ability to take large fields >> of view (2k X 2k pixels) at high resolution instead of having to do tiling. >> Also, with the new cameras that have oodles of small pixels... >> >> I'm considering replacing our 63X with this new 40X. Any experience with >> this, other than the battle of having to explain to other scope users why >> this is not really lower magnification? >> >> Regards, >> >> Michael >> >> >> =========================================================================== >> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU >> Langone Medical Center >> Cell: 914-309-3270 Lab: 212-263-3208 >> http://ocs.med.nyu.edu/microscopy& >> http://www.med.nyu.edu/skirball-lab/dustinlab/ >> >> ------------------------------------------------------------ >> This email message, including any attachments, is for the sole use of the >> intended recipient(s) and may contain information that is proprietary, >> confidential, and exempt from disclosure under applicable law. Any >> unauthorized review, use, disclosure, or distribution is prohibited. If you >> have received this email in error please notify the sender by return email >> and delete the original message. Please note, the recipient should check >> this email and any attachments for the presence of viruses. The organization >> accepts no liability for any damage caused by any virus transmitted by this >> email. >> ================================= >> >> > > > > -- > > > > George McNamara, Ph.D. > Single Cells Analyst > L.J.N. Cooper Lab > University of Texas M.D. Anderson Cancer Center > Houston, TX 77054 > Tattletales http://works.bepress.com/gmcnamara/42 |
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Mark Cannell-2 |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Re: “ But if you want to observe really up close organels and such I would go to 63x NA1,4” . The beauty of the scanning confocal is that you can use zoom to go from 40x -> 63x or whatever. You do not need an objective of the same NA but different magnification to achieve this. Cheers, Mark On 14/07/2014, at 8:54 am, Miroslav Varecha <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hello, > I would like to react to George. In my humble opinion, best lateral > optical resolution for confocal microscope is around 130 nm, so going > to resolution 60x60 nm is overshoot and it is just wasting your drive > space as you are not collecting any new real information. Resolutions > of 50x50nm and such are area of superresolution microscopy. Our > confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k. > High quality CCDs have even less, but sCMOS can get higher than 2k x > 2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you > that it is the most used objective (usually we observe stem cells) > even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger > field of view of 40x. You still get great detail and many cells in one > image. But if you want to observe really up close organels and such I > would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible > objective for vast majority of observations. > Miroslav > > > 2014-07-14 1:31 GMT+02:00 George McNamara <[hidden email]>: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Hi Michael, >> why 2kx2k? >> If the 40x lens has a 250x250 um field of view, this would be undersampling, >> pixel size 125x125 nm. If even larger field of view, undersampling even >> more. >> I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step 200 >> nm, maybe closer), >> George >> >> On 7/11/2014 8:53 AM, Cammer, Michael wrote: >>> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> Post images on http://www.imgur.com and include the link in your posting. >>> ***** >>> >>> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This >>> is something I've wanted for a long time, the ability to take large fields >>> of view (2k X 2k pixels) at high resolution instead of having to do tiling. >>> Also, with the new cameras that have oodles of small pixels... >>> >>> I'm considering replacing our 63X with this new 40X. Any experience with >>> this, other than the battle of having to explain to other scope users why >>> this is not really lower magnification? >>> >>> Regards, >>> >>> Michael >>> >>> >>> =========================================================================== >>> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU >>> Langone Medical Center >>> Cell: 914-309-3270 Lab: 212-263-3208 >>> http://ocs.med.nyu.edu/microscopy& >>> http://www.med.nyu.edu/skirball-lab/dustinlab/ >>> >>> ------------------------------------------------------------ >>> This email message, including any attachments, is for the sole use of the >>> intended recipient(s) and may contain information that is proprietary, >>> confidential, and exempt from disclosure under applicable law. Any >>> unauthorized review, use, disclosure, or distribution is prohibited. If you >>> have received this email in error please notify the sender by return email >>> and delete the original message. Please note, the recipient should check >>> this email and any attachments for the presence of viruses. The organization >>> accepts no liability for any damage caused by any virus transmitted by this >>> email. >>> ================================= >>> >>> >> >> >> >> -- >> >> >> >> George McNamara, Ph.D. >> Single Cells Analyst >> L.J.N. Cooper Lab >> University of Texas M.D. Anderson Cancer Center >> Houston, TX 77054 >> Tattletales http://works.bepress.com/gmcnamara/42 Mark B. Cannell Ph.D. FRSNZ Professor of Cardiac Cell Biology School of Physiology & Pharmacology Medical Sciences Building University of Bristol Bristol BS8 1TD UK [hidden email] |
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Miroslav Varecha |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** True, but I was speaking about my LSM 700 and we have only 63x at NA1,4. We have only NA 1,3 for 40x. It would be great to have NA 1,4 as 40x is most used lens for us. Miroslav ------------------------------------------------ Miroslav Varecha, Ph.D. Department of Biology, Faculty of Medicine Masaryk University, Brno, Czech Republic 2014-07-14 11:13 GMT+02:00 Mark Cannell <[hidden email]>: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Re: “ But if you want to observe really up close organels and such I would go to 63x NA1,4” . > > The beauty of the scanning confocal is that you can use zoom to go from 40x -> 63x or whatever. You do not need an objective of the same NA but different magnification to achieve this. > > Cheers, > Mark > > > On 14/07/2014, at 8:54 am, Miroslav Varecha <[hidden email]> wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Hello, >> I would like to react to George. In my humble opinion, best lateral >> optical resolution for confocal microscope is around 130 nm, so going >> to resolution 60x60 nm is overshoot and it is just wasting your drive >> space as you are not collecting any new real information. Resolutions >> of 50x50nm and such are area of superresolution microscopy. Our >> confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k. >> High quality CCDs have even less, but sCMOS can get higher than 2k x >> 2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you >> that it is the most used objective (usually we observe stem cells) >> even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger >> field of view of 40x. You still get great detail and many cells in one >> image. But if you want to observe really up close organels and such I >> would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible >> objective for vast majority of observations. >> Miroslav >> >> >> 2014-07-14 1:31 GMT+02:00 George McNamara <[hidden email]>: >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> Post images on http://www.imgur.com and include the link in your posting. >>> ***** >>> >>> Hi Michael, >>> why 2kx2k? >>> If the 40x lens has a 250x250 um field of view, this would be undersampling, >>> pixel size 125x125 nm. If even larger field of view, undersampling even >>> more. >>> I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step 200 >>> nm, maybe closer), >>> George >>> >>> On 7/11/2014 8:53 AM, Cammer, Michael wrote: >>>> >>>> ***** >>>> To join, leave or search the confocal microscopy listserv, go to: >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>>> Post images on http://www.imgur.com and include the link in your posting. >>>> ***** >>>> >>>> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This >>>> is something I've wanted for a long time, the ability to take large fields >>>> of view (2k X 2k pixels) at high resolution instead of having to do tiling. >>>> Also, with the new cameras that have oodles of small pixels... >>>> >>>> I'm considering replacing our 63X with this new 40X. Any experience with >>>> this, other than the battle of having to explain to other scope users why >>>> this is not really lower magnification? >>>> >>>> Regards, >>>> >>>> Michael >>>> >>>> >>>> =========================================================================== >>>> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU >>>> Langone Medical Center >>>> Cell: 914-309-3270 Lab: 212-263-3208 >>>> http://ocs.med.nyu.edu/microscopy& >>>> http://www.med.nyu.edu/skirball-lab/dustinlab/ >>>> >>>> ------------------------------------------------------------ >>>> This email message, including any attachments, is for the sole use of the >>>> intended recipient(s) and may contain information that is proprietary, >>>> confidential, and exempt from disclosure under applicable law. Any >>>> unauthorized review, use, disclosure, or distribution is prohibited. If you >>>> have received this email in error please notify the sender by return email >>>> and delete the original message. Please note, the recipient should check >>>> this email and any attachments for the presence of viruses. The organization >>>> accepts no liability for any damage caused by any virus transmitted by this >>>> email. >>>> ================================= >>>> >>>> >>> >>> >>> >>> -- >>> >>> >>> >>> George McNamara, Ph.D. >>> Single Cells Analyst >>> L.J.N. Cooper Lab >>> University of Texas M.D. Anderson Cancer Center >>> Houston, TX 77054 >>> Tattletales http://works.bepress.com/gmcnamara/42 > > Mark B. Cannell Ph.D. FRSNZ > Professor of Cardiac Cell Biology > School of Physiology & Pharmacology > Medical Sciences Building > University of Bristol > Bristol > BS8 1TD UK > > [hidden email] |
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Feinstein, Timothy |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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In reply to this post by Miroslav Varecha
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Miroslav, You are correct that even in the best case a traditional optical microscope can only separate point sources > 100 nm apart. However the theoretical resolution limit is far from the same thing as the ideal resolution for sampling. As George mentioned if you want to get the most out of an optical image then you should deconvolve out the point spread function. In that case the optimal sampling for proper deconvolution depends on seeing enough of the PSF from a given point source to guess at its center, in which case you need to sample at roughly twice as fine as the theoretical limit of a given NA/wavelength/refractive index. This sampling theorem was worked out by a fellow named Harry Nuqiust during his work with radio signals. In practice I recommend that pretty much everyone who works near the resolution limit of their system take the time to deconvolve their data. Cheers, TF Timothy Feinstein, Ph.D. | Confocal Manager 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503 Phone: 616-234-5819 | Email: [hidden email] On 7/14/14, 3:54 AM, "Miroslav Varecha" <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://scanmail.trustwave.com/?c=129&d=yZPD0xJAtv-GdZ33VVkWaDRlPiHp_igFiBn >BHX8_EA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic >roscopy >Post images on >http://scanmail.trustwave.com/?c=129&d=yZPD0xJAtv-GdZ33VVkWaDRlPiHp_igFiEz >AT3VrQA&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your >posting. >***** > >Hello, >I would like to react to George. In my humble opinion, best lateral >optical resolution for confocal microscope is around 130 nm, so going >to resolution 60x60 nm is overshoot and it is just wasting your drive >space as you are not collecting any new real information. Resolutions >of 50x50nm and such are area of superresolution microscopy. Our >confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k. >High quality CCDs have even less, but sCMOS can get higher than 2k x >2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you >that it is the most used objective (usually we observe stem cells) >even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger >field of view of 40x. You still get great detail and many cells in one >image. But if you want to observe really up close organels and such I >would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible >objective for vast majority of observations. >Miroslav > > >2014-07-14 1:31 GMT+02:00 George McNamara <[hidden email]>: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> >>http://scanmail.trustwave.com/?c=129&d=yZPD0xJAtv-GdZ33VVkWaDRlPiHp_igFiB >>nBHX8_EA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm >>icroscopy >> Post images on >>http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4DfB >>tS15fOrw&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your >>posting. >> ***** >> >> Hi Michael, >> why 2kx2k? >> If the 40x lens has a 250x250 um field of view, this would be >>undersampling, >> pixel size 125x125 nm. If even larger field of view, undersampling even >> more. >> I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step >>200 >> nm, maybe closer), >> George >> >> On 7/11/2014 8:53 AM, Cammer, Michael wrote: >>> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> >>>http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df >>>E5ThZ2a_w&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfoca >>>lmicroscopy >>> Post images on >>>http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df >>>BtS15fOrw&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your >>>posting. >>> ***** >>> >>> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? >>>This >>> is something I've wanted for a long time, the ability to take large >>>fields >>> of view (2k X 2k pixels) at high resolution instead of having to do >>>tiling. >>> Also, with the new cameras that have oodles of small pixels... >>> >>> I'm considering replacing our 63X with this new 40X. Any experience >>>with >>> this, other than the battle of having to explain to other scope users >>>why >>> this is not really lower magnification? >>> >>> Regards, >>> >>> Michael >>> >>> >>> >>>======================================================================== >>>=== >>> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU >>> Langone Medical Center >>> Cell: 914-309-3270 Lab: 212-263-3208 >>> >>>http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df >>>BsGgZDJrw&u=http%3a%2f%2focs%2emed%2enyu%2eedu%2fmicroscopy%26 >>> >>>http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df >>>EBS0pSW-w&u=http%3a%2f%2fwww%2emed%2enyu%2eedu%2fskirball-lab%2fdustinla >>>b%2f >>> >>> ------------------------------------------------------------ >>> This email message, including any attachments, is for the sole use of >>>the >>> intended recipient(s) and may contain information that is proprietary, >>> confidential, and exempt from disclosure under applicable law. Any >>> unauthorized review, use, disclosure, or distribution is prohibited. >>>If you >>> have received this email in error please notify the sender by return >>> and delete the original message. Please note, the recipient should >>>check >>> this email and any attachments for the presence of viruses. The >>>organization >>> accepts no liability for any damage caused by any virus transmitted by >>>this >>> email. >>> ================================= >>> >>> >> >> >> >> -- >> >> >> >> George McNamara, Ph.D. >> Single Cells Analyst >> L.J.N. Cooper Lab >> University of Texas M.D. Anderson Cancer Center >> Houston, TX 77054 >> Tattletales >>http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4DfE >>FXhMSb-w&u=http%3a%2f%2fworks%2ebepress%2ecom%2fgmcnamara%2f42 |
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Miroslav Varecha |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Oh, thanks for explanation. I work with scanning confocal only for short time. I was using spinning disc microscope with EMCCD before and resolution was pretty much set by camera. It is very interesting to use deconvolution to reach even higher resolution with confocal. Unfortunately noone here wanted very high resolutions for their data so far. I will do some test images then ☺ I apologize. This approach is new to me. Miroslav Dne 14. 7. 2014 16:28 "Feinstein, Timothy" <[hidden email]> napsal(a): > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Miroslav, > > You are correct that even in the best case a traditional optical > microscope can only separate point sources > 100 nm apart. However the > theoretical resolution limit is far from the same thing as the ideal > resolution for sampling. As George mentioned if you want to get the most > out of an optical image then you should deconvolve out the point spread > function. In that case the optimal sampling for proper deconvolution > depends on seeing enough of the PSF from a given point source to guess at > its center, in which case you need to sample at roughly twice as fine as > the theoretical limit of a given NA/wavelength/refractive index. This > sampling theorem was worked out by a fellow named Harry Nuqiust during his > work with radio signals. In practice I recommend that pretty much > everyone who works near the resolution limit of their system take the time > to deconvolve their data. > > Cheers, > > > TF > > Timothy Feinstein, Ph.D. | Confocal Manager > 333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503 > Phone: 616-234-5819 | Email: [hidden email] > > > > > > > > On 7/14/14, 3:54 AM, "Miroslav Varecha" <[hidden email]> wrote: > > >***** > >To join, leave or search the confocal microscopy listserv, go to: > > > http://scanmail.trustwave.com/?c=129&d=yZPD0xJAtv-GdZ33VVkWaDRlPiHp_igFiBn > >BHX8_EA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic > >roscopy > >Post images on > > > http://scanmail.trustwave.com/?c=129&d=yZPD0xJAtv-GdZ33VVkWaDRlPiHp_igFiEz > >AT3VrQA&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your > >posting. > >***** > > > >Hello, > >I would like to react to George. In my humble opinion, best lateral > >optical resolution for confocal microscope is around 130 nm, so going > >to resolution 60x60 nm is overshoot and it is just wasting your drive > >space as you are not collecting any new real information. Resolutions > >of 50x50nm and such are area of superresolution microscopy. Our > >confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k. > >High quality CCDs have even less, but sCMOS can get higher than 2k x > >2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you > >that it is the most used objective (usually we observe stem cells) > >even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger > >field of view of 40x. You still get great detail and many cells in one > >image. But if you want to observe really up close organels and such I > >would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible > >objective for vast majority of observations. > >Miroslav > > > > > >2014-07-14 1:31 GMT+02:00 George McNamara <[hidden email]>: > >> ***** > >> To join, leave or search the confocal microscopy listserv, go to: > >> > >> > http://scanmail.trustwave.com/?c=129&d=yZPD0xJAtv-GdZ33VVkWaDRlPiHp_igFiB > >>nBHX8_EA&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalm > >>icroscopy > >> Post images on > >> > http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4DfB > >>tS15fOrw&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your > >>posting. > >> ***** > >> > >> Hi Michael, > >> why 2kx2k? > >> If the 40x lens has a 250x250 um field of view, this would be > >>undersampling, > >> pixel size 125x125 nm. If even larger field of view, undersampling even > >> more. > >> I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step > >>200 > >> nm, maybe closer), > >> George > >> > >> On 7/11/2014 8:53 AM, Cammer, Michael wrote: > >>> > >>> ***** > >>> To join, leave or search the confocal microscopy listserv, go to: > >>> > >>> > http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df > >>>E5ThZ2a_w&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfoca > >>>lmicroscopy > >>> Post images on > >>> > http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df > >>>BtS15fOrw&u=http%3a%2f%2fwww%2eimgur%2ecom and include the link in your > >>>posting. > >>> ***** > >>> > >>> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? > >>>This > >>> is something I've wanted for a long time, the ability to take large > >>>fields > >>> of view (2k X 2k pixels) at high resolution instead of having to do > >>>tiling. > >>> Also, with the new cameras that have oodles of small pixels... > >>> > >>> I'm considering replacing our 63X with this new 40X. Any experience > >>>with > >>> this, other than the battle of having to explain to other scope users > >>>why > >>> this is not really lower magnification? > >>> > >>> Regards, > >>> > >>> Michael > >>> > >>> > >>> > >>>======================================================================== > >>>=== > >>> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU > >>> Langone Medical Center > >>> Cell: 914-309-3270 Lab: 212-263-3208 > >>> > >>> > http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df > >>>BsGgZDJrw&u=http%3a%2f%2focs%2emed%2enyu%2eedu%2fmicroscopy%26 > >>> > >>> > http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4Df > >>>EBS0pSW-w&u=http%3a%2f%2fwww%2emed%2enyu%2eedu%2fskirball-lab%2fdustinla > >>>b%2f > >>> > >>> ------------------------------------------------------------ > >>> This email message, including any attachments, is for the sole use of > >>>the > >>> intended recipient(s) and may contain information that is proprietary, > >>> confidential, and exempt from disclosure under applicable law. Any > >>> unauthorized review, use, disclosure, or distribution is prohibited. > >>>If you > >>> have received this email in error please notify the sender by return > >>> and delete the original message. Please note, the recipient should > >>>check > >>> this email and any attachments for the presence of viruses. The > >>>organization > >>> accepts no liability for any damage caused by any virus transmitted by > >>>this > >>> email. > >>> ================================= > >>> > >>> > >> > >> > >> > >> -- > >> > >> > >> > >> George McNamara, Ph.D. > >> Single Cells Analyst > >> L.J.N. Cooper Lab > >> University of Texas M.D. Anderson Cancer Center > >> Houston, TX 77054 > >> Tattletales > >> > http://scanmail.trustwave.com/?c=129&d=ypPD068thzYagx2Iq28ffbXdwsGOCB4DfE > >>FXhMSb-w&u=http%3a%2f%2fworks%2ebepress%2ecom%2fgmcnamara%2f42 > |
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In reply to this post by mcammer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Michael, because I did not see it mentioned in the discussion so far: To achieve the high aperture and resolution, you have to make sure - refractive properties of all media in the light path including immersion and embedding allow. - overfill the back aperture of the 40x lens which is considerably wider than that of a 63x or 100x, so collimation must fit. I propose to compare the system PSF with the different lenses, before you approach your facilitiy users. A further potential drawback: Looking down the eyepiece, samples will look much brighter than with higher-x lenses, but using suitable sampling, you will easily burn the sample and saturate detectors, trying to achieve the brightness seen down the eyepiece. Cheers Jens http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/ Skype Jens.Rietdorf On Fri, Jul 11, 2014 at 10:53 AM, Cammer, Michael < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This > is something I've wanted for a long time, the ability to take large fields > of view (2k X 2k pixels) at high resolution instead of having to do tiling. > Also, with the new cameras that have oodles of small pixels... > > I'm considering replacing our 63X with this new 40X. Any experience with > this, other than the battle of having to explain to other scope users why > this is not really lower magnification? > > Regards, > > Michael > > =========================================================================== > Michael Cammer, Microscopy Core & Dustin Lab , Skirball Institute, NYU > Langone Medical Center > Cell: 914-309-3270 Lab: 212-263-3208 > http://ocs.med.nyu.edu/microscopy & > http://www.med.nyu.edu/skirball-lab/dustinlab/ > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the > intended recipient(s) and may contain information that is proprietary, > confidential, and exempt from disclosure under applicable law. Any > unauthorized review, use, disclosure, or distribution is prohibited. If you > have received this email in error please notify the sender by return email > and delete the original message. Please note, the recipient should check > this email and any attachments for the presence of viruses. The > organization accepts no liability for any damage caused by any virus > transmitted by this email. > ================================= > |
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George McNamara |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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In reply to this post by Miroslav Varecha
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Miroslav, "highest resolution of 2k x 2k." is number of pixels in an image, resolution is set by that and field of view (zoom). With the LSM 700 -- or other point scanning confocals -- you can choose the zoom and number of pixels to get whatever pixel size you want. Data acquired on a confocal microscope can be used to obtain "superresolution", as for the ~10% increase in resolution (ex. from ~214 nm to <200 nm for 500 nm light, Airy disk 1.0) by deconvolving at 50 nm pixel size. A pixel size of 130 nm is not going to give you a resolution improvement (and the optical resolution of visible light confocal is ~200nm, not 130 nm). There are also other ways to process images - confocal or widefield - to get a lot more out of the source data. Two examples: PiMP http://jcs.biologists.org/content/125/9/2257.long ... which I preferred using 30 nm pixel size acquisition on the confocals I managed in Miami (LSM710, SP5). 3B ... http://www.coxphysics.com/3b/ and http://www.superresolved.com/ (later is an online forum hosted by Susan cox and Ed Rosten for all superduperres). SOFI / bSOFI etc (which I've not used) see http://www.ncbi.nlm.nih.gov/pubmed/22711840 for Peter Dedecker et al's entry point. Finally, not required by law (or Guy Cox or Jim Pawley or Alby Diaspro) to set the confocal pinhole at 1.0 Airy units (for one thing, the physical size for that varies with wavelength). With some specimens it is useful to use a smaller pinhole. Zeiss has a nice PDF online on different pinhole settings - if you cannot find it on an internet search, ask your Zeiss confocal rep to find and send it. As for Nyquist - he had a thing for sine waves. anyone imaging sine waves perfectly aligned in X or Y (or rotate scan view to perfectly align) can use the Nyquist sampling value (around 2.3). So, to sample correctly for 2D Nyquist, need to sample to account for the worse case sine wave at 45 degrees. Few biological specimens viewed on a confocal or widefield fluorescence microscope are sine waves (pattern A at http://argolight.com/product-micro/ comes close ... with modest NA objective lens probably close enough, but then that is a calibration slide, not a cell ... muscle fibers can come close).So, stop thinking Nyquist, and start thinking cells. Enjoy, George p.s. a heavily used objective will generally not perform as well as a brand new objective lens. On 7/14/2014 2:54 AM, Miroslav Varecha wrote: > Hello, > I would like to react to George. In my humble opinion, best lateral > optical resolution for confocal microscope is around 130 nm, so going > to resolution 60x60 nm is overshoot and it is just wasting your drive > space as you are not collecting any new real information. Resolutions > of 50x50nm and such are area of superresolution microscopy. Our > confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k. > High quality CCDs have even less, but sCMOS can get higher than 2k x > 2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you > that it is the most used objective (usually we observe stem cells) > even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger > field of view of 40x. You still get great detail and many cells in one > image. But if you want to observe really up close organels and such I > would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible > objective for vast majority of observations. > Miroslav > > > 2014-07-14 1:31 GMT+02:00 George McNamara<[hidden email]>: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Hi Michael, >> why 2kx2k? >> If the 40x lens has a 250x250 um field of view, this would be undersampling, >> pixel size 125x125 nm. If even larger field of view, undersampling even >> more. >> I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step 200 >> nm, maybe closer), >> George >> >> On 7/11/2014 8:53 AM, Cammer, Michael wrote: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> Post images on http://www.imgur.com and include the link in your posting. >>> ***** >>> >>> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? This >>> is something I've wanted for a long time, the ability to take large fields >>> of view (2k X 2k pixels) at high resolution instead of having to do tiling. >>> Also, with the new cameras that have oodles of small pixels... >>> >>> I'm considering replacing our 63X with this new 40X. Any experience with >>> this, other than the battle of having to explain to other scope users why >>> this is not really lower magnification? >>> >>> Regards, >>> >>> Michael >>> >>> >>> =========================================================================== >>> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU >>> Langone Medical Center >>> Cell: 914-309-3270 Lab: 212-263-3208 >>> http://ocs.med.nyu.edu/microscopy& >>> http://www.med.nyu.edu/skirball-lab/dustinlab/ >>> >>> ------------------------------------------------------------ >>> This email message, including any attachments, is for the sole use of the >>> intended recipient(s) and may contain information that is proprietary, >>> confidential, and exempt from disclosure under applicable law. Any >>> unauthorized review, use, disclosure, or distribution is prohibited. If you >>> have received this email in error please notify the sender by return email >>> and delete the original message. Please note, the recipient should check >>> this email and any attachments for the presence of viruses. The organization >>> accepts no liability for any damage caused by any virus transmitted by this >>> email. >>> ================================= >>> >>> >>> >> >> >> -- >> >> >> >> George McNamara, Ph.D. >> Single Cells Analyst >> L.J.N. Cooper Lab >> University of Texas M.D. Anderson Cancer Center >> Houston, TX 77054 >> Tattletales http://works.bepress.com/gmcnamara/42 >> > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42 |
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Lutz Schaefer |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi George, sorry, as non-biologist I have to (at least partially) disagree on your last comment that read: > > Few biological specimens viewed on a confocal or widefield fluorescence > microscope are sine waves (pattern A at > http://argolight.com/product-micro/ comes close ... with modest NA > objective lens probably close enough, but then that is a calibration > slide, not a cell ... muscle fibers can come close).So, stop thinking > Nyquist, and start thinking cells. > According to Fourier synthesis any signal, whether it being originating from the fluorescence of a cell, other material or just as a 1D signal can be represented by sinusoidal functions. For the "survival" of their highest frequency components after discretization it is common to use the Nyquist-Shannon sampling theorem. The factor of two thereby is only the critical period from which on aliasing begins to recede. Sampling at (even) higher than Nyquist rates is always favorable but mostly impractical, because restoring these higher frequencies is subject to a low SNR, set aside the storage needs. Additionally, for 2D or 3D the theorem is separable. In other words an exemplary diagonal, 1D signal inside a 2D image can be represented by the two orthogonal XY sampling directions. Having said that, of course it is always practical to know the shape of the objects you are looking at, even though their emissions or signals are by nature composed of sine waves... Regards Lutz __________________________________ L u t z S c h a e f e r Sen. Scientist Mathematical modeling / Computational microscopy Advanced Imaging Methodology Consultation 16-715 Doon Village Rd. Kitchener, ON, N2P 2A2, Canada Phone/Fax: +1 519 894 8870 Mobile: +1 519 722 8870 Email: [hidden email] Website: http://home.golden.net/~lschafer/ ___________________________________ -----Original Message----- From: George McNamara Sent: Monday, July 14, 2014 9:39 PM To: [hidden email] Subject: Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X? ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Miroslav, "highest resolution of 2k x 2k." is number of pixels in an image, resolution is set by that and field of view (zoom). With the LSM 700 -- or other point scanning confocals -- you can choose the zoom and number of pixels to get whatever pixel size you want. Data acquired on a confocal microscope can be used to obtain "superresolution", as for the ~10% increase in resolution (ex. from ~214 nm to <200 nm for 500 nm light, Airy disk 1.0) by deconvolving at 50 nm pixel size. A pixel size of 130 nm is not going to give you a resolution improvement (and the optical resolution of visible light confocal is ~200nm, not 130 nm). There are also other ways to process images - confocal or widefield - to get a lot more out of the source data. Two examples: PiMP http://jcs.biologists.org/content/125/9/2257.long ... which I preferred using 30 nm pixel size acquisition on the confocals I managed in Miami (LSM710, SP5). 3B ... http://www.coxphysics.com/3b/ and http://www.superresolved.com/ (later is an online forum hosted by Susan cox and Ed Rosten for all superduperres). SOFI / bSOFI etc (which I've not used) see http://www.ncbi.nlm.nih.gov/pubmed/22711840 for Peter Dedecker et al's entry point. Finally, not required by law (or Guy Cox or Jim Pawley or Alby Diaspro) to set the confocal pinhole at 1.0 Airy units (for one thing, the physical size for that varies with wavelength). With some specimens it is useful to use a smaller pinhole. Zeiss has a nice PDF online on different pinhole settings - if you cannot find it on an internet search, ask your Zeiss confocal rep to find and send it. As for Nyquist - he had a thing for sine waves. anyone imaging sine waves perfectly aligned in X or Y (or rotate scan view to perfectly align) can use the Nyquist sampling value (around 2.3). So, to sample correctly for 2D Nyquist, need to sample to account for the worse case sine wave at 45 degrees. Few biological specimens viewed on a confocal or widefield fluorescence microscope are sine waves (pattern A at http://argolight.com/product-micro/ comes close ... with modest NA objective lens probably close enough, but then that is a calibration slide, not a cell ... muscle fibers can come close).So, stop thinking Nyquist, and start thinking cells. Enjoy, George p.s. a heavily used objective will generally not perform as well as a brand new objective lens. On 7/14/2014 2:54 AM, Miroslav Varecha wrote: > Hello, > I would like to react to George. In my humble opinion, best lateral > optical resolution for confocal microscope is around 130 nm, so going > to resolution 60x60 nm is overshoot and it is just wasting your drive > space as you are not collecting any new real information. Resolutions > of 50x50nm and such are area of superresolution microscopy. Our > confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k. > High quality CCDs have even less, but sCMOS can get higher than 2k x > 2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you > that it is the most used objective (usually we observe stem cells) > even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger > field of view of 40x. You still get great detail and many cells in one > image. But if you want to observe really up close organels and such I > would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible > objective for vast majority of observations. > Miroslav > > > 2014-07-14 1:31 GMT+02:00 George McNamara<[hidden email]>: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> Post images on http://www.imgur.com and include the link in your posting. >> ***** >> >> Hi Michael, >> why 2kx2k? >> If the 40x lens has a 250x250 um field of view, this would be >> undersampling, >> pixel size 125x125 nm. If even larger field of view, undersampling even >> more. >> I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step >> 200 >> nm, maybe closer), >> George >> >> On 7/11/2014 8:53 AM, Cammer, Michael wrote: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> Post images on http://www.imgur.com and include the link in your >>> posting. >>> ***** >>> >>> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? >>> This >>> is something I've wanted for a long time, the ability to take large >>> fields >>> of view (2k X 2k pixels) at high resolution instead of having to do >>> tiling. >>> Also, with the new cameras that have oodles of small pixels... >>> >>> I'm considering replacing our 63X with this new 40X. Any experience >>> with >>> this, other than the battle of having to explain to other scope users >>> why >>> this is not really lower magnification? >>> >>> Regards, >>> >>> Michael >>> >>> >>> =========================================================================== >>> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU >>> Langone Medical Center >>> Cell: 914-309-3270 Lab: 212-263-3208 >>> http://ocs.med.nyu.edu/microscopy& >>> http://www.med.nyu.edu/skirball-lab/dustinlab/ >>> >>> ------------------------------------------------------------ >>> This email message, including any attachments, is for the sole use of >>> the >>> intended recipient(s) and may contain information that is proprietary, >>> confidential, and exempt from disclosure under applicable law. Any >>> unauthorized review, use, disclosure, or distribution is prohibited. If >>> you >>> have received this email in error please notify the sender by return >>> and delete the original message. Please note, the recipient should check >>> this email and any attachments for the presence of viruses. The >>> organization >>> accepts no liability for any damage caused by any virus transmitted by >>> this >>> email. >>> ================================= >>> >>> >>> >> >> >> -- >> >> >> >> George McNamara, Ph.D. >> Single Cells Analyst >> L.J.N. Cooper Lab >> University of Texas M.D. Anderson Cancer Center >> Houston, TX 77054 >> Tattletales http://works.bepress.com/gmcnamara/42 >> > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42 |
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Miroslav Varecha |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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In reply to this post by George McNamara
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi George, thanks a lot for interesting information. I was interested in superresolution microscopy like PALM, GSD, but I had no idea there are novel approaches not requiring expensive new supperresolution microscope. Thank you Miroslav -------------------------------------------------------------- Miroslav Varecha, Ph.D. research scientist, technical specialist Department of Biology, Faculty of Medicine Masaryk University, Brno, Czech Republic 2014-07-15 3:39 GMT+02:00 George McNamara <[hidden email]>: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Miroslav, > > > "highest resolution of 2k x 2k." > > is number of pixels in an image, resolution is set by that and field of view > (zoom). > > With the LSM 700 -- or other point scanning confocals -- you can choose the > zoom and number of pixels to get whatever pixel size you want. > > Data acquired on a confocal microscope can be used to obtain > "superresolution", as for the ~10% increase in resolution (ex. from ~214 nm > to <200 nm for 500 nm light, Airy disk 1.0) by deconvolving at 50 nm pixel > size. A pixel size of 130 nm is not going to give you a resolution > improvement (and the optical resolution of visible light confocal is ~200nm, > not 130 nm). > There are also other ways to process images - confocal or widefield - to get > a lot more out of the source data. Two examples: > PiMP http://jcs.biologists.org/content/125/9/2257.long ... which I > preferred using 30 nm pixel size acquisition on the confocals I managed in > Miami (LSM710, SP5). > 3B ... http://www.coxphysics.com/3b/ and http://www.superresolved.com/ > (later is an online forum hosted by Susan cox and Ed Rosten for all > superduperres). > SOFI / bSOFI etc (which I've not used) see > http://www.ncbi.nlm.nih.gov/pubmed/22711840 for Peter Dedecker et al's entry > point. > > Finally, not required by law (or Guy Cox or Jim Pawley or Alby Diaspro) to > set the confocal pinhole at 1.0 Airy units (for one thing, the physical size > for that varies with wavelength). With some specimens it is useful to use a > smaller pinhole. Zeiss has a nice PDF online on different pinhole settings - > if you cannot find it on an internet search, ask your Zeiss confocal rep to > find and send it. > > As for Nyquist - he had a thing for sine waves. anyone imaging sine waves > perfectly aligned in X or Y (or rotate scan view to perfectly align) can use > the Nyquist sampling value (around 2.3). So, to sample correctly for 2D > Nyquist, need to sample to account for the worse case sine wave at 45 > degrees. Few biological specimens viewed on a confocal or widefield > fluorescence microscope are sine waves (pattern A at > http://argolight.com/product-micro/ comes close ... with modest NA objective > lens probably close enough, but then that is a calibration slide, not a cell > ... muscle fibers can come close).So, stop thinking Nyquist, and start > thinking cells. > > Enjoy, > > George > p.s. a heavily used objective will generally not perform as well as a brand > new objective lens. > > > On 7/14/2014 2:54 AM, Miroslav Varecha wrote: >> >> Hello, >> I would like to react to George. In my humble opinion, best lateral >> optical resolution for confocal microscope is around 130 nm, so going >> to resolution 60x60 nm is overshoot and it is just wasting your drive >> space as you are not collecting any new real information. Resolutions >> of 50x50nm and such are area of superresolution microscopy. Our >> confocal Zeiss LSM 700 we have also highest resolution of 2k x 2k. >> High quality CCDs have even less, but sCMOS can get higher than 2k x >> 2k for sure. We have objective Zeiss 40x but NA 1,3 and I can tell you >> that it is the most used objective (usually we observe stem cells) >> even tho it is not NA1,4. We have also 63x1,4 NA but ppl prefer larger >> field of view of 40x. You still get great detail and many cells in one >> image. But if you want to observe really up close organels and such I >> would go to 63x NA1,4. 40x NA 1,4 seems to me like most flexible >> objective for vast majority of observations. >> Miroslav >> >> >> 2014-07-14 1:31 GMT+02:00 George McNamara<[hidden email]>: >> >>> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> Post images on http://www.imgur.com and include the link in your posting. >>> ***** >>> >>> Hi Michael, >>> why 2kx2k? >>> If the 40x lens has a 250x250 um field of view, this would be >>> undersampling, >>> pixel size 125x125 nm. If even larger field of view, undersampling even >>> more. >>> I suggest pixel size of 50x50 or 60x60 nm, and 3D deconvolution (Z step >>> 200 >>> nm, maybe closer), >>> George >>> >>> On 7/11/2014 8:53 AM, Cammer, Michael wrote: >>> >>>> >>>> ***** >>>> To join, leave or search the confocal microscopy listserv, go to: >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>>> Post images on http://www.imgur.com and include the link in your >>>> posting. >>>> ***** >>>> >>>> Does anyone have experience with the new Zeiss 40X N.A. 1.4 PlanApo? >>>> This >>>> is something I've wanted for a long time, the ability to take large >>>> fields >>>> of view (2k X 2k pixels) at high resolution instead of having to do >>>> tiling. >>>> Also, with the new cameras that have oodles of small pixels... >>>> >>>> I'm considering replacing our 63X with this new 40X. Any experience >>>> with >>>> this, other than the battle of having to explain to other scope users >>>> why >>>> this is not really lower magnification? >>>> >>>> Regards, >>>> >>>> Michael >>>> >>>> >>>> >>>> =========================================================================== >>>> Michael Cammer, Microscopy Core& Dustin Lab , Skirball Institute, NYU >>>> Langone Medical Center >>>> Cell: 914-309-3270 Lab: 212-263-3208 >>>> http://ocs.med.nyu.edu/microscopy& >>>> http://www.med.nyu.edu/skirball-lab/dustinlab/ >>>> >>>> ------------------------------------------------------------ >>>> This email message, including any attachments, is for the sole use of >>>> the >>>> intended recipient(s) and may contain information that is proprietary, >>>> confidential, and exempt from disclosure under applicable law. Any >>>> unauthorized review, use, disclosure, or distribution is prohibited. If >>>> you >>>> have received this email in error please notify the sender by return >>>> and delete the original message. Please note, the recipient should check >>>> this email and any attachments for the presence of viruses. The >>>> organization >>>> accepts no liability for any damage caused by any virus transmitted by >>>> this >>>> email. >>>> ================================= >>>> >>>> >>>> >>> >>> >>> >>> -- >>> >>> >>> >>> George McNamara, Ph.D. >>> Single Cells Analyst >>> L.J.N. Cooper Lab >>> University of Texas M.D. Anderson Cancer Center >>> Houston, TX 77054 >>> Tattletales http://works.bepress.com/gmcnamara/42 >>> >> >> > > > > -- > > > > George McNamara, Ph.D. > Single Cells Analyst > L.J.N. Cooper Lab > University of Texas M.D. Anderson Cancer Center > Houston, TX 77054 > Tattletales http://works.bepress.com/gmcnamara/42 |
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Steffen Dietzel |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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In reply to this post by Feinstein, Timothy
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Am 14.07.2014 16:28, schrieb Feinstein, Timothy: > You are correct that even in the best case a traditional optical > microscope can only separate point sources > 100 nm apart. Except of course the two point sources are in different color channels. Then you can get the two much closer together and still measure the distance between their intensity centers very accurately. In the two-digit nm range, depending only on your positioning accuracy. Provided you correct for chromatic aberration. Cheers Steffen -- ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room: Marchioninistr. 15, D-81377 München Building location: Marchioninistr. 27, München-Großhadern |
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Müller,Tobias |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi, The 40x/1.4 also has some drawbacks. It has a working distance of only 130 microns (40x/1.3 Apo: 210 microns, 63x/1.4 Apo: 190 microns, even a 100x/1.4 Apo has 170 microns). Not a problem when you're using cell monolayers (provided they're on the coverslip), but in 2P or with cleared samples, it might become a factor. More importantly, our 40x/1.4 Apos are a bit worse than the 63x/1.4 Apos when it comes to chromatic aberration. I generally do not suggest it for colocalization studies. Of course you can correct with a calibration slide, but this is always more accurate if the error is as small as possible to begin with. In my opinion, chromatic aberration is the only reason to have a 100x objective on a confocal, because it tends to have the smallest lateral chromatic shift. Anyway, I've tested the 40x/1.4 objective for transmission of laser power (with a powermeter), and found the power (very roughly in the focal plane) to be 2.1 times higher with the 40x/1.4 Apo as compared to a 63x/1.4 Apo. I think this is mostly because of the much larger rear aperture. Because of the powermeter I used there is an uncertainty as to the validity of these results. I will repeat this, as soon as I have the microscope slide power meter head from Thorlabs. After adjusting the AOTF to achieve the same excitation power for both objectives, the 40x was not brighter anymore. In fact, it was a bit darker, although that might have been true for that individual objective only. So my impression is that the 40x will only provide stronger excitation, at the same AOTF setting, than a 63x. This is nice, but hardly remarkable. In my opinion, the rise of NA from 1.3 (previous 40x) to 1.4 is counterbalanced by the reduced working distance and the non-optimal chromatic aberrations. It's a nice enough objective, but far from being a gamechanger. Disclaimer: Of course, it was only one objective for the transmission testing, and four for the tests of chromatic aberration, in which one was okay, two were so-so (but worse than 63x/1.4 in general), and one sucked the big one. Note that all brightness tests were done with PMT point detection. For cameras, magnification plays an important role with respect to resolution as well as brightness, and it's a whole different matter. Best, Tobias BTW: Why "new"? We're talking about product number 420762-9900-000 (https://www.micro-shop.zeiss.com/?s=118967537d39b70&l=en&p=us&f=o&a=v&m=s&id=420762-9900-000), right? Not all that new, if you ask me. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel Sent: Wednesday, July 16, 2014 7:03 PM To: [hidden email] Subject: Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X? ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Am 14.07.2014 16:28, schrieb Feinstein, Timothy: > You are correct that even in the best case a traditional optical > microscope can only separate point sources > 100 nm apart. Except of course the two point sources are in different color channels. Then you can get the two much closer together and still measure the distance between their intensity centers very accurately. In the two-digit nm range, depending only on your positioning accuracy. Provided you correct for chromatic aberration. Cheers Steffen -- ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room: Marchioninistr. 15, D-81377 München Building location: Marchioninistr. 27, München-Großhadern |
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Andreas Bruckbauer |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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In reply to this post by mcammer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Does the collimator move when changing the objective? Otherwise this would cause a big difference in laser power as you are unferfilling the back aperture and more of the beam passes through. Resolution might then also be worse. Best wishes Andreas -----Original Message----- From: "Müller,Tobias" <[hidden email]> Sent: 17/07/2014 17:38 To: "[hidden email]" <[hidden email]> Subject: Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X? ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi, The 40x/1.4 also has some drawbacks. It has a working distance of only 130 microns (40x/1.3 Apo: 210 microns, 63x/1.4 Apo: 190 microns, even a 100x/1.4 Apo has 170 microns). Not a problem when you're using cell monolayers (provided they're on the coverslip), but in 2P or with cleared samples, it might become a factor. More importantly, our 40x/1.4 Apos are a bit worse than the 63x/1.4 Apos when it comes to chromatic aberration. I generally do not suggest it for colocalization studies. Of course you can correct with a calibration slide, but this is always more accurate if the error is as small as possible to begin with. In my opinion, chromatic aberration is the only reason to have a 100x objective on a confocal, because it tends to have the smallest lateral chromatic shift. Anyway, I've tested the 40x/1.4 objective for transmission of laser power (with a powermeter), and found the power (very roughly in the focal plane) to be 2.1 times higher with the 40x/1.4 Apo as compared to a 63x/1.4 Apo. I think this is mostly because of the much larger rear aperture. Because of the powermeter I used there is an uncertainty as to the validity of these results. I will repeat this, as soon as I have the microscope slide power meter head from Thorlabs. After adjusting the AOTF to achieve the same excitation power for both objectives, the 40x was not brighter anymore. In fact, it was a bit darker, although that might have been true for that individual objective only. So my impression is that the 40x will only provide stronger excitation, at the same AOTF setting, than a 63x. This is nice, but hardly remarkable. In my opinion, the rise of NA from 1.3 (previous 40x) to 1.4 is counterbalanced by the reduced working distance and the non-optimal chromatic aberrations. It's a nice enough objective, but far from being a gamechanger. Disclaimer: Of course, it was only one objective for the transmission testing, and four for the tests of chromatic aberration, in which one was okay, two were so-so (but worse than 63x/1.4 in general), and one sucked the big one. Note that all brightness tests were done with PMT point detection. For cameras, magnification plays an important role with respect to resolution as well as brightness, and it's a whole different matter. Best, Tobias BTW: Why "new"? We're talking about product number 420762-9900-000 (https://www.micro-shop.zeiss.com/?s=118967537d39b70&l=en&p=us&f=o&a=v&m=s&id=420762-9900-000), right? Not all that new, if you ask me. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel Sent: Wednesday, July 16, 2014 7:03 PM To: [hidden email] Subject: Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X? ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Am 14.07.2014 16:28, schrieb Feinstein, Timothy: > You are correct that even in the best case a traditional optical > microscope can only separate point sources > 100 nm apart. Except of course the two point sources are in different color channels. Then you can get the two much closer together and still measure the distance between their intensity centers very accurately. In the two-digit nm range, depending only on your positioning accuracy. Provided you correct for chromatic aberration. Cheers Steffen -- ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room: Marchioninistr. 15, D-81377 München Building location: Marchioninistr. 27, München-Großhadern |
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James Pawley |
Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X?
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In reply to this post by Müller,Tobias
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi all, Good discussion and many important points have been raised. I would like to add a couple more. The larger field of the 40x might make it easier to find the cell you are interested in but remember all objective lenses are optical compromises. The manufacturers try to correct for chromatic and spherical aberration, flatness of field, coma, astigmatism, distortion etc, by judicious choices of the shape, material and position of the 10-20 elements in each objective. But they are never quite successful. True, they can transmit an image of a point object that looks a lot like an Airy figure, but it's central peak brightness is seldom close to 60 times greater than the brightness of the first bright ring, as the mathematics suggests it should be. OK, you know all this. But is it might be relevant here because it is just a lot harder to correct for all these things over the larger field of view of the 40x. It may be easier to understand this if you have a chance to take a good look at figures 11-9 and 11-10 in Chapter 11 of the Handbook (by Rimas Juskaitis) . Here you will be able to get some idea of the loss of resolution that occurs as one moves off 5 and 10mm off-axis in the intermediate image plane, with any objective. And you will see how much more severe this effect is on low-mag, high-NA objectives. Part of the loss with the 10x 0.5NA shown here is due to the fact that it is not a plan- objective (so there is also a focus shift) but the image shoudl still be symmetrical (and it isn't). From 11-9 you can see that another reason is vignetting. The glass is just not big enough to transmit all the high-NA rays once one gets off axis So my message is that with the 63x and even more so with the 40x NA 1.4s, use the wide field of view for surveys, but plan to do any high resolution work in the center of this field of view. And yes, at a given NA, the diameter of the back focal plane is inversely proportional to the magnification. So, as long as the laser beam has been spread out enough to fill the BFP (uniformly!) you should get about 6.25x more light from the 40x as the 100x. However, you probably won't quite do this, not only because it is hard to make a large uniform laser beam but because the transmission losses of the the objectives may differ and, more importantly, more light will probably be lost to vignetting in the 40x. Happy summer, Jim Pawley >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >Post images on http://www.imgur.com and include the link in your posting. >***** > >Hi, > >The 40x/1.4 also has some drawbacks. It has a >working distance of only 130 microns (40x/1.3 >Apo: 210 microns, 63x/1.4 Apo: 190 microns, even >a 100x/1.4 Apo has 170 microns). Not a problem >when you're using cell monolayers (provided >they're on the coverslip), but in 2P or with >cleared samples, it might become a factor. > >More importantly, our 40x/1.4 Apos are a bit >worse than the 63x/1.4 Apos when it comes to >chromatic aberration. I generally do not suggest >it for colocalization studies. Of course you can >correct with a calibration slide, but this is >always more accurate if the error is as small as >possible to begin with. In my opinion, chromatic >aberration is the only reason to have a 100x >objective on a confocal, because it tends to >have the smallest lateral chromatic shift. > >Anyway, I've tested the 40x/1.4 objective for >transmission of laser power (with a powermeter), >and found the power (very roughly in the focal >plane) to be 2.1 times higher with the 40x/1.4 >Apo as compared to a 63x/1.4 Apo. I think this >is mostly because of the much larger rear >aperture. Because of the powermeter I used there >is an uncertainty as to the validity of these >results. I will repeat this, as soon as I have >the microscope slide power meter head from >Thorlabs. > >After adjusting the AOTF to achieve the same >excitation power for both objectives, the 40x >was not brighter anymore. In fact, it was a bit >darker, although that might have been true for >that individual objective only. So my impression >is that the 40x will only provide stronger >excitation, at the same AOTF setting, than a >63x. This is nice, but hardly remarkable. In my >opinion, the rise of NA from 1.3 (previous 40x) >to 1.4 is counterbalanced by the reduced working >distance and the non-optimal chromatic >aberrations. It's a nice enough objective, but >far from being a gamechanger. > >Disclaimer: Of course, it was only one objective >for the transmission testing, and four for the >tests of chromatic aberration, in which one was >okay, two were so-so (but worse than 63x/1.4 in >general), and one sucked the big one. >Note that all brightness tests were done with >PMT point detection. For cameras, magnification >plays an important role with respect to >resolution as well as brightness, and it's a >whole different matter. > >Best, >Tobias > > >BTW: Why "new"? We're talking about product >number 420762-9900-000 >(https://www.micro-shop.zeiss.com/?s=118967537d39b70&l=en&p=us&f=o&a=v&m=s&id=420762-9900-000), >right? Not all that new, if you ask me. > > > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On >Behalf Of Steffen Dietzel >Sent: Wednesday, July 16, 2014 7:03 PM >To: [hidden email] >Subject: Re: Zeiss 40X N.A. 1.4 Plan APo as replacement for 63X? > >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >Post images on http://www.imgur.com and include the link in your posting. >***** > >Am 14.07.2014 16:28, schrieb Feinstein, Timothy: >> You are correct that even in the best case a traditional optical >> microscope can only separate point sources > 100 nm apart. > >Except of course the two point sources are in different color channels. >Then you can get the two much closer together >and still measure the distance between their >intensity centers very accurately. In the >two-digit nm range, depending only on your >positioning accuracy. >Provided you correct for chromatic aberration. > >Cheers >Steffen > > >-- >------------------------------------------------------------ >Steffen Dietzel, PD Dr. rer. nat >Ludwig-Maximilians-Universität München >Walter-Brendel-Zentrum für experimentelle >Medizin (WBex) Head of light microscopy > >Mail room: >Marchioninistr. 15, D-81377 München > >Building location: >Marchioninistr. 27, München-Großhadern -- **************************************** James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone 604-885-0840, email <[hidden email]> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146 |
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