Coverslips
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Not strictly a confocal question, but I'm sure someone out there will be able to help. We are about to start using a SIM super res system and have been advised that one of the critical factors in acquiring optimal images is the cover glass. Specifcally, the thickness of the glass needs to be consistant across the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between coverslips. One option is to measure each individual coverslip before use, but this seems rather impractical to me. Has anyone looked into this, and if so, are there any manufacturers out there who can provide cover glass with this high specification? Thanks, Simon |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Simon I used coverslip from Zeiss 170 +/- 5 um (not for super resolution though) http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E524C89 1852575A200721A7C Bertrand -- Bertrand Vernay, PhD Microscopy Senior Research Associate Developmental Biology Unit UCL Institute of Child Health 30 Guilford Street London WC1N 1EH, UK Tel : (+44) 020 7905 2224 (direct line) (+44) 020 7242 9789 (ICH switchboard) Fax: (+44) 020 7831 4366 E-mail: [hidden email] https://www.ucl.ac.uk/ich/services/lab-services/cmcf -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: 06 October 2011 09:43 To: [hidden email] Subject: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Not strictly a confocal question, but I'm sure someone out there will be able to help. We are about to start using a SIM super res system and have been advised that one of the critical factors in acquiring optimal images is the cover glass. Specifcally, the thickness of the glass needs to be consistant across the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between coverslips. One option is to measure each individual coverslip before use, but this seems rather impractical to me. Has anyone looked into this, and if so, are there any manufacturers out there who can provide cover glass with this high specification? Thanks, Simon |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I think Marienfeld does. I have ordered them, so not tried yet. But they seem to guarantee tight specs Axel CIF, IMCB -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Bertrand Vernay Sent: Thursday, 6 October, 2011 4:48 PM To: [hidden email] Subject: Re: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Simon I used coverslip from Zeiss 170 +/- 5 um (not for super resolution though) http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E524C89 1852575A200721A7C Bertrand -- Bertrand Vernay, PhD Microscopy Senior Research Associate Developmental Biology Unit UCL Institute of Child Health 30 Guilford Street London WC1N 1EH, UK Tel : (+44) 020 7905 2224 (direct line) (+44) 020 7242 9789 (ICH switchboard) Fax: (+44) 020 7831 4366 E-mail: [hidden email] https://www.ucl.ac.uk/ich/services/lab-services/cmcf -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: 06 October 2011 09:43 To: [hidden email] Subject: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Not strictly a confocal question, but I'm sure someone out there will be able to help. We are about to start using a SIM super res system and have been advised that one of the critical factors in acquiring optimal images is the cover glass. Specifcally, the thickness of the glass needs to be consistant across the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between coverslips. One option is to measure each individual coverslip before use, but this seems rather impractical to me. Has anyone looked into this, and if so, are there any manufacturers out there who can provide cover glass with this high specification? Thanks, Simon Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you. |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Simon, We are using Marienfeld glass coverslip #1.5 H. The tolerance is not 20um as the normal #1.5 category is, but 5um. To my knowledge, this is the best tolerance. Reference are : #0117500 : 12mm round coverslips #0117580 : 18mm round # 0107052 : 22x22mm square #0107222 : 24x50mm You can get some square 18x18mm from Zeiss as well, but these coverslips are from Marienfeld anyway. The french coverslip market is a bit weird as among 40 reference in any provider's catalog, only 2 are #1.5 I have to request these Marienfeld reference to my coverslip provider as they are not available in their catalog. The difference compared to the wrong #1 coverslip is amazing, and compared to regular #1.5 is quite noticeable. The price is roughly twice as expensive compared to regular #1.5, but we are talking about a few euro cents per coverslip, so its better not wasting a hundreds of euros experiment with crap coverslips. We have a precision ruler for measuring the thickness of the coverslips, but you don't want to waste your time doing this do you? These coverslips are the one we used for our OMX SIM microscope and they are of course fine. Another crucial parameter is the refraction index potential mismatch between immersion medium and mounting medium, but I am sure you are aware of this. Hope it helped. Best regards Julien Le 06/10/2011 10:43, Simon Walker a écrit : > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Not strictly a confocal question, but I'm sure someone out there will be able to > help. We are about to start using a SIM super res system and have been > advised that one of the critical factors in acquiring optimal images is the cover > glass. Specifcally, the thickness of the glass needs to be consistant across > the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between > coverslips. One option is to measure each individual coverslip before use, but > this seems rather impractical to me. Has anyone looked into this, and if so, > are there any manufacturers out there who can provide cover glass with this > high specification? > Thanks, > Simon > -- ____________________________________________ */Julien Cau, PhD./* /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ Montpellier RIO Imaging Montpellier BIOCAMPUS, UMS3426 Arnaud de Villeneuve Campus Imaging Facility Institut de Génétique Humaine-CNRS 141, rue de la Cardonille F-34396 Montpellier(France) e-mail: [hidden email].fr_ <mailto:[hidden email]> phone: +33.4.34.35.99.90 mobile: +33.6.50.19.27.49 fax: +33.4.34.35.99.01 URL: _http://www.mri.cnrs.fr/_ ____________________________________________ |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Simon, We use selected Menzel-glaser cover slips that are guaranteed 0.17mm +/- 0.01. Below i have pasted a response to these questions that was on the list a while ago from Stephen Cody (hope you don't mind Steve). Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal G'day List, Probably most of you are already using Menzel coverslips just re-branded by your suppliers. It's a matter of getting the suppliers to find out how to order this new sorting of coverslips. In the meantime you can order them through our supplier here in Australia if you wish. I've just spoken to them and last time they prepared for an influx of orders international orders. I order "0.170 +/- 0.01mm" coverslips for confocal through Lomb Scientific www.lomb.com.au <http://www.lomb.com.au/> . This is an Australian distributor of Menzel Glass. Lomb will also cut coverslips to special custom sizes (larger bulk orders are more economical). Peter Bigelow is the fantastic fellow who negotiated on my behalf to have this grading established. You can contact Peter directly on [hidden email] for product information, order codes etc.. Or as Peter suggests he is not always available [hidden email] may work better sometimes. I only have a catalogue code for the 25mm Round coverslips I order. This is a Lomb catalogue code rather than a Menzel one. Code: CSC25(170)GP Desc: 25mm Coverslip Circle 170 microns (+/- 0.01mm) Unit: Pack/100 No commercial affiliation with Lomb or Menzel. Cheers Stephen H. Cody Microscopy Manager Cheers Cam Cameron J. Nowell Microscopy Manager Centre for Advanced Microscopy Ludwig Institute for Cancer Research Melbourne - Parkville Branch PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 Facility Website -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Thursday, 6 October 2011 7:43 PM To: [hidden email] Subject: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Not strictly a confocal question, but I'm sure someone out there will be able to help. We are about to start using a SIM super res system and have been advised that one of the critical factors in acquiring optimal images is the cover glass. Specifcally, the thickness of the glass needs to be consistant across the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between coverslips. One option is to measure each individual coverslip before use, but this seems rather impractical to me. Has anyone looked into this, and if so, are there any manufacturers out there who can provide cover glass with this high specification? Thanks, Simon |
 
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George McNamara |
Re: Coverslips ... 170 +/- 1 um sounds good ... next question: how flat does it need to be for best image quality?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Re: Coverslips ... 170 +/- 1 um sounds good. as for the question of measuring each one: if you are not willing to take the time and/or spend the money to select optimal coverglasses, you might as well go back to using a confocal scope. ... next question: how flat does it need to be for best image quality? That is, most slide-specimen-coverglass preparations are hand made - I do not see how anyone can make them perfectly flat. Has anyone deliberately (1) perfectly refractive indexed matched everything, and (2) deliberately tilted the specimen, then quantify image quality? On the K-inserts, turning the screws on one side might be enough. thanks in advance, George On 10/6/2011 6:41 PM, Cameron Nowell wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Simon, > > We use selected Menzel-glaser cover slips that are guaranteed 0.17mm +/- > 0.01. Below i have pasted a response to these questions that was on the > list a while ago from Stephen Cody (hope you don't mind Steve). > > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > G'day List, > > > > Probably most of you are already using Menzel coverslips just re-branded > by your suppliers. It's a matter of getting the suppliers to find out > how to order this new sorting of coverslips. In the meantime you can > order them through our supplier here in Australia if you wish. I've just > spoken to them and last time they prepared for an influx of orders > international orders. > > > > I order "0.170 +/- 0.01mm" coverslips for confocal through Lomb > Scientific www.lomb.com.au<http://www.lomb.com.au/> . This is an > Australian distributor of Menzel Glass. Lomb will also cut coverslips to > special custom sizes (larger bulk orders are more economical). > > > > Peter Bigelow is the fantastic fellow who negotiated on my behalf to > have this grading established. > > You can contact Peter directly on [hidden email] for product > information, order codes etc.. > > > > Or as Peter suggests he is not always available [hidden email] may > work better sometimes. > > > > I only have a catalogue code for the 25mm Round coverslips I order. This > is a Lomb catalogue code rather than a Menzel one. > > > > Code: CSC25(170)GP > > Desc: 25mm Coverslip Circle 170 microns (+/- 0.01mm) > > Unit: Pack/100 > > > > No commercial affiliation with Lomb or Menzel. > > > > Cheers > > Stephen H. Cody > Microscopy Manager > > > > > > > Cheers > > Cam > > > Cameron J. Nowell > Microscopy Manager > Centre for Advanced Microscopy > Ludwig Institute for Cancer Research Melbourne - Parkville Branch > PO Box 2008 > Royal Melbourne Hospital > Victoria, 3050 > AUSTRALIA > Office: +61 3 9341 3155 > Mobile: +61422882700 > Fax: +61 3 9341 3104 > Facility Website > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Simon Walker > Sent: Thursday, 6 October 2011 7:43 PM > To: [hidden email] > Subject: Coverslips > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Not strictly a confocal question, but I'm sure someone out there will be > able to > help. We are about to start using a SIM super res system and have been > advised that one of the critical factors in acquiring optimal images is > the cover > glass. Specifcally, the thickness of the glass needs to be consistant > across > the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between > coverslips. One option is to measure each individual coverslip before > use, but > this seems rather impractical to me. Has anyone looked into this, and > if so, > are there any manufacturers out there who can provide cover glass with > this > high specification? > Thanks, > Simon > > -- George McNamara, PhD Analytical Imaging Core Facility University of Miami |
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Ryan Schreiner |
Re: Coverslips ... 170 +/- 1 um sounds good ... next question: how flat does it need to be for best image quality?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** It is 170µm +/- 10µm. On Thu, Oct 6, 2011 at 9:16 PM, George McNamara <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > ***** > > Re: Coverslips ... 170 +/- 1 um sounds good. > > as for the question of measuring each one: if you are not willing to take > the time and/or spend the money to select optimal coverglasses, you might as > well go back to using a confocal scope. > > > ... next question: how flat does it need to be for best image quality? > > That is, most slide-specimen-coverglass preparations are hand made - I do > not see how anyone can make them perfectly flat. Has anyone deliberately (1) > perfectly refractive indexed matched everything, and (2) deliberately tilted > the specimen, then quantify image quality? On the K-inserts, turning the > screws on one side might be enough. > > thanks in advance, > > George > > > On 10/6/2011 6:41 PM, Cameron Nowell wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> >> ***** >> >> Hi Simon, >> >> We use selected Menzel-glaser cover slips that are guaranteed 0.17mm +/- >> 0.01. Below i have pasted a response to these questions that was on the >> list a while ago from Stephen Cody (hope you don't mind Steve). >> >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.**edu/cgi-bin/wa?S1=confocal<http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal> >> >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.**edu/cgi-bin/wa?S1=confocal<http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal> >> >> G'day List, >> >> >> >> Probably most of you are already using Menzel coverslips just re-branded >> by your suppliers. It's a matter of getting the suppliers to find out >> how to order this new sorting of coverslips. In the meantime you can >> order them through our supplier here in Australia if you wish. I've just >> spoken to them and last time they prepared for an influx of orders >> international orders. >> >> >> >> I order "0.170 +/- 0.01mm" coverslips for confocal through Lomb >> Scientific www.lomb.com.au<http://www.**lomb.com.au/<http://www.lomb.com.au/>> >> . This is an >> Australian distributor of Menzel Glass. Lomb will also cut coverslips to >> special custom sizes (larger bulk orders are more economical). >> >> >> >> Peter Bigelow is the fantastic fellow who negotiated on my behalf to >> have this grading established. >> >> You can contact Peter directly on [hidden email] for product >> information, order codes etc.. >> >> >> >> Or as Peter suggests he is not always available [hidden email] may >> work better sometimes. >> >> >> >> I only have a catalogue code for the 25mm Round coverslips I order. This >> is a Lomb catalogue code rather than a Menzel one. >> >> >> >> Code: CSC25(170)GP >> >> Desc: 25mm Coverslip Circle 170 microns (+/- 0.01mm) >> >> Unit: Pack/100 >> >> >> >> No commercial affiliation with Lomb or Menzel. >> >> >> >> Cheers >> >> Stephen H. Cody >> Microscopy Manager >> >> >> >> >> >> >> Cheers >> >> Cam >> >> >> Cameron J. Nowell >> Microscopy Manager >> Centre for Advanced Microscopy >> Ludwig Institute for Cancer Research Melbourne - Parkville Branch >> PO Box 2008 >> Royal Melbourne Hospital >> Victoria, 3050 >> AUSTRALIA >> Office: +61 3 9341 3155 >> Mobile: +61422882700 >> Fax: +61 3 9341 3104 >> Facility Website >> >> >> >> -----Original Message----- >> From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@**LISTS.UMN.EDU<[hidden email]> >> ] >> On Behalf Of Simon Walker >> Sent: Thursday, 6 October 2011 7:43 PM >> To: [hidden email].**EDU <[hidden email]> >> Subject: Coverslips >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> >> ***** >> >> Hello, >> Not strictly a confocal question, but I'm sure someone out there will be >> able to >> help. We are about to start using a SIM super res system and have been >> advised that one of the critical factors in acquiring optimal images is >> the cover >> glass. Specifcally, the thickness of the glass needs to be consistant >> across >> the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between >> coverslips. One option is to measure each individual coverslip before >> use, but >> this seems rather impractical to me. Has anyone looked into this, and >> if so, >> are there any manufacturers out there who can provide cover glass with >> this >> high specification? >> Thanks, >> Simon >> >> >> > > > -- > > > George McNamara, PhD > Analytical Imaging Core Facility > University of Miami > |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** On 10/6/2011 3:43 AM, Simon Walker wrote: > Not strictly a confocal question, but I'm sure someone out there will be able to > help. We are about to start using a SIM super res system and have been > advised that one of the critical factors in acquiring optimal images is the cover > glass. Specifcally, the thickness of the glass needs to be consistant across > the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between > coverslips. One option is to measure each individual coverslip before use, but > this seems rather impractical to me. Has anyone looked into this, and if so, > are there any manufacturers out there who can provide cover glass with this > high specification? My ignorance is showing here. Why are higher-quality coverslips needed for SIM? Or is this for live-cell imaging? Best wishes-- Martin Wessendorf -- Martin Wessendorf, Ph.D. office: (612) 626-0145 Assoc Prof, Dept Neuroscience lab: (612) 624-2991 University of Minnesota Preferred FAX: (612) 624-8118 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 Minneapolis, MN 55455 e-mail: [hidden email] |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello: My guess is that this is required to have more homogeneous and reliable data, maybe you have to ask for the convenience of using sapphire coverslips, this will help you improving your light output. Best Regards Gabriel OH > Date: Thu, 6 Oct 2011 20:26:39 -0500 > From: [hidden email] > Subject: Re: Coverslips > To: [hidden email] > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > On 10/6/2011 3:43 AM, Simon Walker wrote: > > > Not strictly a confocal question, but I'm sure someone out there will be able to > > help. We are about to start using a SIM super res system and have been > > advised that one of the critical factors in acquiring optimal images is the cover > > glass. Specifcally, the thickness of the glass needs to be consistant across > > the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between > > coverslips. One option is to measure each individual coverslip before use, but > > this seems rather impractical to me. Has anyone looked into this, and if so, > > are there any manufacturers out there who can provide cover glass with this > > high specification? > > My ignorance is showing here. Why are higher-quality coverslips needed > for SIM? Or is this for live-cell imaging? > > Best wishes-- > > Martin Wessendorf > -- > Martin Wessendorf, Ph.D. office: (612) 626-0145 > Assoc Prof, Dept Neuroscience lab: (612) 624-2991 > University of Minnesota Preferred FAX: (612) 624-8118 > 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009 > Minneapolis, MN 55455 e-mail: [hidden email] |
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In reply to this post by Martin Wessendorf-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Martin, You are absolutely right and SIM does not need better coverslips than any other 0.17 objective-based microscopy technique. Our users use these coverslips for any experiment (widefield, confocal, SIM) and they appreciated the difference. The SIM point is more "don't use a fancy device that promiss you superesolution if you waste this potential with crap coverslips". If you buy a wonderfull coffee machine, will you put in it moldy coffee beans? Wrong thickness coverslips induce decreased light collection, spherical aberrations and then reduced resolution. It is worth using them for any type of experiment using a 0.17 lens. Alternatively, you can buy an expensive lens with a correction collar that can counteract the effects of the coverslip thickness error. Best regards PS : see for instance http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E524C891852575A200721A7C for a direct comparison of imaging with #1.5 vs #2 coverglass. Le 07/10/2011 03:26, Martin Wessendorf a écrit : > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > On 10/6/2011 3:43 AM, Simon Walker wrote: > >> Not strictly a confocal question, but I'm sure someone out there will >> be able to >> help. We are about to start using a SIM super res system and have been >> advised that one of the critical factors in acquiring optimal images >> is the cover >> glass. Specifcally, the thickness of the glass needs to be >> consistant across >> the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between >> coverslips. One option is to measure each individual coverslip >> before use, but >> this seems rather impractical to me. Has anyone looked into this, >> and if so, >> are there any manufacturers out there who can provide cover glass >> with this >> high specification? > > My ignorance is showing here. Why are higher-quality coverslips > needed for SIM? Or is this for live-cell imaging? > > Best wishes-- > > Martin Wessendorf -- ____________________________________________ */Julien Cau, PhD./* /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ Montpellier RIO Imaging Montpellier BIOCAMPUS, UMS3426 Arnaud de Villeneuve Campus Imaging Facility Institut de Génétique Humaine-CNRS 141, rue de la Cardonille F-34396 Montpellier(France) e-mail: [hidden email].fr_ <mailto:[hidden email]> phone: +33.4.34.35.99.90 mobile: +33.6.50.19.27.49 fax: +33.4.34.35.99.01 URL: _http://www.mri.cnrs.fr/_ ____________________________________________ |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thanks for everyone's input on this, it's really appreciated. Julien - although it doesn't mention it specifically I think the Zeiss link makes the point about the requirement for precise cover glass thickness with SIM. If you're to achieve the best resolution possible you have to use glass of a thickness that most closely matches that for which the objective is designed (usually 170 um). Any deviation from this will introduce sperical aberration. My guess is that because the resolution of a confocal system is lower, you won't notice much of a difference between an image acquired using 170 and a 180 um glass, whereas on a SIM system you will. Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julien Cau Sent: 07 October 2011 08:26 To: [hidden email] Subject: Re: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Martin, You are absolutely right and SIM does not need better coverslips than any other 0.17 objective-based microscopy technique. Our users use these coverslips for any experiment (widefield, confocal, SIM) and they appreciated the difference. The SIM point is more "don't use a fancy device that promiss you superesolution if you waste this potential with crap coverslips". If you buy a wonderfull coffee machine, will you put in it moldy coffee beans? Wrong thickness coverslips induce decreased light collection, spherical aberrations and then reduced resolution. It is worth using them for any type of experiment using a 0.17 lens. Alternatively, you can buy an expensive lens with a correction collar that can counteract the effects of the coverslip thickness error. Best regards PS : see for instance http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E524C891852575A200721A7C for a direct comparison of imaging with #1.5 vs #2 coverglass. Le 07/10/2011 03:26, Martin Wessendorf a écrit : > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > On 10/6/2011 3:43 AM, Simon Walker wrote: > >> Not strictly a confocal question, but I'm sure someone out there will >> be able to >> help. We are about to start using a SIM super res system and have been >> advised that one of the critical factors in acquiring optimal images >> is the cover >> glass. Specifcally, the thickness of the glass needs to be >> consistant across >> the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between >> coverslips. One option is to measure each individual coverslip >> before use, but >> this seems rather impractical to me. Has anyone looked into this, >> and if so, >> are there any manufacturers out there who can provide cover glass >> with this >> high specification? > > My ignorance is showing here. Why are higher-quality coverslips > needed for SIM? Or is this for live-cell imaging? > > Best wishes-- > > Martin Wessendorf -- ____________________________________________ */Julien Cau, PhD./* /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ Montpellier RIO Imaging Montpellier BIOCAMPUS, UMS3426 Arnaud de Villeneuve Campus Imaging Facility Institut de Génétique Humaine-CNRS 141, rue de la Cardonille F-34396 Montpellier(France) e-mail: [hidden email].fr_ <mailto:[hidden email]> phone: +33.4.34.35.99.90 mobile: +33.6.50.19.27.49 fax: +33.4.34.35.99.01 URL: _http://www.mri.cnrs.fr/_ ____________________________________________ |
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In reply to this post by Julien Cau
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** SIM is more sensitive to refractive index mismatch (including that produced by incorrect coverslip thickness) than more traditional microscopy techniques. This is partly because the improved resolution makes differences that are there for all microscopy types more visible, but also because refractive index (potentiall in the form of coverslip thickness) variations can distort the illumination pattern and lead to artefacts during the reconstruction process. Thus it probably is fair to say that SIM does require tighter tolerances than, e.g., confocal microscopy. Although moderately painful, testing each coverslip is not actually too bad once you get into the swing of it. When I was using a 4Pi microscope (which has a similar sensitivity to refractive index) I'd check each coverslip in about 3 places using a digital micrometer and it only took around a minute/coverslip. Coverslip thickness is much more important if you are using water immersion. cheers, David ________________________________ From: Julien Cau <[hidden email]> To: [hidden email] Sent: Friday, 7 October 2011 8:26 PM Subject: Re: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Martin, You are absolutely right and SIM does not need better coverslips than any other 0.17 objective-based microscopy technique. Our users use these coverslips for any experiment (widefield, confocal, SIM) and they appreciated the difference. The SIM point is more "don't use a fancy device that promiss you superesolution if you waste this potential with crap coverslips". If you buy a wonderfull coffee machine, will you put in it moldy coffee beans? Wrong thickness coverslips induce decreased light collection, spherical aberrations and then reduced resolution. It is worth using them for any type of experiment using a 0.17 lens. Alternatively, you can buy an expensive lens with a correction collar that can counteract the effects of the coverslip thickness error. Best regards PS : see for instance http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E524C891852575A200721A7C for a direct comparison of imaging with #1.5 vs #2 coverglass. Le 07/10/2011 03:26, Martin Wessendorf a écrit : > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > On 10/6/2011 3:43 AM, Simon Walker wrote: > >> Not strictly a confocal question, but I'm sure someone out there will be able to >> help. We are about to start using a SIM super res system and have been >> advised that one of the critical factors in acquiring optimal images is the cover >> glass. Specifcally, the thickness of the glass needs to be consistant across >> the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between >> coverslips. One option is to measure each individual coverslip before use, but >> this seems rather impractical to me. Has anyone looked into this, and if so, >> are there any manufacturers out there who can provide cover glass with this >> high specification? > > My ignorance is showing here. Why are higher-quality coverslips needed for SIM? Or is this for live-cell imaging? > > Best wishes-- > > Martin Wessendorf -- ____________________________________________ */Julien Cau, PhD./* /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ Montpellier RIO Imaging Montpellier BIOCAMPUS, UMS3426 Arnaud de Villeneuve Campus Imaging Facility Institut de Génétique Humaine-CNRS 141, rue de la Cardonille F-34396 Montpellier(France) e-mail: [hidden email].fr_ <mailto:[hidden email]> phone: +33.4.34.35.99.90 mobile: +33.6.50.19.27.49 fax: +33.4.34.35.99.01 URL: _http://www.mri.cnrs.fr/_ ____________________________________________ |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Thanks for everyone's input on this, it's really >appreciated. Julien - although it doesn't >mention it specifically I think the Zeiss link >makes the point about the requirement for >precise cover glass thickness with SIM. >If you're to achieve the best resolution >possible you have to use glass of a thickness >that most closely matches that for which the >objective is designed (usually 170 um). Any >deviation from this will introduce sperical >aberration. My guess is that because the >resolution of a confocal system is lower, you >won't notice much of a difference between an >image acquired using 170 and a 180 um glass, >whereas on a SIM system you will. >Simon > Just one more point: It makes a lot of difference whether we are talking about oil or water lenses. As immersion oil is almost the same RI as the BK-7 glass that I think is used for coverslips, changes in thickness from the optimum are much less important than if you are using a water lens. In the latter case, (RI = 1.515 vs Ri 1.334?) even a 2µm error in thickness is easily noticeable by eye with an NA 1.2 objective. Other factors that affect the PSF (such as the temperature of the immersion oil!) are well covered in Chapter 11 of the Handbook. SIM is indeed curcially dependent of having a known and unaberrated PSF and this is only possible if you re free from SA. To check this, always have some sub-resolution beads in your preparation and ensure that they "go out of focus" in a manner that looks the same whether you focus up or focus down. JP > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On >Behalf Of Julien Cau >Sent: 07 October 2011 08:26 >To: [hidden email] >Subject: Re: Coverslips > >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Hi Martin, > >You are absolutely right and SIM does not need better coverslips than >any other 0.17 objective-based microscopy technique. >Our users use these coverslips for any experiment (widefield, confocal, >SIM) and they appreciated the difference. >The SIM point is more "don't use a fancy device that promiss you >superesolution if you waste this potential with crap coverslips". If you >buy a wonderfull coffee machine, will you put in it moldy coffee beans? >Wrong thickness coverslips induce decreased light collection, spherical >aberrations and then reduced resolution. > >It is worth using them for any type of experiment using a 0.17 lens. >Alternatively, you can buy an expensive lens with a correction collar >that can counteract the effects of the coverslip thickness error. > >Best regards > >PS : see for instance >http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E524C891852575A200721A7C >for a direct comparison of imaging with #1.5 vs #2 coverglass. > >Le 07/10/2011 03:26, Martin Wessendorf a écrit : >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> On 10/6/2011 3:43 AM, Simon Walker wrote: >> >>> Not strictly a confocal question, but I'm sure someone out there will >>> be able to >>> help. We are about to start using a SIM super res system and have been >>> advised that one of the critical factors in acquiring optimal images >>> is the cover >>> glass. Specifcally, the thickness of the glass needs to be >>> consistant across >>> the whole coverslip (e.g. 170 um +/- 2 um), and reproducible between >>> coverslips. One option is to measure each individual coverslip >>> before use, but >>> this seems rather impractical to me. Has anyone looked into this, >>> and if so, >>> are there any manufacturers out there who can provide cover glass >>> with this >>> high specification? >> >> My ignorance is showing here. Why are higher-quality coverslips > > needed for SIM? Or is this for live-cell imaging? >> >> Best wishes-- >> >> Martin Wessendorf > >-- > >____________________________________________ > >*/Julien Cau, PhD./* > >/Montpellier RIO Imaging Facility manager/Responsable technique MRI/ > >Montpellier RIO Imaging > >Montpellier BIOCAMPUS, UMS3426 > >Arnaud de Villeneuve Campus Imaging Facility > >Institut de Génétique Humaine-CNRS > >141, rue de la Cardonille > >F-34396 Montpellier(France) > >e-mail: [hidden email].fr_ <mailto:[hidden email]> > >phone: +33.4.34.35.99.90 > >mobile: +33.6.50.19.27.49 > >fax: +33.4.34.35.99.01 > >URL: _http://www.mri.cnrs.fr/_ > >____________________________________________ -- James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726 Phone: 608-238-3953 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** A couple of things I have noticed about coverslips. Firstly, if you ever do measure commercial coverslips, you will notice that within a batch, most are within a 5 um tolerance with a few outliers. From batch to batch you will notice far more differences (I had one batch of #1.5's that was all almost 190 um). Secondly, the comment about oil vs. water objectives is a very important one. Oil objectives are designed to image just above the coverslip. Since the oil, coverslip, and front lens are all of the same refractive index, it makes no difference how thick the coverslip is--that ends up being compensated by the increased oil thickness necessary to achieve the same focal depth into the sample. For water, I have never noticed a 2 um difference by eye, but fluorescence correlation spectroscopy is uniquely sensitive to these things and I don't see a significant difference as long as I stay within a 5 um tolerance. I used to think that the solution was to carefully adjust the correction collar on water objectives and soon discovered that the lag in the correction collar is as much as 100 um! As a result, the numbers on the correction collar are essentially meaningless unless you always adjust from the same direction (note that I have only tested this for Zeiss objectives). The best practice is to adjust the collar with a sensitive sample and then leave it and use measured or high precision coverslips for the remainder of the experiment. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley Sent: Friday, October 07, 2011 10:32 AM To: [hidden email] Subject: Re: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Thanks for everyone's input on this, it's really appreciated. Julien - >although it doesn't mention it specifically I think the Zeiss link >makes the point about the requirement for precise cover glass thickness >with SIM. >If you're to achieve the best resolution possible you have to use glass >of a thickness that most closely matches that for which the objective >is designed (usually 170 um). Any deviation from this will introduce >sperical aberration. My guess is that because the resolution of a >confocal system is lower, you won't notice much of a difference between >an image acquired using 170 and a 180 um glass, whereas on a SIM system >you will. >Simon > Just one more point: It makes a lot of difference whether we are talking about oil or water lenses. As immersion oil is almost the same RI as the BK-7 glass that I think is used for coverslips, changes in thickness from the optimum are much less important than if you are using a water lens. In the latter case, (RI = 1.515 vs Ri 1.334?) even a 2µm error in thickness is easily noticeable by eye with an NA 1.2 objective. Other factors that affect the PSF (such as the temperature of the immersion oil!) are well covered in Chapter 11 of the Handbook. SIM is indeed curcially dependent of having a known and unaberrated PSF and this is only possible if you re free from SA. To check this, always have some sub-resolution beads in your preparation and ensure that they "go out of focus" in a manner that looks the same whether you focus up or focus down. JP > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of Julien Cau >Sent: 07 October 2011 08:26 >To: [hidden email] >Subject: Re: Coverslips > >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Hi Martin, > >You are absolutely right and SIM does not need better coverslips than >any other 0.17 objective-based microscopy technique. >Our users use these coverslips for any experiment (widefield, confocal, >SIM) and they appreciated the difference. >The SIM point is more "don't use a fancy device that promiss you >superesolution if you waste this potential with crap coverslips". If >you buy a wonderfull coffee machine, will you put in it moldy coffee beans? >Wrong thickness coverslips induce decreased light collection, spherical >aberrations and then reduced resolution. > >It is worth using them for any type of experiment using a 0.17 lens. >Alternatively, you can buy an expensive lens with a correction collar >that can counteract the effects of the coverslip thickness error. > >Best regards > >PS : see for instance >http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E5 >24C891852575A200721A7C for a direct comparison of imaging with #1.5 vs >#2 coverglass. > >Le 07/10/2011 03:26, Martin Wessendorf a écrit : >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> On 10/6/2011 3:43 AM, Simon Walker wrote: >> >>> Not strictly a confocal question, but I'm sure someone out there >>> will be able to help. We are about to start using a SIM super res >>> system and have been advised that one of the critical factors in >>> acquiring optimal images is the cover glass. Specifcally, the >>> thickness of the glass needs to be consistant across the whole >>> coverslip (e.g. 170 um +/- 2 um), and reproducible between >>> coverslips. One option is to measure each individual coverslip >>> before use, but this seems rather impractical to me. Has anyone >>> looked into this, and if so, are there any manufacturers out there >>> who can provide cover glass with this high specification? >> >> My ignorance is showing here. Why are higher-quality coverslips > > needed for SIM? Or is this for live-cell imaging? >> >> Best wishes-- >> >> Martin Wessendorf > >-- > >____________________________________________ > >*/Julien Cau, PhD./* > >/Montpellier RIO Imaging Facility manager/Responsable technique MRI/ > >Montpellier RIO Imaging > >Montpellier BIOCAMPUS, UMS3426 > >Arnaud de Villeneuve Campus Imaging Facility > >Institut de Génétique Humaine-CNRS > >141, rue de la Cardonille > >F-34396 Montpellier(France) > >e-mail: [hidden email].fr_ <mailto:[hidden email]> > >phone: +33.4.34.35.99.90 > >mobile: +33.6.50.19.27.49 > >fax: +33.4.34.35.99.01 > >URL: _http://www.mri.cnrs.fr/_ > >____________________________________________ -- James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726 Phone: 608-238-3953 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I can understand the argument for not needing exact coverslip thickness for oil objectives when imaging near the coverslip surface. Just out of curiosity then, why are there some oil objectives sold with correction collars as well? Are these collars there just for adjustment when imaging with an oil immersion objective at a temperature other than room temperature? John Oreopoulos Research Assistant Spectral Applied Research Richmond Hill, Ontario Canada www.spectral.ca On 2011-10-07, at 12:02 PM, Unruh, Jay wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > A couple of things I have noticed about coverslips. Firstly, if you ever do measure commercial coverslips, you will notice that within a batch, most are within a 5 um tolerance with a few outliers. From batch to batch you will notice far more differences (I had one batch of #1.5's that was all almost 190 um). Secondly, the comment about oil vs. water objectives is a very important one. Oil objectives are designed to image just above the coverslip. Since the oil, coverslip, and front lens are all of the same refractive index, it makes no difference how thick the coverslip is--that ends up being compensated by the increased oil thickness necessary to achieve the same focal depth into the sample. For water, I have never noticed a 2 um difference by eye, but fluorescence correlation spectroscopy is uniquely sensitive to these things and I don't see a significant difference as long as I stay within a 5 um tolerance. I used to think that the solution was to carefully adjust the correction collar on water objectives and soon discovered that the lag in the correction collar is as much as 100 um! As a result, the numbers on the correction collar are essentially meaningless unless you always adjust from the same direction (note that I have only tested this for Zeiss objectives). The best practice is to adjust the collar with a sensitive sample and then leave it and use measured or high precision coverslips for the remainder of the experiment. > > Jay > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley > Sent: Friday, October 07, 2011 10:32 AM > To: [hidden email] > Subject: Re: Coverslips > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Thanks for everyone's input on this, it's really appreciated. Julien - >> although it doesn't mention it specifically I think the Zeiss link >> makes the point about the requirement for precise cover glass thickness >> with SIM. >> If you're to achieve the best resolution possible you have to use glass >> of a thickness that most closely matches that for which the objective >> is designed (usually 170 um). Any deviation from this will introduce >> sperical aberration. My guess is that because the resolution of a >> confocal system is lower, you won't notice much of a difference between >> an image acquired using 170 and a 180 um glass, whereas on a SIM system >> you will. >> Simon >> > > > Just one more point: It makes a lot of difference whether we are talking about oil or water lenses. > > As immersion oil is almost the same RI as the > BK-7 glass that I think is used for coverslips, changes in thickness from the optimum are much less important than if you are using a water lens. In the latter case, (RI = 1.515 vs Ri > 1.334?) even a 2µm error in thickness is easily noticeable by eye with an NA 1.2 objective. Other factors that affect the PSF (such as the temperature of the immersion oil!) are well covered in Chapter 11 of the Handbook. > > SIM is indeed curcially dependent of having a known and unaberrated PSF and this is only possible if you re free from SA. To check this, always have some sub-resolution beads in your preparation and ensure that they "go out of focus" in a manner that looks the same whether you focus up or focus down. > > JP > > >> >> -----Original Message----- >> From: Confocal Microscopy List >> [mailto:[hidden email]] On Behalf Of Julien Cau >> Sent: 07 October 2011 08:26 >> To: [hidden email] >> Subject: Re: Coverslips >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi Martin, >> >> You are absolutely right and SIM does not need better coverslips than >> any other 0.17 objective-based microscopy technique. >> Our users use these coverslips for any experiment (widefield, confocal, >> SIM) and they appreciated the difference. >> The SIM point is more "don't use a fancy device that promiss you >> superesolution if you waste this potential with crap coverslips". If >> you buy a wonderfull coffee machine, will you put in it moldy coffee beans? >> Wrong thickness coverslips induce decreased light collection, spherical >> aberrations and then reduced resolution. >> >> It is worth using them for any type of experiment using a 0.17 lens. >> Alternatively, you can buy an expensive lens with a correction collar >> that can counteract the effects of the coverslip thickness error. >> >> Best regards >> >> PS : see for instance >> http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E5 >> 24C891852575A200721A7C for a direct comparison of imaging with #1.5 vs >> #2 coverglass. >> >> Le 07/10/2011 03:26, Martin Wessendorf a écrit : >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> On 10/6/2011 3:43 AM, Simon Walker wrote: >>> >>>> Not strictly a confocal question, but I'm sure someone out there >>>> will be able to help. We are about to start using a SIM super res >>>> system and have been advised that one of the critical factors in >>>> acquiring optimal images is the cover glass. Specifcally, the >>>> thickness of the glass needs to be consistant across the whole >>>> coverslip (e.g. 170 um +/- 2 um), and reproducible between >>>> coverslips. One option is to measure each individual coverslip >>>> before use, but this seems rather impractical to me. Has anyone >>>> looked into this, and if so, are there any manufacturers out there >>>> who can provide cover glass with this high specification? >>> >>> My ignorance is showing here. Why are higher-quality coverslips >>> needed for SIM? Or is this for live-cell imaging? >>> >>> Best wishes-- >>> >>> Martin Wessendorf >> >> -- >> >> ____________________________________________ >> >> */Julien Cau, PhD./* >> >> /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ >> >> Montpellier RIO Imaging >> >> Montpellier BIOCAMPUS, UMS3426 >> >> Arnaud de Villeneuve Campus Imaging Facility >> >> Institut de Génétique Humaine-CNRS >> >> 141, rue de la Cardonille >> >> F-34396 Montpellier(France) >> >> e-mail: [hidden email].fr_ <mailto:[hidden email]> >> >> phone: +33.4.34.35.99.90 >> >> mobile: +33.6.50.19.27.49 >> >> fax: +33.4.34.35.99.01 >> >> URL: _http://www.mri.cnrs.fr/_ >> >> ____________________________________________ > > > -- > James and Christine Pawley, 21 N. Prospect Ave. > Madison, WI, 53726 Phone: 608-238-3953 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Most of the oil correction collars I have seen are to correct for glycerol immersion/mounting media. The temperature is an interesting point. I haven't seen this advertised by the microscope companies but that wouldn't be too surprising. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos Sent: Friday, October 07, 2011 11:16 AM To: [hidden email] Subject: Re: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I can understand the argument for not needing exact coverslip thickness for oil objectives when imaging near the coverslip surface. Just out of curiosity then, why are there some oil objectives sold with correction collars as well? Are these collars there just for adjustment when imaging with an oil immersion objective at a temperature other than room temperature? John Oreopoulos Research Assistant Spectral Applied Research Richmond Hill, Ontario Canada www.spectral.ca On 2011-10-07, at 12:02 PM, Unruh, Jay wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > A couple of things I have noticed about coverslips. Firstly, if you ever do measure commercial coverslips, you will notice that within a batch, most are within a 5 um tolerance with a few outliers. From batch to batch you will notice far more differences (I had one batch of #1.5's that was all almost 190 um). Secondly, the comment about oil vs. water objectives is a very important one. Oil objectives are designed to image just above the coverslip. Since the oil, coverslip, and front lens are all of the same refractive index, it makes no difference how thick the coverslip is--that ends up being compensated by the increased oil thickness necessary to achieve the same focal depth into the sample. For water, I have never noticed a 2 um difference by eye, but fluorescence correlation spectroscopy is uniquely sensitive to these things and I don't see a significant difference as long as I stay within a 5 um tolerance. I used to think that the solution was to carefully adjust the correction collar on water objectives and soon discovered that the lag in the correction collar is as much as 100 um! As a result, the numbers on the correction collar are essentially meaningless unless you always adjust from the same direction (note that I have only tested this for Zeiss objectives). The best practice is to adjust the collar with a sensitive sample and then leave it and use measured or high precision coverslips for the remainder of the experiment. > > Jay > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of James Pawley > Sent: Friday, October 07, 2011 10:32 AM > To: [hidden email] > Subject: Re: Coverslips > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Thanks for everyone's input on this, it's really appreciated. Julien >> - although it doesn't mention it specifically I think the Zeiss link >> makes the point about the requirement for precise cover glass >> thickness with SIM. >> If you're to achieve the best resolution possible you have to use >> glass of a thickness that most closely matches that for which the >> objective is designed (usually 170 um). Any deviation from this will >> introduce sperical aberration. My guess is that because the >> resolution of a confocal system is lower, you won't notice much of a >> difference between an image acquired using 170 and a 180 um glass, >> whereas on a SIM system you will. >> Simon >> > > > Just one more point: It makes a lot of difference whether we are talking about oil or water lenses. > > As immersion oil is almost the same RI as the > BK-7 glass that I think is used for coverslips, changes in thickness > from the optimum are much less important than if you are using a water > lens. In the latter case, (RI = 1.515 vs Ri > 1.334?) even a 2µm error in thickness is easily noticeable by eye with an NA 1.2 objective. Other factors that affect the PSF (such as the temperature of the immersion oil!) are well covered in Chapter 11 of the Handbook. > > SIM is indeed curcially dependent of having a known and unaberrated PSF and this is only possible if you re free from SA. To check this, always have some sub-resolution beads in your preparation and ensure that they "go out of focus" in a manner that looks the same whether you focus up or focus down. > > JP > > >> >> -----Original Message----- >> From: Confocal Microscopy List >> [mailto:[hidden email]] On Behalf Of Julien Cau >> Sent: 07 October 2011 08:26 >> To: [hidden email] >> Subject: Re: Coverslips >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi Martin, >> >> You are absolutely right and SIM does not need better coverslips than >> any other 0.17 objective-based microscopy technique. >> Our users use these coverslips for any experiment (widefield, >> confocal, >> SIM) and they appreciated the difference. >> The SIM point is more "don't use a fancy device that promiss you >> superesolution if you waste this potential with crap coverslips". If >> you buy a wonderfull coffee machine, will you put in it moldy coffee beans? >> Wrong thickness coverslips induce decreased light collection, >> spherical aberrations and then reduced resolution. >> >> It is worth using them for any type of experiment using a 0.17 lens. >> Alternatively, you can buy an expensive lens with a correction collar >> that can counteract the effects of the coverslip thickness error. >> >> Best regards >> >> PS : see for instance >> http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521 >> E5 24C891852575A200721A7C for a direct comparison of imaging with >> #1.5 vs >> #2 coverglass. >> >> Le 07/10/2011 03:26, Martin Wessendorf a écrit : >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> On 10/6/2011 3:43 AM, Simon Walker wrote: >>> >>>> Not strictly a confocal question, but I'm sure someone out there >>>> will be able to help. We are about to start using a SIM super >>>> res system and have been advised that one of the critical factors >>>> in acquiring optimal images is the cover glass. Specifcally, the >>>> thickness of the glass needs to be consistant across the whole >>>> coverslip (e.g. 170 um +/- 2 um), and reproducible between >>>> coverslips. One option is to measure each individual coverslip >>>> before use, but this seems rather impractical to me. Has anyone >>>> looked into this, and if so, are there any manufacturers out >>>> there who can provide cover glass with this high specification? >>> >>> My ignorance is showing here. Why are higher-quality coverslips >>> needed for SIM? Or is this for live-cell imaging? >>> >>> Best wishes-- >>> >>> Martin Wessendorf >> >> -- >> >> ____________________________________________ >> >> */Julien Cau, PhD./* >> >> /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ >> >> Montpellier RIO Imaging >> >> Montpellier BIOCAMPUS, UMS3426 >> >> Arnaud de Villeneuve Campus Imaging Facility >> >> Institut de Génétique Humaine-CNRS >> >> 141, rue de la Cardonille >> >> F-34396 Montpellier(France) >> >> e-mail: [hidden email].fr_ <mailto:[hidden email]> >> >> phone: +33.4.34.35.99.90 >> >> mobile: +33.6.50.19.27.49 >> >> fax: +33.4.34.35.99.01 >> >> URL: _http://www.mri.cnrs.fr/_ >> >> ____________________________________________ > > > -- > James and Christine Pawley, 21 N. Prospect Ave. > Madison, WI, 53726 Phone: 608-238-3953 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Nikon's 1.45 and 1.49 NA's objectives have two separate sets of markings: One for room temp (the exact temp is printed and I do not remember) and a separate one for 37.C. Hank Adams Genetics U.T.M.D.Anderson Cancer Center -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Unruh, Jay Sent: Friday, October 07, 2011 11:35 AM To: [hidden email] Subject: Re: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Most of the oil correction collars I have seen are to correct for glycerol immersion/mounting media. The temperature is an interesting point. I haven't seen this advertised by the microscope companies but that wouldn't be too surprising. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos Sent: Friday, October 07, 2011 11:16 AM To: [hidden email] Subject: Re: Coverslips ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I can understand the argument for not needing exact coverslip thickness for oil objectives when imaging near the coverslip surface. Just out of curiosity then, why are there some oil objectives sold with correction collars as well? Are these collars there just for adjustment when imaging with an oil immersion objective at a temperature other than room temperature? John Oreopoulos Research Assistant Spectral Applied Research Richmond Hill, Ontario Canada www.spectral.ca On 2011-10-07, at 12:02 PM, Unruh, Jay wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > A couple of things I have noticed about coverslips. Firstly, if you ever do measure commercial coverslips, you will notice that within a batch, most are within a 5 um tolerance with a few outliers. From batch to batch you will notice far more differences (I had one batch of #1.5's that was all almost 190 um). Secondly, the comment about oil vs. water objectives is a very important one. Oil objectives are designed to image just above the coverslip. Since the oil, coverslip, and front lens are all of the same refractive index, it makes no difference how thick the coverslip is--that ends up being compensated by the increased oil thickness necessary to achieve the same focal depth into the sample. For water, I have never noticed a 2 um difference by eye, but fluorescence correlation spectroscopy is uniquely sensitive to these things and I don't see a significant difference as long as I stay within a 5 um tolerance. I used to think that the solution was to carefully adjust the correction collar on water objectives and soon discovered that the lag in the correction collar is as much as 100 um! As a result, the numbers on the correction collar are essentially meaningless unless you always adjust from the same direction (note that I have only tested this for Zeiss objectives). The best practice is to adjust the collar with a sensitive sample and then leave it and use measured or high precision coverslips for the remainder of the experiment. > > Jay > > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of James Pawley > Sent: Friday, October 07, 2011 10:32 AM > To: [hidden email] > Subject: Re: Coverslips > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Thanks for everyone's input on this, it's really appreciated. Julien >> - although it doesn't mention it specifically I think the Zeiss link >> makes the point about the requirement for precise cover glass >> thickness with SIM. >> If you're to achieve the best resolution possible you have to use >> glass of a thickness that most closely matches that for which the >> objective is designed (usually 170 um). Any deviation from this will >> introduce sperical aberration. My guess is that because the >> resolution of a confocal system is lower, you won't notice much of a >> difference between an image acquired using 170 and a 180 um glass, >> whereas on a SIM system you will. >> Simon >> > > > Just one more point: It makes a lot of difference whether we are talking about oil or water lenses. > > As immersion oil is almost the same RI as the > BK-7 glass that I think is used for coverslips, changes in thickness > from the optimum are much less important than if you are using a water > lens. In the latter case, (RI = 1.515 vs Ri > 1.334?) even a 2µm error in thickness is easily noticeable by eye with an NA 1.2 objective. Other factors that affect the PSF (such as the temperature of the immersion oil!) are well covered in Chapter 11 of the Handbook. > > SIM is indeed curcially dependent of having a known and unaberrated PSF and this is only possible if you re free from SA. To check this, always have some sub-resolution beads in your preparation and ensure that they "go out of focus" in a manner that looks the same whether you focus up or focus down. > > JP > > >> >> -----Original Message----- >> From: Confocal Microscopy List >> [mailto:[hidden email]] On Behalf Of Julien Cau >> Sent: 07 October 2011 08:26 >> To: [hidden email] >> Subject: Re: Coverslips >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi Martin, >> >> You are absolutely right and SIM does not need better coverslips than >> any other 0.17 objective-based microscopy technique. >> Our users use these coverslips for any experiment (widefield, >> confocal, >> SIM) and they appreciated the difference. >> The SIM point is more "don't use a fancy device that promiss you >> superesolution if you waste this potential with crap coverslips". If >> you buy a wonderfull coffee machine, will you put in it moldy coffee beans? >> Wrong thickness coverslips induce decreased light collection, >> spherical aberrations and then reduced resolution. >> >> It is worth using them for any type of experiment using a 0.17 lens. >> Alternatively, you can buy an expensive lens with a correction collar >> that can counteract the effects of the coverslip thickness error. >> >> Best regards >> >> PS : see for instance >> http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521 >> E5 24C891852575A200721A7C for a direct comparison of imaging with >> #1.5 vs >> #2 coverglass. >> >> Le 07/10/2011 03:26, Martin Wessendorf a écrit : >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> On 10/6/2011 3:43 AM, Simon Walker wrote: >>> >>>> Not strictly a confocal question, but I'm sure someone out there >>>> will be able to help. We are about to start using a SIM super >>>> res system and have been advised that one of the critical factors >>>> in acquiring optimal images is the cover glass. Specifcally, the >>>> thickness of the glass needs to be consistant across the whole >>>> coverslip (e.g. 170 um +/- 2 um), and reproducible between >>>> coverslips. One option is to measure each individual coverslip >>>> before use, but this seems rather impractical to me. Has anyone >>>> looked into this, and if so, are there any manufacturers out >>>> there who can provide cover glass with this high specification? >>> >>> My ignorance is showing here. Why are higher-quality coverslips >>> needed for SIM? Or is this for live-cell imaging? >>> >>> Best wishes-- >>> >>> Martin Wessendorf >> >> -- >> >> ____________________________________________ >> >> */Julien Cau, PhD./* >> >> /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ >> >> Montpellier RIO Imaging >> >> Montpellier BIOCAMPUS, UMS3426 >> >> Arnaud de Villeneuve Campus Imaging Facility >> >> Institut de Génétique Humaine-CNRS >> >> 141, rue de la Cardonille >> >> F-34396 Montpellier(France) >> >> e-mail: [hidden email].fr_ <mailto:[hidden email]> >> >> phone: +33.4.34.35.99.90 >> >> mobile: +33.6.50.19.27.49 >> >> fax: +33.4.34.35.99.01 >> >> URL: _http://www.mri.cnrs.fr/_ >> >> ____________________________________________ > > > -- > James and Christine Pawley, 21 N. Prospect Ave. > Madison, WI, 53726 Phone: 608-238-3953 |
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In reply to this post by Unruh, Jay
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Most of the oil correction collars I have seen >are to correct for glycerol immersion/mounting >media. The temperature is an interesting point. >I haven't seen this advertised by the microscope >companies but that wouldn't be too surprising. > >Jay > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On >Behalf Of John Oreopoulos >Sent: Friday, October 07, 2011 11:16 AM >To: [hidden email] >Subject: Re: Coverslips > >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >I can understand the argument for not needing >exact coverslip thickness for oil objectives >when imaging near the coverslip surface. Just >out of curiosity then, why are there some oil >objectives sold with correction collars as well? >Are these collars there just for adjustment when >imaging with an oil immersion objective at a >temperature other than room temperature? On, for instance, the Zeiss 1.2 cAPO, you will find two set point marks opposite the coverslip-thickness gradations on the rotatable collar. The black one is said to be for room temp (20 degC) and the red one for 37 degC. It is discussed further in Chapter 11. JP > >John Oreopoulos >Research Assistant >Spectral Applied Research >Richmond Hill, Ontario >Canada >www.spectral.ca > > >On 2011-10-07, at 12:02 PM, Unruh, Jay wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> A couple of things I have noticed about >>coverslips. Firstly, if you ever do measure >>commercial coverslips, you will notice that >>within a batch, most are within a 5 um >>tolerance with a few outliers. From batch to >>batch you will notice far more differences (I >>had one batch of #1.5's that was all almost 190 >>um). Secondly, the comment about oil vs. water >>objectives is a very important one. Oil >>objectives are designed to image just above the >>coverslip. Since the oil, coverslip, and front >>lens are all of the same refractive index, it >>makes no difference how thick the coverslip >>is--that ends up being compensated by the >>increased oil thickness necessary to achieve >>the same focal depth into the sample. For >>water, I have never noticed a 2 um difference >>by eye, but fluorescence correlation >>spectroscopy is uniquely sensitive to these >>things and I don't see a significant difference >>as long as I stay within a 5 um tolerance. I >>used to think that the solution was to >>carefully adjust the correction collar on water >>objectives and soon discovered that the lag in >>the correction collar is as much as 100 um! As >>a result, the numbers on the correction collar >>are essentially meaningless unless you always >>adjust from the same direction (note that I >>have only tested this for Zeiss objectives). >>The best practice is to adjust the collar with >>a sensitive sample and then leave it and use >>measured or high precision coverslips for the >>remainder of the experiment. > > >> Jay >> >> -----Original Message----- >> From: Confocal Microscopy List >> [mailto:[hidden email]] On Behalf Of James Pawley >> Sent: Friday, October 07, 2011 10:32 AM >> To: [hidden email] >> Subject: Re: Coverslips >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Thanks for everyone's input on this, it's really appreciated. Julien >>> - although it doesn't mention it specifically I think the Zeiss link >>> makes the point about the requirement for precise cover glass >>> thickness with SIM. >>> If you're to achieve the best resolution possible you have to use > >> glass of a thickness that most closely matches that for which the >>> objective is designed (usually 170 um). Any deviation from this will >>> introduce sperical aberration. My guess is that because the >>> resolution of a confocal system is lower, you won't notice much of a >>> difference between an image acquired using 170 and a 180 um glass, >>> whereas on a SIM system you will. >>> Simon >>> >> >> >> Just one more point: It makes a lot of >>difference whether we are talking about oil or >>water lenses. >> >> As immersion oil is almost the same RI as the >> BK-7 glass that I think is used for coverslips, changes in thickness >> from the optimum are much less important than if you are using a water >> lens. In the latter case, (RI = 1.515 vs Ri >> 1.334?) even a 2µm error in thickness is >>easily noticeable by eye with an NA 1.2 >>objective. Other factors that affect the PSF >>(such as the temperature of the immersion oil!) >>are well covered in Chapter 11 of the Handbook. >> >> SIM is indeed curcially dependent of having a >>known and unaberrated PSF and this is only >>possible if you re free from SA. To check this, >>always have some sub-resolution beads in your >>preparation and ensure that they "go out of >>focus" in a manner that looks the same whether >>you focus up or focus down. >> >> JP >> >> >>> >>> -----Original Message----- >>> From: Confocal Microscopy List >>> [mailto:[hidden email]] On Behalf Of Julien Cau >>> Sent: 07 October 2011 08:26 >>> To: [hidden email] >>> Subject: Re: Coverslips >>> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Hi Martin, >>> >>> You are absolutely right and SIM does not need better coverslips than >>> any other 0.17 objective-based microscopy technique. >>> Our users use these coverslips for any experiment (widefield, >>> confocal, >>> SIM) and they appreciated the difference. >>> The SIM point is more "don't use a fancy device that promiss you >>> superesolution if you waste this potential with crap coverslips". If >>> you buy a wonderfull coffee machine, will you put in it moldy coffee beans? >>> Wrong thickness coverslips induce decreased light collection, >>> spherical aberrations and then reduced resolution. >>> >>> It is worth using them for any type of experiment using a 0.17 lens. >>> Alternatively, you can buy an expensive lens with a correction collar >>> that can counteract the effects of the coverslip thickness error. >>> >>> Best regards >>> >>> PS : see for instance >>> http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521 >>> E5 24C891852575A200721A7C for a direct comparison of imaging with >>> #1.5 vs >>> #2 coverglass. >>> >>> Le 07/10/2011 03:26, Martin Wessendorf a écrit : >>>> ***** >>>> To join, leave or search the confocal microscopy listserv, go to: >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>>> ***** >>>> >>>> On 10/6/2011 3:43 AM, Simon Walker wrote: >>>> >>>>> Not strictly a confocal question, but I'm sure someone out there >>>>> will be able to help. We are about to start using a SIM super >>>>> res system and have been advised that one of the critical factors >>>>> in acquiring optimal images is the cover glass. Specifcally, the > >>>> thickness of the glass needs to be consistant across the whole >>>>> coverslip (e.g. 170 um +/- 2 um), and reproducible between >>>>> coverslips. One option is to measure each individual coverslip >>>>> before use, but this seems rather impractical to me. Has anyone >>>>> looked into this, and if so, are there any manufacturers out >>>>> there who can provide cover glass with this high specification? >>>> >>>> My ignorance is showing here. Why are higher-quality coverslips >>>> needed for SIM? Or is this for live-cell imaging? >>>> >>>> Best wishes-- >>>> >>>> Martin Wessendorf >>> >>> -- >>> >>> ____________________________________________ >>> >>> */Julien Cau, PhD./* >>> >>> /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ >>> >>> Montpellier RIO Imaging >>> >>> Montpellier BIOCAMPUS, UMS3426 >>> >>> Arnaud de Villeneuve Campus Imaging Facility > >> >>> Institut de Génétique Humaine-CNRS >>> >>> 141, rue de la Cardonille >>> >>> F-34396 Montpellier(France) >>> >>> e-mail: [hidden email].fr_ <mailto:[hidden email]> >>> >>> phone: +33.4.34.35.99.90 >>> >>> mobile: +33.6.50.19.27.49 >>> >>> fax: +33.4.34.35.99.01 >>> >>> URL: _http://www.mri.cnrs.fr/_ >>> >>> ____________________________________________ >> >> >> -- >> James and Christine Pawley, 21 N. Prospect Ave. >> Madison, WI, 53726 Phone: 608-238-3953 -- James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726 Phone: 608-238-3953 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi there, A main advantage of the SIM technique is that you need few if any modifications in your sample preparation (but you need strong signal). SIM, as any confocal/widefield, needs microscopy best practices. That's said, then of course, it is more sensitive (as STED is), to coverslip thickness errors as it needs the pattern to be well projected for instance. But the coverslips are not any different from coverslips you would use with confocal and listers should not believe SIM needs fancy preparation of the sample. It just need care and the right coverslips, as any nice confocal image deserves as well. My understanding so far, and I might be wrong, is that the right coverslip thickness is very important with good dry/water objectives (and we noticed the difference) as well as oil objectives. Even if you use a mounting medium with the right refractive index, you never really have no RI mismatch in your preparation. And our experience with these high precision coverslips shows, when using oil objectives, improved light collection and image quality in sample like drosophila embryos, even if we used the right refractive index mounting medium. So my personal conclusion is it is important in any case anyway and not using right coverslips (which cost a few more cents per item) is of no advantage. If it doesn't help, it doesn't harm. To answer another question, we usually recommand liquid vectashield or hardening Prolong Gold as mounting media for oil lenses. Six years ago, we requested users to bring their mounting media and measured their RI with an Abbe refractometer. Among a dozen of immersion media tested (including home-made mowiol), liquid vectashield (and glycerol) had the right refraction index (hardening vectashield had a lower RI, although it is hard to measure has you have to leave it harden in the refractometer and I don't know whether the protocol is really correct or not). Then, later on, when released, we tested Prolong Gold and basically reproduced the RI over time ( a week!) chart the manufacturer claimed. The advantage of Prolong Gold is it hardens (no idea whether it modifies the geometry of the sample when hardening). Of course, there might be other mounting media we did not test, or our measures might be not precise enough, but we quite like them. We find both vectashield and Prolong Gold are doing ok to protect sample from severe photobleaching. Of note, at the time we tested several mounting solutions, from all commercial media specs sheets provided by users, the only one to mention the refractive index was vectashield. Again, this might be different now, but in a "right refractive index mounting medium chase" I would suggest the following criterion : does the specs sheets indicate the RI? Then most of them usually include antifading reagents. Hope it helped Julien -- ____________________________________________ */Julien Cau, PhD./* /Montpellier RIO Imaging Facility manager/Responsable technique MRI/ Montpellier RIO Imaging Montpellier BIOCAMPUS, UMS3426 Arnaud de Villeneuve Campus Imaging Facility Institut de Génétique Humaine-CNRS 141, rue de la Cardonille F-34396 Montpellier(France) e-mail: [hidden email].fr_ <mailto:[hidden email]> phone: +33.4.34.35.99.90 mobile: +33.6.50.19.27.49 fax: +33.4.34.35.99.01 URL: _http://www.mri.cnrs.fr/_ ____________________________________________ |
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In reply to this post by Unruh, Jay
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >A couple of things I have noticed about >coverslips. Firstly, if you ever do measure >commercial coverslips, you will notice that >within a batch, most are within a 5 um tolerance >with a few outliers. From batch to batch you >will notice far more differences (I had one >batch of #1.5's that was all almost 190 um). >Secondly, the comment about oil vs. water >objectives is a very important one. Oil >objectives are designed to image just above the >coverslip. Since the oil, coverslip, and front >lens are all of the same refractive index, it >makes no difference how thick the coverslip >is--that ends up being compensated by the >increased oil thickness necessary to achieve the >same focal depth into the sample. For water, I >have never noticed a 2 um difference by eye, but >fluorescence correlation spectroscopy is >uniquely sensitive to these things and I don't >see a significant difference as long as I stay >within a 5 um tolerance. I used to think that >the solution was to carefully adjust the >correction collar on water objectives and soon >discovered that the lag in the correction collar >is as much as 100 um! As a result, the numbers >on the correction collar are essentially >meaningless unless you always adjust from the >same direction (note that I have only tested >this for Zeiss objectives). The best practice >is to adjust the collar with a sensitive sample >and then leave it and use measured or high >precision coverslips for the remainder of the >experiment. > >Jay To see it by eye, you need only to have a bright, small object, a sensitive detector and lots of magnification. Adjust the collar, focus up and down. Look for symmetry (same "blobby-ring" image 1 µm above best focus as 1µm below). If it isn't z-symmetrical, adjust the collar and try again until it is z-symmetrical (Forget trying to guess if the spot is smaller. You can't, and z-symmetry is what you want.). After having done this a number of times, you will find you have adjusted the collar to the exactly same setting +- 2 µm (the small spacings on the collar of the Zeiss 1.2 cAPO). Such images are seen in FIg. 20.3 of the Handbook. The collar setting for series D was 2µm of glass-replaced-by-water different from that used in series A. The image is of 3 small holes in a mirror film. Cheers, JP >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On >Behalf Of James Pawley >Sent: Friday, October 07, 2011 10:32 AM >To: [hidden email] >Subject: Re: Coverslips > >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >>***** >>To join, leave or search the confocal microscopy listserv, go to: >>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>***** >> >>Thanks for everyone's input on this, it's really appreciated. Julien - >>although it doesn't mention it specifically I think the Zeiss link >>makes the point about the requirement for precise cover glass thickness >>with SIM. >>If you're to achieve the best resolution possible you have to use glass >>of a thickness that most closely matches that for which the objective >>is designed (usually 170 um). Any deviation from this will introduce >>sperical aberration. My guess is that because the resolution of a > >confocal system is lower, you won't notice much of a difference between >>an image acquired using 170 and a 180 um glass, whereas on a SIM system >>you will. >>Simon >> > > >Just one more point: It makes a lot of >difference whether we are talking about oil or >water lenses. > >As immersion oil is almost the same RI as the >BK-7 glass that I think is used for coverslips, >changes in thickness from the optimum are much >less important than if you are using a water >lens. In the latter case, (RI = 1.515 vs Ri >1.334?) even a 2µm error in thickness is easily >noticeable by eye with an NA 1.2 objective. >Other factors that affect the PSF (such as the >temperature of the immersion oil!) are well >covered in Chapter 11 of the Handbook. > >SIM is indeed curcially dependent of having a >known and unaberrated PSF and this is only >possible if you re free from SA. To check this, >always have some sub-resolution beads in your >preparation and ensure that they "go out of >focus" in a manner that looks the same whether >you focus up or focus down. > >JP > > >> >>-----Original Message----- >>From: Confocal Microscopy List >>[mailto:[hidden email]] On Behalf Of Julien Cau >>Sent: 07 October 2011 08:26 >>To: [hidden email] >>Subject: Re: Coverslips >> >>***** >>To join, leave or search the confocal microscopy listserv, go to: >>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>***** >> >>Hi Martin, >> >>You are absolutely right and SIM does not need better coverslips than >>any other 0.17 objective-based microscopy technique. >>Our users use these coverslips for any experiment (widefield, confocal, >>SIM) and they appreciated the difference. >>The SIM point is more "don't use a fancy device that promiss you >>superesolution if you waste this potential with crap coverslips". If >>you buy a wonderfull coffee machine, will you put in it moldy coffee beans? >>Wrong thickness coverslips induce decreased light collection, spherical >>aberrations and then reduced resolution. >> >>It is worth using them for any type of experiment using a 0.17 lens. >>Alternatively, you can buy an expensive lens with a correction collar >>that can counteract the effects of the coverslip thickness error. >> >>Best regards >> >>PS : see for instance >>http://www.meditec.zeiss.com/4125681F004CA025/Contents-Frame/C66A2521E5 >>24C891852575A200721A7C for a direct comparison of imaging with #1.5 vs >>#2 coverglass. >> >>Le 07/10/2011 03:26, Martin Wessendorf a écrit : >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> On 10/6/2011 3:43 AM, Simon Walker wrote: >>> >>>> Not strictly a confocal question, but I'm sure someone out there >>>> will be able to help. We are about to start using a SIM super res >>>> system and have been advised that one of the critical factors in >>>> acquiring optimal images is the cover glass. Specifcally, the >>>> thickness of the glass needs to be consistant across the whole >>>> coverslip (e.g. 170 um +/- 2 um), and reproducible between >>>> coverslips. One option is to measure each individual coverslip >>>> before use, but this seems rather impractical to me. Has anyone >>>> looked into this, and if so, are there any manufacturers out there >>>> who can provide cover glass with this high specification? >>> >>> My ignorance is showing here. Why are higher-quality coverslips >> > needed for SIM? Or is this for live-cell imaging? >>> >>> Best wishes-- >>> >>> Martin Wessendorf >> >>-- >> >>____________________________________________ >> >>*/Julien Cau, PhD./* >> >>/Montpellier RIO Imaging Facility manager/Responsable technique MRI/ >> >>Montpellier RIO Imaging >> >>Montpellier BIOCAMPUS, UMS3426 >> >>Arnaud de Villeneuve Campus Imaging Facility >> >>Institut de Génétique Humaine-CNRS >> >>141, rue de la Cardonille >> >>F-34396 Montpellier(France) >> >>e-mail: [hidden email].fr_ <mailto:[hidden email]> >> >>phone: +33.4.34.35.99.90 >> >>mobile: +33.6.50.19.27.49 >> >>fax: +33.4.34.35.99.01 >> >>URL: _http://www.mri.cnrs.fr/_ >> >>____________________________________________ > > >-- >James and Christine Pawley, 21 N. Prospect Ave. >Madison, WI, 53726 Phone: 608-238-3953 -- James and Christine Pawley, 21 N. Prospect Ave. Madison, WI, 53726 Phone: 608-238-3953 |
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